Cancer immunotherapy for promoting hyperacute rejection reaction

Abstract

The present application relates to a bifunctional therapeutic substance for treating cancer, comprising a targeting component that targets a tumor-associated antigen and an enzyme that, when delivered to a tumor by the targeting component, converts the tumor phenotype into the phenotype of a mismatched allograft or xenograft. The enzyme is connected to the targeting component. Also disclosed is a method for treating cancer, comprising the step of administering the bifunctional therapeutic substance.

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 347,710, filed on June 1, 2022. U.S. Provisional Patent Application No. 63 / 347,710 is hereby incorporated by reference in its entirety.

[0002] Field The present disclosure relates to cancer immunotherapy for promoting hyperacute rejection.

Background Art

[0003] Background Combination therapy is a commonly accepted and common treatment approach for substantially all types of cancer and has been the standard treatment approach for decades. The basis for the adoption of combination therapy was the early experience in chemotherapy, which confirmed that due to the high mutation rate of cancer, resistant strains of tumor cells rapidly develop when only one type of drug is used. The goal of combination therapy is to increase efficacy and minimize the development of tumor resistance or escape. This is generally achieved by using two or more anticancer drugs, each with a different mechanism of action, thereby making it difficult and less likely for resistant tumor cells to develop. The additive or synergistic effect of combining two or more drugs can make the difference between the success and failure of a patient's treatment.

[0004] Many combination treatment regimens are well known in the field of oncology. As an example, MOPP (the acronym for mechlorethamine, vincristine, procarbazine, prednisone) is a curative treatment regimen for Hodgkin's disease. In the treatment of testicular cancer, several different combination regimens (all including cisplatin, vinblastine, and bleomycin) are accepted, and testicular cancer is curable in up to 98% of diagnosed cases. More than 300 different combination regimens have been used.

[0005] A major drawback of combination therapies is that they often also cause an increase in toxicity. For example, most forms of non-surgical cancer therapies such as external beam radiation and chemotherapy have limited efficacy due to toxic side effects on normal tissues and cells and limited specificity of these treatment modalities for cancer cells. This limitation is particularly important even when anti-cancer antibodies are used to target toxic agents such as isotopes, drugs, and toxins to the cancer site, because anti-cancer antibodies also circulate as systemic agents in sensitive cell compartments such as the bone marrow. In acute radiation injury, destruction of the lymphoid and hematopoietic compartments is a major cause of the development of sepsis and subsequent death. Thus, there is a great need for ways to reduce the toxic effects of cancer therapies while maintaining, and even enhancing, efficacy.

[0006] Another option for combination therapies is that recent advances in immunotherapy have clearly established that the immune system can be involved in responses to cancer, and that these responses can be highly effective and long-lasting. From considerable experience using immune checkpoint inhibition, it has been suggested that its greatest benefit lies in its application to cancers with a relatively high amount of mutations. However, even in such cases, only a minority of patients respond. Some cancers, such as prostate cancer, have no immune cells in the tumor microenvironment. This absence of immune cells is sometimes referred to as a "cold" microenvironment or an immune "desert," which significantly limits the ability to activate the immune system. Chimeric antigen receptor T (CAR-T) cells and bispecific T cell engagers (BiTEs) utilize antibody targeting of tumor-associated antigens to induce the T cell lytic machinery to lyse cancer cells. However, to date, the anti-tumor activity of CAR-T and BiTE has been limited to hematological cancers rather than common solid tumors. Clearly, further methods for treating various cancers are still needed.

[0007] The present disclosure relates to overcoming these and other deficiencies in the art. SUMMARY OF THE INVENTION

[0008] Summary One aspect of the present disclosure relates to a bifunctional therapeutic substance for treating cancer, comprising a targeting component that targets a tumor-associated antigen and an enzyme that enzymatically converts a tumor phenotype into the phenotype of an incompatible allograft or xenograft when delivered to a tumor by the targeting component. The enzyme is connected to the targeting component.

[0009] Another aspect of the present disclosure relates to a method for treating cancer. The method includes the steps of selecting a subject having cancer; providing a bifunctional therapeutic substance according to the present disclosure; and administering the bifunctional therapeutic substance to the selected subject under conditions effective to treat the cancer.

[0010] A novel immunotherapy approach is presented in which a tumor-targeted glycosyltransferase adds a non-self histo-blood group antigen (HBGA) or an α-gal glycotope to alter the glycan phenotype of a tumor and / or its blood vessels. This effectively converts the tumor into an HBGA-incompatible allograft or xenograft. Exemplary embodiments of this multifunctional agent can target PSMA / FOLH1 to convert the tumor's neovasculature into an incompatible HBGA or xenograft, thereby initiating a hyperacute rejection response. In half a century of transplantation experience, it has been recorded that HBGA-incompatible allografts or α-gal-expressing xenografts stimulate an active immune rejection process.

[0011] As described herein, in order to cause the expression of heterologous or homologous antigens by tumors, heterologous or homologous glycosyltransferases, such as α-galactosyltransferase (αgalT) or homologous glycosyltransferase A and / or B enzymes, although these are normally all in the Golgi, are delivered to the tumor cell surface. That is, in effect, it becomes a molecular-scale allograft / xenograft. In addition to the targeting of glycosyltransferases (αgalT, glycosyltransferase A and / or B enzymes), the respective sugar-nucleotide donors (UDP-gal or UDP-NAcGal) are supplied. When glycosyltransferase is present in the tumor, sugar (gal or NAcGal) is added to existing glycoproteins and glycolipids, including products secreted by target cells, to generate homologous or heterologous antigens, thereby inducing a strong immune response. The converted homologous / heterologous proteins secreted into the microenvironment bind to a large amount of natural antibodies that induce complement activation, immune response, and antibody-dependent cell cytotoxicity (ADCC), and help convert the "cold" microenvironment into a "hot" microenvironment.

[0012] Glycosyltransferase A and glycosyltransferase B enzymes differ by only four out of 353 amino acid residues (the entirety of which is incorporated herein by reference, Hakomori, "Antigen Structure and Genetic Basis of Histo-Blood Groups A, B and O: Their Changes Associated With Human Cancer", Biochimica et Biophysica Acta 1473:247-266 (1999); Seto et al., "Sequential Interchange of Four Amino Acids From Blood Group B to Blood Group A Glycosyltransferase Boosts Catalytic Activity and Progressively Modifies Substrate Recognition in Human Recombinant Enzymes", J. Biol. Chem. 272:14133-14138 (1997)), and thus are unlikely to be immunogenic. Studies of patient sera have confirmed that these enzymes are not immunogenic as expected. Indeed, while these HBGA carbohydrate products are highly immunogenic, no reports have indicated that transferase A and transferase B enzymes are immunogenic. Targeted delivery of non-immunogenic transferase A or B enzymes provides a means of changing the immune phenotype of tumors or neovessels to one that expresses highly immunogenic non-self HBGA, thereby presenting the phenotype of an incompatible allograft and promoting an active rejection response by the host.

[0013] As described herein, for proof of concept, the above approach was verified using human-derived GTA or GTB. Instead, a heterologous α-gal transferase (α1,3 galactosyltransferase; α-galT) enzyme, which is mutated / non-functional in humans and is responsible for causing rejection of xenotransplanted organs derived from other mammals, could be utilized. The use of the α-galT enzyme may require humanization or de-immunization of α-galT, and there are methods known in the art for accomplishing this, including but not limited to the use of homologous region sequences of other glycosyltransferases that are not immunogenic to humans. Such humanization or de-immunization methods have been widely and successfully used for the purpose of humanizing or de-immunizing foreign-derived antibodies before use as therapeutic agents in humans. However, studies of patient sera have shown that these enzymes are not immunogenic.

[0014] The present disclosure presents a novel immunotherapy approach in which a glycosyltransferase targeted to a tumor alters the expression of the tumor's tissue blood group antigen and / or its blood supply. This effectively converts the tumor into an allograft that is incompatible with HBGA.

[0015] As described herein, a complementary orthogonal immunotherapy approach was developed based on the understanding of the active immune response against xenografts or allografts and the rejection process that has evolved over the past half century. To accomplish this, the most extreme form of the host-graft response, namely hyperacute rejection (HAR), was selected as the model.

[0016] HAR results from ancestral mutations in either of two highly related genes: α1,3 galactosyltransferase (α1,3GalT) in the case of xenografts (Collins, et al., 「Cardiac Xenografts Between Primate Species Provide Evidence for the Importance of the Alpha-Galactosyl Determinant in Hyperacute Rejection」, J. Immunol. 154:5500-5510 (1995), which is hereby incorporated by reference in its entirety), and the well-known histo-blood group antigen (HBGA) locus in the case of allografts (Milland et al., 「ABO Blood Group and Related Antigens, Natural Antibodies and Transplantation」, Tissue Antigens 68:459-466 (2006), which is hereby incorporated by reference in its entirety). These two highly related genes are found on the same chromosome (9q34), have 45% homology, and are thought to be derived from the same ancestral gene (Yamamoto et al., 「Molecular Genetic Basis of the Histo-Blood Group ABO System」, Nature 345:229-233 (1990); Yamamoto et al., 「Sugar-Nucleotide Donor Specificity of Histo-Blood Group A and B Transferases is Based on Amino Acid Substitutions」, J. Biol. Chem. 265:19257-19262 (1990); Yamamoto et al., 「Genomic Organization of Human Histo-Blood Group ABO Genes」, Glycobiology 5:51-58 (1995), which are hereby incorporated by reference in their entirety).These alleles encode glycosyltransferases that add terminal sugar moieties to carbohydrate (CHO) chains present on nascent proteins and lipids destined for cell membrane expression or secretion post-translationally. The human and Old World monkey αGalT enzymes were inactivated approximately 28 million years ago due to mutations, but not in other mammals (Macher et al., "The Gal Alpha1,3Gal Beta1,4GlcNAc-R (Alpha-Gal) Epitope: a Carbohydrate of Unique Evolution and Clinical Relevance", Biochim. Biophys. 1780:75-88 (2008), which is hereby incorporated by reference in its entirety). As a result, xenotransplanted organs and tissues derived from non-primate mammals express the αgal epitope, which is foreign to humans. In the case of the HBGA locus, a few mutations gave rise to the alleles classically known as A, B, and O. The B allele encodes glycosyltransferase B (GTB), which, like its α1,3GalT homolog, adds terminal Gal to the CHO chain. The only difference is that transferase B adds Gal only when 1,2 fucose is present on the adjacent Gal. Transferase A differs functionally from transferase B only in that it adds N-acetylated terminal Gal (NAcGal). The O gene product is inactive due to a frameshift mutation (Figure 1).

[0017] The α-Gal, HBGA A, and HBGA B epitopes generated by these three active enzymes are widely expressed in nature, including bacteria that inhabit the human gastrointestinal tract (Springer et al., 「Blood Group Isoantibody Stimulation in Man by Feeding Blood Group-Active Bacteria」, J. Clin. Invest. 48:1280-1291 (1969), which is hereby incorporated by reference in its entirety). As a result, humans lacking aGalT as well as the A and / or B alleles are constantly immunized by these bacterial-derived epitopes. This results in very high levels of natural antibodies (Abs) against these non-self epitopes, which constitute more than 1% of plasma immunoglobulins (Igs) (Galili et al., 「One Percent of Human Circulating B Lymphocytes are Capable of Producing the Natural Anti-Gal Antibody」, Blood 82:2485-2493 (1993); Galili et al., 「A Unique Natural Human IgG Antibody With Anti-Alpha-Galactosyl Specificity」, J. Exp. Med. 160:1519-1531 (1984), which are hereby incorporated by reference in their entireties). Considering the diversity of the Ab repertoire, which is estimated to be in the billions of different specificities, this represents a very large proportion of endogenous Ig activity.These Abs consist of IgM and IgG, which can activate the complement cascade and then initiate vascular thrombosis (Subramaniam et al., "Distinct Contributions of Complement Factors to Platelet Activation and Fibrin Formation in Venous Thrombus Development", Blood 129(16):2291 - 2302 (2017); Foley et al., "Cross Talk Pathways Between Coagulation and Inflammation", Circ. Res. 118:1392 - 1408 (2016); and Conway EM, "Reincarnation of Ancient Links Between Coagulation and Complement", J. Thromb. Haemost. 13(Suppl. 1):S121 - S32 (2015), which are all incorporated herein by reference). Other immunoglobulin classes such as IgA and IgE can also be made against these glycol - epitopes. In fact, the presence of evolutionary mutations in these two genes results in an immunological state that is maintained at the switch point, primed by a first stimulus, and ready to respond rapidly, aggressively, and destructively to the appearance of any of these non - self epitopes. The immunological effects of these mutations have hindered the success of xenografts in humans, explaining why HBGA matching has been the single most important match in solid organ transplantation since its crucial importance was first recognized by Starzl, Experience In Renal Transplantation. (WB Saunders Company, Philadelphia, PA, chapter 6 (1964), which is incorporated herein by reference) at the dawn of kidney allotransplantation in the 1960s.Since then, the devastating effects of HBGA mismatches in solid organ transplantation have only been recognized in the very rare cases of iatrogenic error (Altman, Doctors Discuss Transplant Mistake. New York Times, Feb 22, 2003, which is hereby incorporated by reference in its entirety). Against this background, it has been the goal to induce the expression of one of these non-self epitopes by host cancer cells and / or by the vascular endothelial cells that supply the tumor.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0019] Detailed Description The present disclosure discloses a bifunctional therapeutic substance for treating cancer, which comprises a targeting component targeting a tumor-associated antigen and an enzyme that enzymatically converts a tumor phenotype into the phenotype of a mismatched allograft or xenograft when delivered to a tumor by the targeting component. The enzyme is connected to the targeting component.

[0020] The present disclosure also relates to a method for treating cancer. The method includes the steps of selecting a subject having cancer and providing a bifunctional therapeutic substance according to the present disclosure. The bifunctional therapeutic substance is administered to the selected subject under conditions effective to treat cancer.

[0021] As used herein, the term "treating" refers to applying or administering to a subject, such as a patient, the bifunctional therapeutic substance of the present disclosure. Treatment may cure, treat, relieve, alleviate, alter, mitigate, remit, soothe, improve, or affect cancer, cancer symptoms, or a predisposition to cancer.

[0022] As used herein, the term "subject" is intended to include humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0023] As used herein, the term "cancer" includes all types of cancer growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, regardless of histological type or stage of invasiveness.

[0024] As used herein, "incompatible allograft" refers to a tissue or tumor that induces hyperacute, acute, and / or chronic immune rejection reactions. Hyperacute rejection reactions occur within minutes to hours after organ transplantation, i.e., after the bifunctional therapeutic substance has been delivered and the tumor or tissue has been transformed as described herein. This rapid rejection reaction is characterized by vascular thrombosis leading to necrosis of the graft / tumor. Hyperacute rejection reactions are caused by the presence of anti-donor antibodies in the recipient prior to transplantation / transformation.

[0025] As used herein, a "targeting component" is a component that can bind to a tumor-associated antigen or otherwise associate with a tumor-associated antigen. Such tumor-associated antigens include, but are not limited to: FOLH1 / PSMA, VEGFR, CD19, CD20, CD25, CD30, CD33, CD38, CD52, B cell maturation antigen (BCMA), CD79, somatostatin receptor, 5T4, gp100, carcinoembryonic antigen (CEA), mammaglobin A, Melan A / MART-1, MAGE, NY-ESO-1, PSA, tyrosinase, HER-2 / neu, HER-3, EGFR, hTERT, mesothelin, nectin-4, TROP-2, tissue factor, MUC-1, CA-125, and peptide fragments thereof, protein MZ2-E, polymorphic epithelial mucin, folate-binding protein, cancer testis protein MAGE-1 or MAGE-3 or NY-ESO-1, human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP), pancreatic oncofetal antigen, CA-15-3, 19-9, 549, 195, squamous cell carcinoma antigen (SCCA), ovarian cancer antigen (OCA), pancreatic cancer-associated antigen (PaA), mutant K-ras protein, mutant p53, wild-type p53, truncated epidermal growth factor receptor (EGFR), chimeric protein p210BCR-ABL, telomerase, survivin, WT1 protein, LMP2 protein, HPV E6 E7Protein, idiotypic protein, PAP protein, ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF binding protein (IGFBP), IGFR ligand family (IGF-1R); platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, VEGF family; HGF receptor family: TRK receptor family; ephrin (EPH) receptor family (e.g., EphA2); AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR) receptor family; discoidin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; transforming growth factor α (TGF-α), TGF-α receptor; transforming growth factor-β (TGF-β), TGF-β receptor; interleukin β receptor α2 chain (IL13Rα2), interleukin-6 (IL-6), 1L-6 receptor, interleukin-4, IL-4 receptor, cytokine receptor, class I (hematopoietin family) and class II (interferon / 1L-10 family) receptors, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily (TNTRSF), death receptor family, TRAIL receptor; cancer testis (CT) antigen, lineage-specific antigen, differentiation antigen, α-actinin-4, ARTC1, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, elongation factor 2 (ELF2), Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low density lipoprotein receptor / GDP-L-fucose:β-D-galactose 2-α-L-fucosyltransferase (LDLR / FUT) fusion protein, HLA-A2, HLA-A11, heat shock protein 70-2 mutant (HSP70-2M), KIAA0205, MART2, melanoma ubiquitous mutant 1, 2, 3 (MUM-1, 2, 3), neoPAP, myosin class 1, NFYC, OGT, OS-9, pml-RARα fusion protein, PRDXS, PTPRK, N-ras (NRAS), HRAS, RBAF600, SIRT12, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, triosephosphate isomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8, GnT-V (abnormal N-acetylglucosaminyltransferase V, MGATS), HERV-K MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognized antigen on melanoma (CAMEL), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, gp100 / Pme117 (S1LV), TRP-1, HAGE, NA-88, NY-ESO-1 / LAGE-2, SAGE, Sp17, SSX-1, 2, 3, 4, TRP2-1NT2, kallikrein 4, mammaglobin-A, OA1, TRP-1 / , 75, TRP-2 adipophilin, interferon-inducible protein 2 not present in melanoma (AIM-2), BING-4, CPSF, cyclin D1, cyclin B1, epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250 intestinal carboxylesterase (iCE), M-CSF, mdm-2, MUCI, PBF, PRAME, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE, TPTE, immunoglobulin idiotype, Bence Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD4, CD3, cancer antigen 72-4 (CA 72-4), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), beta-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96, GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4), carcinoembryonic antigen peptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B), human stromal tumor-2 (HST-2), PR1, claudin family (e.g., claudin 1, claudin 3, claudin 4, claudin 6, claudin 7, claudin 18.2, etc.), GPC3, GD2, EpCam, CD70, CD123, prostate stem cell antigen (PSCA), CD133, ROR1, FAP, EGFRVIII, CA9, ML-IAP, ERG (TMPRSS2 ETS fusion), NA17, PAX3, ALK, MYCN, RhoC, GD3, PLAC1, CD166, LIV1A, CD71, CD228, P-cadherin, LAMP1, Napi2b, etc., and peptide fragments thereof, but not limited thereto. In some embodiments, the tumor-associated antigen is a neoantigen. In such some embodiments, the targeting domain is a TCR that binds to the MHC / neoantigen complex on the surface of tumor cells. In some embodiments, the neoantigen is derived from G12D KRAS mutations, MART-1, gp100, NY-ESO-1, CEA, MAGE-A3, MAGE-A4, or WT1. See, for example, Leko and Rosenberg, Cancer Cell 38: 454 (2020). The foregoing list exemplifies tumor-associated antigens. Additional tumor-associated antigens are known to those of skill in the art.

[0026] The antigen may be, for example, an antigen or epitope present on tumor cells in the lung, breast, esophagus, intestine, stomach, rectum, kidney - urinary system, prostate, bladder, brain, thyroid, liver, pancreas, spleen, skin, connective tissue, heart, blood system, or vascular system. The target antigen may be an antigen or epitope present on the cell membrane, on the surface of a secreted protein, or on the surface of a non - membrane - bound protein. Examples of secreted proteins include, but are not limited to, hormones, enzymes, toxins, and antimicrobial peptides.

[0027] The targeting component may be localized or concentrated at a specific targeted site, such as a tumor, disease site, tissue, organ, cell type, infectious bacterium or virus, etc.

[0028] For example, the intended targeting component includes peptides, polypeptides, proteins, glycoproteins, aptamers, carbohydrates, or lipids. The targeting component may be a natural or synthetic ligand for a cell - surface receptor, such as a growth factor, hormone, LDL, transferrin, etc. The targeting component may be an antibody, and this term is intended to include antibody fragments and derivatives, characteristic parts of the antibody, such as single - chain targeting moieties that can be identified using procedures such as phage display. The targeting component may also be a targeting peptide, targeting peptidomimetic, or small molecule, whether natural or artificially created (e.g., via chemical synthesis).

[0029] In one aspect, the targeting component is selected from the group consisting of an antibody or its antigen - binding fragment, a protein, a peptide, an aptamer, and a small molecule.

[0030] Antibodies against tumor-associated antigens are known. For example, antibodies and antibody fragments that specifically bind to markers produced by or associated with tumors are disclosed in U.S. Patent No. 3,927,193 to Hansen, as well as U.S. Patents Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,818,709, and 4,624,846 to Goldenberg, which are hereby incorporated by reference in their entirety. In particular, antibodies against tumor-associated antigens, such as gastrointestinal tumors, lung tumors, breast tumors, prostate tumors, ovarian tumors, testicular tumors, brain tumors, or lymphoid or hematopoietic tumors, sarcomas or melanomas, are advantageously used. Antibodies against tumor-associated antigens are well known to those skilled in the art.

[0031] The antibodies of the present disclosure can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), antibody fragments (e.g., Fv, Fab, and F(ab)2), half-antibodies, hybrid derivatives, as well as single-chain antibodies (scFv), chimeric antibodies, and deimmunized or humanized antibodies (Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 1999); Houston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli", Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Bird et al, "Single-Chain Antigen-Binding Proteins", Science 242:423-426 (1988). Each of these is hereby incorporated by reference in its entirety).

[0032] The antibodies of the present disclosure may also be prepared using recombinant DNA techniques, such as, for example, antibodies or fragments thereof expressed by bacteriophage. Alternatively, synthetic antibodies are prepared by synthesizing a DNA molecule encoding and expressing the antibody of the present disclosure, or by synthesizing the amino acid sequence specifying the antibody. In this case, the DNA sequence or amino acid sequence is obtained using available and well-known synthetic DNA sequence techniques or amino acid sequence techniques in the art.

[0033] Methods for the production of monoclonal antibodies may be carried out using the techniques described herein or are well known in the art (MONOCLONAL ANTIBODIES - PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman eds., 1995), which is incorporated herein by reference in its entirety). Generally, the process involves obtaining immune cells (lymphocytes) from the spleen of a mammal previously immunized with an antigen of interest, either in vivo or in vitro.

[0034] Alternatively, monoclonal antibodies can be made using recombinant DNA methods as described in U.S. Patent No. 4,816,567 to Cabilly et al., which is hereby incorporated by reference in its entirety. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells by RT-PCR using, for example, oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody. The isolated polynucleotides encoding the heavy and light chains are then cloned into an appropriate expression vector and introduced by transfection into a host cell that does not produce immunoglobulin protein in the absence of the expression vector, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, whereupon the host cells produce the monoclonal antibody. Similarly, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries (McCafferty et al., 「Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains」, Nature 348:552-554 (1990); Clackson et al., 「Making Antibody Fragments using Phage Display Libraries」, Nature 352:624-628 (1991); and Marks et al., 「By-Passing Immunization. Human Antibodies from V-Gene Libraries Displayed on Phage」, J. Mol. Biol. 222:581-597 (1991), which are hereby incorporated by reference in their entirety).

[0035] The polynucleotide encoding the monoclonal antibody can be further modified using recombinant DNA techniques to yield another antibody or derivative. For example, the constant domains of the light and heavy chains of a murine monoclonal antibody can be replaced with the constant domains of the light and heavy chains derived from a human antibody to yield a chimeric antibody. Or, the constant domains of the light and / or heavy chains of the monoclonal antibody can be replaced with a non-immunoglobulin polypeptide to yield a fusion antibody. In other embodiments, the constant region is cleaved or removed to yield a desired antibody fragment of the monoclonal antibody. Furthermore, the specificity and affinity of the monoclonal antibody can be optimized using site-directed mutagenesis or high-density mutagenesis of the variable region.

[0036] The monoclonal antibodies of the present disclosure may be humanized antibodies. A humanized antibody is an antibody that contains a minimal sequence derived from a non-human (e.g., murine) antibody within the variable region. Such antibodies are therapeutically useful for reducing antigenicity and the human anti-mouse antibody response when administered to a human subject. Indeed, a humanized antibody is typically a human antibody that has minimal or no non-human sequence. A human antibody is an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.

[0037] In addition to the whole antibody, the present disclosure includes the antigen-binding portion of such an antibody. Such binding portions include monovalent Fab fragments, Fv fragments (e.g., single-chain antibodies, scFv), as well as single variable V H and V Lincluding domains, as well as F(ab’)2 fragments, Bis-scFv, diabodies, triabodies, minibodies, etc. These antibody fragments may be made by conventional procedures such as proteolytic fragmentation procedures as described in James Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (Academic Press, 1983), and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, 1988), which are hereby incorporated by reference in their entirety, or by other methods known in the art.

[0038] Furthermore, it may be desirable to modify the antibody, particularly in the case of antibody fragments, to extend its half-life in serum. This can be accomplished, for example, by mutating an appropriate region in the antibody fragment, by incorporating a salvage receptor binding epitope into the antibody fragment, or by incorporating this epitope (e.g., by DNA synthesis or peptide synthesis) into a peptide tag and then fusing it at the terminus or in the middle of the antibody fragment.

[0039] Antibody mimetics are also suitable for use according to the present disclosure. The 10th human fibronectin type III domain ( 10Antibody mimetics known as monobodies derived from Fn3) (Koide et al., "The Fibronectin Type III Domain as a Scaffold for Novel Binding Proteins", J. Mol. Biol. 284:1141-1151 (1998); Koide et al., "Probing Protein Conformational Changes in Living Cells by Using Designer Binding Proteins: Application to the Estrogen Receptor", Proc. Natl. Acad. Sci. USA 99:1253-1258 (2002). Each of these is incorporated herein by reference in its entirety); and antibody mimetics known as affibodies derived from the stable α-helical bacterial receptor domain Z of staphylococcal protein A (Nord et al., "Binding Proteins Selected from Combinatorial Libraries of an alpha-helical Bacterial Receptor Domain", Nature Biotechnol. 15(8):772-777 (1997), which is incorporated herein by reference in its entirety), although a number of antibody mimetics are known in the art, including but not limited to these.

[0040] In certain embodiments, the targeting component is a peptide that binds to a tumor-associated antigen. Exemplary peptides include, but are not limited to, glutamate-urea-lysine derivatives such as 2(3-99S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid (ACUPA) that binds to FOLH1 / PSMA, somatostatin derivatives that bind to SSTR2, and Arg-Gly-Asp (RGD) peptides that bind to α-v / β-3 integrin.

[0041] The peptides used in conjunction with the present disclosure may be obtained by known isolation and purification protocols from natural sources, may be synthesized by standard solid-phase peptide synthesis or liquid-phase peptide synthesis according to known peptide sequences of the peptides, or may be obtained from commercially available preparations or peptide libraries. Peptides that exhibit the biological binding properties of natural peptides and retain the specific binding properties of natural peptides are included herein. Derivatives and analogs of the peptides used herein, so long as they retain the specific binding properties of the natural peptides, include modifications to the composition, identity, and derivatization of the individual amino acids of the peptides. Examples of such modifications would include D-stereoisomers, substitutions in the aromatic side chains of aromatic amino acids, derivatization of amino or carboxyl groups in the side chains of amino acids containing amino or carboxyl groups in the side chains, substitutions at the amino or carboxyl termini of the peptides, conjugation of the peptides with a second peptide or a biologically active moiety, and cyclization of the peptides (G. Van Binst and D. Tourwe, 「Backbone Modifications in Somatostatin Analogues: Relation Between Conformation and Activity」, Peptide Research 5:8-13 (1992), which is hereby incorporated by reference in its entirety).

[0042] As used herein, 「small molecule」 typically refers to an organic molecule, peptide molecule, or non-peptide molecule having a molecular weight of less than 10,000 Da, preferably less than 5,000 Da, more preferably less than 1,000 Da, and most preferably less than 500 Da. This class of modulators includes molecules synthesized chemically, e.g., compounds derived from combinatorial chemical libraries.

[0043] In certain embodiments, the targeting component is an aptamer. An aptamer is a small single-stranded DNA or RNA oligonucleotide that specifically binds to a target molecule (e.g., a tumor-associated antigen) with high affinity and specificity. Aptamers are created using an in vitro selection process called systematic evolution of ligands by exponential enrichment (SELEX), which is described in Ellington et al., 「In Vitro Selection of RNA Molecules That Bind Specific Ligands」, Nature 346:818-822 (1990), and Jayasena, 「Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics」, Clin. Chem. 45:1628-1650 (1999), which are hereby incorporated by reference in their entirety. Several aptamers have been developed that can target tumor-associated antigens, including but not limited to MUC1, HER2, HER3, EpCAM, NF-kB, PSMA, CD44, PD-1, CD137, CD134, PDGF, VEGF, and NCL (Jayasena, 「Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics」, Clin. Chem. 45:1628-1650 (1999), which is hereby incorporated by reference in its entirety).

[0044] In some embodiments, the targeting component is a TCR specific for a tumor antigen. Human TCRs contain two variable domains (Vα and Vβ) joined together by constant regions (Cα and Cβ). In some embodiments, the TCR targeting component comprises a Vα domain and a Vβ domain that bind to an MHC / neoantigen complex on the surface of tumor cells. In such some embodiments, the TCR targeting component may be a single-chain TCR (scTCR), in which the Vα domain and the Vβ domain are linked by a flexible peptide. In some embodiments, the targeting component is a single variable domain TCR, for example, a TCR of Vβ only. As used herein, the term "TCR" includes any form of TCR, including Vα / Vβ complexes, single-chain TCRs (scTCRs), and single variable domain TCRs (also referred to as single domain TCRs or sdTCRs).

[0045] KRAS G12D , certain common tumor-associated neoantigens are known in the art, including neoantigens derived from MART-1, gp100, NY-ESO-1, CEA, MAGE-A3, MAGE-A4, and WT1. See, for example, He et al., J. Hematol. & Oncol. 12:139 (2019); Oh et al., Scientific Reports 9:17291 (2019), which are incorporated herein by reference in their entirety.

[0046] In one aspect, the targeting moiety is an Anticalin®. Anticalins are based on human lipocalin proteins, a family of abundant plasma proteins characterized by one central β-barrel and four variable loops that form the binding site. See, e.g., Rothe and Skerra, 2018, BioDrugs 32(3):233-243, which is hereby incorporated by reference in its entirety. Using random library design, Anticalins that bind to various tumor antigens, including Anticalins that bind to CTLA-4, PSMA, VEGFR-3, and Hsp70, have been developed. An Anticalin is a lipocalin family polypeptide in which the amino acid positions in the region of four peptide loops are mutated, and these four peptide loops are arranged at the ends of a cylindrical β-barrel structure surrounding the binding pocket and correspond to segments in a linear polypeptide sequence comprising amino acid positions 28-45, 58-69, 86-99, and 114-129 of the bilin-binding protein of Pieris brassicae. See, e.g., WO2005019255A1; WO2012065978A1; and WO1999016873A1, which are hereby incorporated by reference in their entirety.

[0047] In some embodiments, the targeting moiety may be a single domain antibody (an "sdAb", also known as a heavy chain only antibody) that can be obtained from camelid animals, as described in, for example, Eyer, L., and K. Hruska. "Single-domain antibody fragments derived from heavy-chain antibodies: a review." Veterinarni Medicina 57.9 (2012): 439. Due to the absence of a light chain, the antigen-binding site of a heavy chain antibody is formed by only three complementary determining regions (CDRs), as compared to the six CDRs in a conventional antibody. Single domain antibody fragments exhibit high affinity for binding into crevices and cavities on the protein surface, which offers the possibility of developing selective therapeutic agents for modulating the activity of cell surface proteins such as receptors involved in cancer and inflammatory diseases, ion channels, and leukocyte ectoenzymes. Wei GW, Meng WX, Guo HJ, Pan WQ, Liu JS, Peng T, Chen L, Chen CY (2011): Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein. Plos One 6; Altintas I, Kok RJ, Schiffelers RM (2012): Targeting epidermal growth factor receptor in tumors: From conventional monoclonal antibodies via heavy chain-only antibodies to nanobodies. European Journal of Pharmaceutical Sciences 45, 399-407.

[0048] In certain embodiments, the targeting moiety targets the prostate specific membrane antigen (PSMA) receptor.

[0049] As used herein, the term "PSMA" or "prostate-specific membrane antigen" protein refers to mammalian PSMA, preferably human PSMA protein. PSMA is sometimes also referred to as folate hydrolase 1 (FOLH1) when encoded by the FOLH1 gene. The long transcript of PSMA has sequence homology to the transferrin receptor and encodes a protein product with a molecular weight of approximately 100-120 kDa, characterized as a type II transmembrane receptor with NAALADase activity (Carter et al., "Prostate-Specific Membrane Antigen is a Hydrolase With Substrate and Pharmacologic Characteristics of a Neuropeptidase", Proc. Natl. Acad. Sci. USA 93:749-753 (1996); Israeli et al., "Molecular Cloning of a Complementary DNA Encoding a Prostate-Specific Membrane Antigen", Cancer Research 53:227-230 (1993), which are hereby incorporated by reference in their entirety).

[0050] As used herein, the term "enzyme" includes any enzyme, protein, or peptide that catalyzes the conversion of a tumor or tissue into an allograft when delivered to the tumor or tissue by a targeting component.

[0051] In one aspect, the enzyme is an enzyme involved in post-translational modification and is selected from the group consisting of transferases and glycosyltransferases.

[0052] A transferase is any one of a class of enzymes that transfer a specific functional group (e.g., a methyl group or a glycosyl group) from one molecule (referred to as the donor) to another molecule (referred to as the acceptor).

[0053] Exemplary groups of transferases include, but are not limited to, glycosyltransferases. Glycosyltransferases catalyze the stepwise addition of an activated sugar (donor NDP-sugar) to a protein, glycoprotein, lipid or glycolipid, or to the non-reducing end of a growing oligosaccharide (Lairson et al., 「Glycosyltransferases: Structures, Functions, and Mechanisms」, Annu. Rev. Biochem. 77:521-55 (2008), which is hereby incorporated by reference in its entirety). Glycosyltransferases are well known in the art.

[0054] Mammals utilize nine sugar nucleotide donors for glycosyltransferases: UDP-glucose, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, UDP-xylose, UDP-glucuronic acid, GDP-mannose, GDP-fucose, and CMP-sialic acid.

[0055] For enzymatic oligosaccharide synthesis involving glycosyltransferase reactions, glycosyltransferases can be cloned, i.e., isolated, from any source. Many cloned glycosyltransferases are known, and similarly, their polynucleotide sequences are also known (see, for example, 「The WWW Guide To Cloned Glycosyltransferases」, Taniguchi et al., 2002, Handbook of Glycosyltransferases and Related Genes, Springer, Tokyo, which is hereby incorporated by reference in its entirety). The amino acid sequences of glycosyltransferases, and nucleotide sequences encoding glycosyltransferases from which the amino acid sequences can be deduced, are also well known in the art.

[0056] Glycosyltransferases that can be used in the methods of the present disclosure include, but are not limited to, galactosyltransferase, fucosyltransferase, glucosyltransferase, N-acetylgalactosaminyltransferase, N-acetylglucosaminyltransferase, glucuronyltransferase, sialyltransferase, mannosyltransferase, glucuronic acid transferase, galacturonic acid transferase, and oligosaccharide glycosyltransferase. Suitable glycosyltransferases include glycosyltransferases obtained from eukaryotes as well as prokaryotes.

[0057] Glycosyltransferases have significant implications for the development of the ABO blood group antigen system. As described above, the ABO blood group system is the first important antigen system in blood transfusion and solid organ transplantation. This tissue blood group antigen (HBGA) system is controlled by the activities of GTA and / or GTB glycosyltransferases that attach sugar residues (N-acetylgalactosamine or galactose) to a common substrate (H antigen). There are several phenotypic variants of this enzyme that either change the attached carbohydrate (N-acetylgalactosamine (A) versus galactose (B)) or cause a loss-of-function type of the enzyme so that the H antigen is not modified (O). The A variant A2 has reduced levels of N-acetylgalactosamine activity and NAc-gal addition. These variants are currently distinguished serologically and by lectin binding (defining A1 versus A2). Serology can detect the modification of the H antigen or the presence of natural antibodies made against A and / or B (e.g., a person with a B glycosylation pattern has antibodies made against A).

[0058] In humans, the glycosyltransferase locus, referred to herein as the ABO locus or the ABO glycosyltransferase locus, is located on chromosome 9 and contains seven exons spanning more than 18 kb of genomic DNA. Exon 7 is the largest exon and contains most of the coding sequence. There are three main allelic genotypes at the ABO locus: A, B, and O. The A "allele" (also called A1 or A2) encodes a glycosyltransferase that enzymatically adds N-acetylgalactosamine to the D-galactose terminus of the H antigen, resulting in the so-called A antigen. The B allele encodes a glycosyltransferase that enzymatically adds D-galactose to the D-galactose terminus of the H antigen and thus creates the so-called B antigen. The O allele encodes a non-functional form of glycosyltransferase, and as a result, the H antigen is not modified, resulting in the so-called O antigen phenotype.

[0059] At the genomic level, the ABO glycosyltransferase gene has many alleles (about 300). These natural allelic variants are described in Yip, "Sequence Variation at the Human ABO Locus", Ann. Hum. Genet. 66:1-27 (2002); Hakomori "Antigen Structure and Genetic Basis of Histo-Blood Group A, B, and O: Their Changes Associated with Human Cancer", Biochimica et Biophysica Acta 1473:247-266 (1999); Seto et al., "Sequential Interchange of Four Amino Acids from Blood Group B to Blood Group A Glycosyltransferase Boosts Catalytic Activity and Progressively Modifies Substrate Recognition in Human Recombinant Enzymes", J. Biol. Chem. 272:14133-14138 (1997), which are hereby incorporated by reference in their entirety, and their use is contemplated in the bifunctional therapeutic substances of the present disclosure. The sequence encoding the catalytic site of the enzyme is in exon 7 of the gene. The important amino acid residues 176, 235, 266, and 268 control the specificity of this active site. Furthermore, a common nucleotide deletion in exon 6 results in a stop codon that abrogates the synthesis of full-length glycosyltransferase, thereby giving rise to the O or null phenotype.

[0060] Thus, in some embodiments, the glycosyltransferase is selected from the group consisting of glycosyltransferase A (α1-3-N-acetylgalactosaminyltransferase), glycosyltransferase B (α1-3-galactosyltransferase), α-gal-transferase, and glycosyltransferase A (Gly268Ala). Allelic variants such as those described above are also contemplated.

[0061] In some embodiments, the glycosyltransferase used in the methods of the present disclosure is a fucosyltransferase. Fucosyltransferases are known to those of skill in the art. Exemplary fucosyltransferases include enzymes that transfer L-fucose from GDP-fucose to the hydroxy position of an acceptor sugar. Fucosyltransferases that transfer non-nucleotide sugars to an acceptor are also useful in the present disclosure.

[0062] In some embodiments, the glycosyltransferase is a humanized or deimmunized glycosyltransferase. Methods for humanizing or deimmunizing proteins are known in the art.

[0063] Accordingly, one aspect of the present disclosure relates to the alteration of blood group antigen expression and / or tumor blood supply on a tumor by a tumor-targeted glycosyltransferase. As described above, this effectively converts the tumor phenotype to that of an incompatible allograft or xenograft, thereby initiating a hyperacute rejection response.

[0064] The bifunctional therapeutic agents described herein may be formed such that the targeting component is a protein or peptide linked to the enzyme via a peptide bond.

[0065] In certain embodiments, a protein or peptide targeting component linked to an enzyme via a peptide bond may be referred to as a chimeric protein or fusion protein. As used herein, the terms "chimeric protein" or "fusion protein" refer to a single continuous polypeptide chain, i.e., a continuous series of contiguous amino acids linked by peptide bonds, or at least a portion of the full-length sequence of a first polypeptide sequence and at least a portion of the full-length sequence of a second polypeptide sequence, wherein the first polypeptide and the second polypeptide are different polypeptides, and a continuous series of polypeptide chains (i.e., a polypeptide complex) linked to each other by covalent or non-covalent bonds. Chimeric polypeptides also include polypeptides that contain two or more non-contiguous portions derived from the same polypeptide. Chimeric polypeptides or proteins also include polypeptides having at least one substitution, and a chimeric polypeptide includes a first polypeptide sequence in which a portion of the first polypeptide sequence is replaced by a portion of a second polypeptide sequence. The continuous series of polypeptide chains may be covalently linked using a suitable biochemical linker or disulfide bond.

[0066] The connection between the targeting component and the enzyme may also be prepared using chemical bonds (Brennan et al., 「Preparation of Bispecific Antibodies by Chemical Recombination of Monoclonal Immunoglobulin G1 Fragments」, Science 229:81-3 (1985), which is incorporated herein by reference in its entirety), or may be prepared using chemical coupling (Shalaby et al., 「Development of Humanized Bispecific Antibodies Reactive With Cytotoxic Lymphocytes and Tumor Cells Overexpressing the HER2 Protooncogene」, J. Exp. Med. 175:217-225 (1992), which is incorporated herein by reference in its entirety).

[0067] In other embodiments, the targeting component and the enzyme may be linked via non-covalent bonds including, but not limited to, hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0068] Accordingly, the fusion or linkage between the targeting component (e.g., an antibody) and the enzyme can be accomplished by conventional covalent or ionic bonds, protein fusion by genetic engineering, or hetero-bifunctional cross-linkers such as carbodiimide, glutaraldehyde, etc. Conventionally, an inert linker sequence (e.g., a peptide linker) that simply provides a desired amount of space between the targeting component and the enzyme may also be used. The design of such linkers is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 8,580,922; 5,525,491; and 6,165,476, which are hereby incorporated by reference in their entirety. Various coupling agents or cross-linking agents can be used for protein conjugation by covalent bonds. Examples of cross-linking agents include Protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al., 「Production of Target-Specific Effector Cells Using Hetero-Cross-Linked Aggregates Containing Anti-Target Cell and Anti-Fc Gamma Receptor Antibodies」, J. Exp. Med. 160(6):1686-701 (1984); Liu et al., 「Heteroantibody Duplexes Target Cells for Lysis by Cytotoxic T Lymphocytes」, Proc. Natl. Acad. Sci. USA 82(24):8648-52 (1985), which are hereby incorporated by reference in their entirety).Other methods include those described in Paulus, Behring Ins Mitt No 78, 118-132 (1985); Brennan et al., 「Preparation of Bispecific Antibodies by Chemical Recombination of Monoclonal Immunoglobulin G1 Fragments」, Science 229:81-83 (1985); Glennie et al., 「Preparation and Performance of Bispecific F(ab’ gamma)2 Antibody Containing Thioether-Linked Fab’ Gamma Fragments」, J. Immunol. 139:2367-2375 (1987), which are hereby incorporated by reference in their entirety.

[0069] A number of other linkers can be used to connect the targeting component to the enzyme. For example, disulfide bonds can be used as described in Saito et al., Adv. Drug Delivery Reviews 55:199-215 (2003), which is hereby incorporated by reference in its entirety. Linkers sensitive to low pH found in the endosomal or tumor environment, including hydrazone, ketal, and / or aconitic acid, can also be used. Hybrid linkers, for example, linkers having two or more possible cleavage sites, such as disulfide and hydrazone, can also be used. Peptidase-sensitive linkers, for example, linkers sensitive to cleavage by tumor-specific peptidases, such as PSA, can also be used. PEG linkers can also be used (Wiiest et al., Oncogene 21:4257-4265 (2002), which is hereby incorporated by reference in its entirety). Exemplary linkers include hydrazone and disulfide hybrid linkers (see Hamann et al., Bioconjugate Chem. 13:47-58 (2002); Hamann et al., Bioconjug Chem. 13(1):40-6 (2002), which are hereby incorporated by reference in their entirety); SPP (immunogen); and various linkers available from Pierce Biotechnology, Inc. In some embodiments, the linker is SSP (disulfide linker, available from Immunogen), and the ratio of linker to antibody can be, for example, 7:1 to 4:1. A variety of spacer and linker sequences are known in the art and are described in Chen et al., 「Fusion Protein Linkers: Property, Design and Functionality」, Adv. Drug Deliv. Rev. 65(10):1357-69 (2013), which is hereby incorporated by reference in its entirety.

[0070] The term "peptide linker" or spacer refers to a short peptide fragment that connects or joins the targeting component of a polypeptide of a bifunctional therapeutic substance to the enzyme moiety. The linker is preferably composed of amino acids linked by peptide bonds. For example, the peptide linker may contain small amino acid residues or hydrophilic amino acid residues (e.g., glycine, serine, threonine, proline, aspartic acid, asparagine, etc.). For example, the peptide linker is a peptide having an amino acid sequence of at least 5 amino acids in length, or about 5 to about 100 amino acids in length, or about 10 to 50 amino acids in length, or about 10 to 15 amino acids in length.

[0071] In one example, the linker is composed of a majority of sterically hindered amino acids such as glycine and alanine. Thus, in a further example, the linker is polyglycine, polyalanine, or polyserine.

[0072] One of ordinary skill in the art will understand that many commonly used peptide linkers can be used in the aspects of the present disclosure. In certain aspects, a short peptide linker may include repeating units, e.g., a 2-repeat linker, a 3-repeat linker, or a 4-repeat linker, to increase the linker length. In one example, the linker includes the formula (Gly-Gly-Gly-Gly-Ser) n (SEQ ID NO:1) or includes the formula (Ser-Gly-Gly-Gly-Gly) n Ser (SEQ ID NO:2), wherein n is a number from 3 to 6. In some aspects, the linker is a (G4S)3 linker (SEQ ID NO:67).

[0073] Non-peptide linkers or spacers are also possible. For example, an alkyl linker, e.g., -NH-(CH2) where s = 2 - 20 s-C(O)- can be used. These alkyl linkers can be further substituted with any non - sterically hindered group such as lower alkyl (e.g., C1 - C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. Exemplary non - peptide linkers are PEG linkers or spacers having a molecular weight of 100 - 5000 kD, preferably 1000 - 2000 kD, more preferably 1500 kD.

[0074] For the bifunctional therapeutic substances according to the present disclosure, the N - terminus may be connected to the C - terminus. The N - terminus and the C - terminus are used herein to refer to the N - terminal region or portion and the C - terminal region or portion of the bifunctional therapeutic protein of the present disclosure, respectively. In some aspects of the present disclosure, the C - terminal portion and the N - terminal portion of the bifunctional therapeutic substance of the present disclosure are connected adjacently. In another aspect, the C - terminal portion and the N - terminal portion of the bifunctional therapeutic substance of the present disclosure are connected by an intervening spacer. In one aspect, the spacer may be a polypeptide sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues. In some aspects, the C - terminal portion and / or the N - terminal portion of the bifunctional therapeutic substance of the present disclosure may each include a further portion connected to the C - terminal residue and / or the N - terminal residue of the chimeric protein of the present disclosure. In some aspects, the further portion may be a polypeptide sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues. In some aspects, the C - terminal portion and / or the N - terminal portion having such a further portion each maintain the activity of the corresponding native N - terminal portion of the targeting component and / or the C - terminal portion of the enzyme. In some aspects, the N - terminal portion and / or the C - terminal portion having such a further portion each have improved and / or extended activity compared to the corresponding native N - terminal portion of the targeting component and / or the C - terminal portion of the enzyme. In other aspects, the C - terminal portion and / or the N - terminal portion of the bifunctional therapeutic substance of the present disclosure do not include any further portion connected to the C - terminal residue and / or the N - terminal residue of the chimeric protein of the present disclosure.

[0075] In one aspect, the N-terminal region comprises a targeting component. In certain aspects, the targeting component is an antibody or an antigen-binding portion thereof, including, but not limited to, monomeric single-chain antibodies, Fab fragments, Fab’2, scFv, and other antibody fragment derivatives such as minibodies, diabodies, and triabodies. The antibody or antigen-binding fragment may or may not maintain the FcRn-binding domain. In certain embodiments, the targeting component is a TCR, a single-domain antibody, or an Anticalin.

[0076] In some aspects, the C-terminal region comprises an enzyme.

[0077] In one aspect, the bifunctional therapeutic substance comprises the catalytic domain of glucosyltransferase B (GTB) and has the amino acid sequence of SEQ ID NO:7 (GenBank accession number AM423112.1, which is hereby incorporated by reference in its entirety) or a portion thereof as follows. TIFF2025518735000001.tif53138

[0078] In another aspect, the bifunctional therapeutic substance comprises a "cisA,B" sequence that results in a hybrid sequence of GTB and GTA and has the amino acid sequence of SEQ ID NO:8 (GenBank accession number ABL75287.1, which is hereby incorporated by reference in its entirety) or a portion thereof as follows. TIFF2025518735000002.tif32138

[0079] In another aspect, the bifunctional therapeutic substance comprises the catalytic domain of glucosyltransferase A (GTA) and has the amino acid sequence of SEQ ID NO:9 (GenBank accession number AFB74122.1, which is hereby incorporated by reference in its entirety) or a portion thereof as follows. TIFF2025518735000003.tif53138

[0080] In certain embodiments, tumors having tumor - associated antigens express the H antigen. As used herein, the "H antigen" is an oligosaccharide chain having a terminal disaccharide fucose - galactose, where the fucose has an α-(1 - 2)-bond. The H antigen is produced by fucosyltransferase and is a basic element for producing the A antigen or B antigen in the ABO blood group system.

[0081] Accordingly, the present disclosure also relates to a method of treating cancer. The method includes the steps of selecting a subject having cancer and providing a bifunctional therapeutic substance according to the present disclosure. The bifunctional therapeutic substance is administered to the selected subject under conditions effective to treat cancer.

[0082] Using the bifunctional therapeutic substances described herein, prostate tumors, adrenocortical carcinoma tumors, anal tumors, appendix tumors, astrocytomas (pediatric cerebellum or cerebrum), basal cell carcinomas, bile duct tumors, bladder tumors, bone tumors, osteosarcoma / malignant fibrous histiocytoma, brainstem gliomas, ependymomas, medulloblastomas, breast tumors, bronchial adenomas / carcinoids, Burkitt lymphoma, carcinoid tumors, cervical tumors, pediatric tumors, chondrosarcomas, colon tumors, cutaneous T - cell lymphomas, desmoplastic small round cell tumors, endometrial tumors, esophageal tumors, Ewing sarcoma, retinoblastomas, gallbladder tumors, gastric (stomach) tumors, gastrointestinal stromal tumors, germ cell tumors, gestational trophoblastic tumors, head and neck tumors, heart tumors, hepatocellular (liver) tumors, Hodgkin lymphoma, hypopharyngeal tumors, islet cell carcinomas (endocrine pancreas), Kaposi sarcoma, kidney tumors, laryngeal tumors, lip and oral tumors, non - small cell lung tumors, small cell lung tumors, lymphomas, melanomas, Merkel cell tumors, mesotheliomas, multiple endocrine neoplasia, multiple myeloma, nasopharyngeal tumors, neuroblastomas, oligodendrogliomas, oral tumors, oropharyngeal tumors, ovarian tumors, pancreatic tumors, pleuropulmonary, primary central nervous system lymphomas, retinoblastomas, rhabdomyosarcomas, salivary gland tumors, soft tissue sarcomas, uterine sarcomas, skin tumors (non - melanoma), small intestine tumors, squamous cell carcinomas, stomach tumors, testicular tumors, pharyngeal tumors, thymomas and thymic carcinomas, thyroid tumors, trophoblastic tumors, and urethral tumors, among others, can be treated, and substantially any H - antigen - expressing tumor can be treated.

[0083] Some cancers, including, but not limited to, hematopoietic cancers or lymphatic cancers, mesoderm-derived cancers, sarcomas, and neuroectodermal cancers, may not express the H antigen. This can be easily confirmed by flow cytometry or immunohistochemistry of tumor samples using Ulex lectin binding to reveal the presence or absence of H. When H is absent, the treatment using the present application can be achieved in two ways. That is, as previously described, a fucosyltransferase targeted to add the H antigen before or simultaneously with the targeted glycosyltransferase may be used. Or, an αgalT enzyme that can add terminal galactose and does not require the presence of 1,2 fucose (H antigen) may be targeted.

[0084] In one aspect, the targeting component of the bifunctional therapeutic substance targets the PSMA receptor on tumor vascular endothelium. PSMA expression has been reported in the tumor neovessels of various tumors but is not present in normal tissue vasculature. Exemplary tissue types with PSMA-positive vascular endothelium include, but are not limited to, tumors of the kidney, lung, colon, stomach, breast, brain, pancreas, liver, bladder, esophagus, adrenal gland, head and neck, melanoma, and brain. Other aspects include targeting of PSMA expressed on the surface of prostate cancer cells, targeting of HER2 on the surface of breast and other HER2-positive cancers, targeting of CD19 on the surface of B-cell lineage cancers, and targeting of CEA on the surface of colorectal cancers. Other applicable targets are described above.

[0085] In some aspects, the heavy chain variable region and / or light chain variable region of the antibody-based molecules described herein further comprise, respectively, a human or humanized immunoglobulin heavy chain framework region and / or light chain framework region.

[0086] Suitable amino acid modifications to the heavy chain CDR sequences and / or light chain CDR sequences of the targeting domains disclosed herein include conservative substitutions or functionally equivalent amino acid residue substitutions that result in variant CDR sequences having binding properties similar to or improved from the binding properties of the CDR sequences disclosed herein, such as those described above. Conservative substitutions are substitutions that occur within amino acid families that are related in terms of their side chains. The amino acids encoded by genes can be divided into four families: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. Alternatively, the amino acid repertoire can be classified as (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine). Serine and threonine can optionally be classified separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981, which is hereby incorporated by reference in its entirety). Non-conservative substitutions can also be added. Non-conservative substitutions involve substituting one or more amino acid residues of the CDR with one or more amino acid residues from a different amino acid class to improve or enhance the binding properties of the CDR. The amino acid sequence of the heavy chain variable region CDR and / or the amino acid sequence of the light chain variable region CDR may further include one or more internal neutral amino acid insertions or deletions that maintain or improve target binding.

[0087] The targeting domain of the present disclosure can be described or specified in terms of binding affinity. Thus, in some embodiments, the targeting domain of the present disclosure has a dissociation constant or K of less than 1 μM, 500 nM, 250 nM, 200 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. D and includes a targeting domain having the same.

[0088] In some embodiments, the targeting component optionally includes a signaling peptide, which has the sequence of amino acids 1 to 19 of SEQ ID NO: 34.

[0089] In some embodiments, the glycosyltransferase is selected from glycosyltransferase A (α1-3-N-acetylgalactosaminyltransferase) and glycosyltransferase B (α1-3-galactosyltransferase).

[0090] In some embodiments, the glycosyltransferase is glycosyltransferase A (“GTA”) and has the amino acid sequence of SEQ ID NO: 64 or a portion thereof as follows. TIFF2025518735000004.tif32163

[0091] In some embodiments, the glycosyltransferase is glycosyltransferase B (“GTB”) and has the amino acid sequence of SEQ ID NO: 65 or a portion thereof as follows. TIFF2025518735000005.tif32163

[0092] Suitable additional glycosylases are described below. In some embodiments, the glycosyltransferase is marmoset α-1,3 galactosyltransferase (aa90-376) and has the amino acid sequence of SEQ ID NO: 66 or a portion thereof as follows. TIFF2025518735000006.tif32163

[0093] Another aspect of the present disclosure relates to a bifunctional therapeutic substance for treating cancer, comprising a targeting component that targets a human epidermal growth factor receptor (HER) family member and a glycosyltransferase that enzymatically converts a tumor phenotype to that of a mismatched allograft or xenograft when delivered to a tumor by the targeting component, the glycosyltransferase being connected to the targeting component. This aspect of the present disclosure is useful in the treatment of subjects having breast cancer or any HER2-expressing cancer.

[0094] It is understood that the exact dosage of the bifunctional therapeutic substance of the present disclosure is selected by the individual physician in view of the patient to be treated. Generally, the dosage and administration are adjusted to provide an effective amount of the agent to the patient being treated. As used herein, an "effective amount" of a bifunctional therapeutic substance refers to the amount necessary to induce a desired biological response. As will be understood by those skilled in the art, the effective amount of the bifunctional therapeutic substance of the present disclosure may vary depending on factors such as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, and the like. For example, an effective amount of a bifunctional therapeutic substance may be an amount that reduces the tumor size by a desired amount over a desired period. Additional factors that may be considered include the severity of the disease state; the age, weight, and gender of the patient being treated; diet, time and frequency of administration; drug combination; responsiveness; and tolerance / response to the therapy.

[0095] "Effective amount" may also refer to a "prophylactically effective amount" of the bifunctional therapeutic substance described herein that is effective when administered to a subject once or multiple times to prevent or delay the occurrence or recurrence of a disorder, such as cancer, or to treat its symptoms.

[0096] Generally, the dosage may be about 25% to about 100% of the maximum tolerated dose (MTD) of the bifunctional therapeutic substance when administered as a single agent. Based on the composition, the dosage may be delivered once, continuously, for example, continuously by a continuous pump, or delivered at regular intervals. The dosage may be appropriately adjusted locally or systemically to reach the desired drug level. At such dosages, if the response in the subject is insufficient, higher dosages (or different, more locally effective dosages via a more local delivery route) may be used to the extent tolerated by the patient. To reach an appropriate systemic level of the compound, continuous IV dosing, for example, continuous IV dosing over 24 hours, or multiple dosing per day is also contemplated. By way of example, the dosing schedule can be changed such that the bifunctional therapeutic substance is administered once, twice, three times, or more than three times per week over any number of weeks, or the bifunctional therapeutic substance is administered multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, or 24 times) and the dosing is done once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks. For example, the bifunctional therapeutic substance can be administered at least two, three, or four times at the dosage levels cited above, and the dosing is done once every 4 - 8 weeks. If the subject does not exhibit an adverse reaction to the bifunctional therapeutic substance and / or one or more symptoms of cancer improve or remain the same, one or more additional dosages can be administered. In some embodiments, the amount of the bifunctional therapeutic substance can be increased as the period between dosings is extended.

[0097] The biodistribution and pharmacokinetics of bifunctional therapeutic substances may vary for different targeting components. As an example, large bifunctional therapeutic substances composed of full-length intact antibodies have long plasma and whole-body half-lives and tend to remain in circulation. Such bifunctional therapeutic substances are also likely to be excreted through the liver and have a low potential to penetrate normal tissues. Conversely, for example, small bifunctional therapeutic substances composed of targeting peptides or small molecule ligands have shorter half-lives, are excreted through the kidneys / urinary tract, and tend to rapidly penetrate normal tissues and tumors.

[0098] In practicing the methods of the present disclosure, an administration step is performed to treat cancer in a subject. In one aspect, prior to administration, a subject having cancer is selected. Such administration may be performed systemically, or via direct administration to the tumor site or local administration. As examples, suitable systemic administration methods include, but are not limited to, oral administration, topical administration, transdermal administration, parenteral administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, subcutaneous administration, or administration by nasal, intracavitary, or intravesical instillation, intravitreal administration, intraarterial administration, intralesional administration, or application to a mucosa. Suitable local administration methods include, but are not limited to, catheter placement, implantation, direct injection, cutaneous / transdermal application, or portal vein administration to related tissues, or any other local administration technique, method, or procedure generally known in the art. The method of delivering the bifunctional therapeutic substance varies depending on the type of bifunctional therapeutic substance (e.g., having an antibody targeting component or a peptide targeting component) and the disease to be treated.

[0099] The two-functional therapeutic substance of the present disclosure may be orally administered, for example, together with an inert diluent or an assimilable edible carrier, may be enclosed in a hard shell or soft shell capsule, may be compressed into tablets, or may be directly incorporated with food in a diet. The two-functional therapeutic substance of the present disclosure may also be incorporated into a device such as a sustained-release capsule or nanotube for sustained-release administration. Such devices provide flexibility with respect to time and dosage. In the case of oral therapeutic administration, the agents of the present disclosure may be incorporated with excipients and may be used in the form of tablets, capsules, elixirs, suspensions, syrups, etc. Such compositions and formulations must contain at least 0.1% of the agent, although lower concentrations may be effective and may actually be optimal. The percentage of the agent in these compositions may, of course, be varied and may conveniently be about 2% to about 60% by unit weight. The amount of the two-functional therapeutic substance of the present disclosure in such therapeutically useful compositions is an amount such that an appropriate dosage is obtained.

[0100] When the two-functional therapeutic substance of the present disclosure is administered parenterally, a solution or suspension of the agent can be prepared by dissolving it in water appropriately mixed with a surfactant such as hydroxypropylcellulose. The dispersion can also be prepared by dissolving it in glycerol, liquid polyethylene glycol, and a mixture dissolved in oil. Exemplary oils are oils derived from petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, or mineral oil. Generally, water, saline, aqueous dextrose solution and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are particularly preferred liquid carriers for injection solutions. Under normal storage and use conditions, these formulations contain preservatives to prevent the growth of microorganisms.

[0101] Pharmaceutical preparations suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and must be liquid to the extent that it is readily injectable. The dosage form must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0102] When it is desirable to systemically deliver the bifunctional therapeutic substance of the present disclosure, the bifunctional therapeutic substance of the present disclosure may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations are in unit dosage form and may be presented, for example, in ampoules or in multi-dose containers together with an added preservative. The composition may take the form of a suspension, solution, or emulsion dissolved in an oily or aqueous vehicle and may contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents.

[0103] Intraperitoneal or intrathecal administration of the bifunctional therapeutic substance of the present disclosure can also be accomplished using an infusion pump device. Using such a device allows for the continuous infusion of the desired compound without multiple injections and multiple manipulations.

[0104] In addition to the formulations described above, the bifunctional therapeutic substance may be formulated as a depot formulation. Such long-acting formulations may be formulated using suitable polymeric materials or hydrophobic materials (such as an emulsion in a tolerated oil) or ion exchange resins and may be formulated as a somewhat less soluble derivative, for example, as a somewhat less soluble salt.

[0105] Another aspect of the present disclosure relates to a pharmaceutical composition comprising the bifunctional therapeutic substance of the present disclosure and a pharmaceutically acceptable carrier.

[0106] The dual-functional therapeutic substance has been described above.

[0107] The pharmaceutical composition containing the dual-functional therapeutic substance for use in the method of the present disclosure may include a pharmaceutically acceptable carrier as described below, one or more active agents, and a suitable delivery vehicle. Suitable delivery vehicles include, but are not limited to, viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen minipellets, and cochleates.

[0108] In one aspect of the present disclosure, the pharmaceutical composition or formulation is encapsulated in a lipid formulation to form nucleic acid-lipid particles as described in Semple et al., 「Rational Design of Cationic Lipids for siRNA Delivery」, Nature Biotech. 28:172-176 (2010), WO2011 / 034798 to Bumcrot et al., WO2009 / 111658 to Bumcrot et al., and WO2010 / 105209 to Bumcrot et al., which are hereby incorporated by reference in their entirety.

[0109] In another aspect of the disclosure, the delivery vehicle is a nanoparticle. A variety of nanoparticle delivery vehicles are known in the art and are suitable for delivering the bifunctional therapeutic agent of the present disclosure (see, for example, van Vlerken et al., 「Multi-functional Polymeric Nanoparticles for Tumour-Targeted Drug Delivery」, Expert Opin. Drug Deliv. 3(2):205-216 (2006), which is incorporated herein by reference in its entirety).Suitable nanoparticles include, but are not limited to, poly(β - amino ester) (Sawicki et al., "Nanoparticle Delivery of Suicide DNA for Epithelial Ovarian Cancer Cell Therapy", Adv. Exp. Med. Biol. 622:209 - 219 (2008), which is hereby incorporated by reference in its entirety), polyethyleneimine - alt - poly(ethylene glycol) copolymer (Park et al., "Degradable Polyethylenimine - alt - Poly(ethylene glycol) Copolymers As Novel Gene Carriers", J. Control Release 105(3):367 - 80 (2005), and Park et al., "Intratumoral Administration of Anti - KITENIN shRNA - Loaded PEI - alt - PEG Nanoparticles Suppressed Colon Carcinoma Established Subcutaneously in Mice", J Nanosci. Nanotechnology 10(5):3280 - 3 (2010), which are hereby incorporated by reference in their entirety), and siRNA nanoparticles encapsulated in liposomes (Kenny et al., "Novel Multifunctional Nanoparticle Mediates siRNA Tumor Delivery, Visualization and Therapeutic Tumor Reduction In Vivo", J. Control Release 149(2): 111 - 116 (2011), which is hereby incorporated by reference in its entirety). Other nanoparticle delivery vehicles suitable for use in the present disclosure include the microcapsule nanotube device disclosed in U.S. Patent Application Publication No. 2010 / 0215724 to Prakash et al., which is hereby incorporated by reference in its entirety.

[0110] In another aspect of the disclosure, the pharmaceutical composition is contained within a liposomal delivery vehicle. The term "liposome" means a vesicle composed of amphiphilic lipids arranged in the form of one or more spherical bilayers. Liposomes are unilamellar or multilamellar vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with cell walls. Non-cationic liposomes cannot fuse efficiently with cell walls but are taken up by macrophages in vivo.

[0111] Some of the advantages of liposomes include biocompatibility and biodegradability, the ability to incorporate a wide variety of water-soluble and lipid-soluble drugs, and the protection of encapsulated drugs from metabolism and degradation. Important considerations in the preparation of liposomal formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

[0112] Liposomes are useful for transporting and delivering the active ingredient to the site of action. Since the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to tissues, they begin to integrate with cell membranes, and as the integration of liposomes and cells progresses, the contents of the liposomes are poured into cells where the active drug can act.

[0113] Methods for preparing liposomes for use in the present disclosure include the methods disclosed in Bangham et al., "Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids", J. Mol. Biol. 13:238-52 (1965); U.S. Patent No. 5,653,996 to Hsu; U.S. Patent No. 5,643,599 to Lee et al.; U.S. Patent No. 5,885,613 to Holland et al.; U.S. Patent No. 5,631,237 to Dzau & Kaneda, and U.S. Patent No. 5,059,421 to Loughrey et al., which are hereby incorporated by reference in their entirety.

[0114] In another aspect of the disclosure, the delivery vehicle is a viral vector. Viral vectors are particularly suitable for the delivery of nucleic acid molecules, but can also be used to deliver molecules encoding bifunctional therapeutic substances. Suitable gene therapy vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and herpes viral vectors.

[0115] As described in Berkner, "Development of Adenovirus Vectors for the Expression of Heterologous Genes", Biotechniques 6:616-627 (1988), Rosenfeld et al., "Adenovirus-Mediated Transfer of a Recombinant Alpha 1-Antitrypsin Gene to the Lung Epithelium In Vivo", Science 252:431-434 (1991), WO 93 / 07283 to Curiel et al., WO 93 / 06223 to Perricaudet et al., and WO 93 / 07282 to Curiel et al., all of which are hereby incorporated by reference in their entirety, adenovirus viral vector delivery vehicles can be readily prepared and utilized.As described in Shi et al., "Therapeutic Expression of an Anti-Death Receptor-5 Single-Chain Fixed Variable Region Prevents Tumor Growth in Mice", Cancer Res. 66:11946-53 (2006); Fukuchi et al., "Anti-Aβ Single-Chain Antibody Delivery via Adeno-Associated Virus for Treatment of Alzheimer's Disease", Neurobiol. Dis. 23:502-511 (2006); Chatterjee et al., "Dual-Target Inhibition of HIV-1 In Vitro by Means of an Adeno-Associated Virus Antisense Vector", Science 258:1485-1488 (1992); Ponnazhagan et al., "Suppression of Human Alpha-Globin Gene Expression Mediated by the Recombinant Adeno-Associated Virus 2-Based Antisense Vectors", J. Exp. Med. 179:733-738 (1994); and Zhou et al., "Adeno-associated Virus 2-Mediated Transduction and Erythroid Cell-Specific Expression of a Human Beta-Globin Gene", Gene Ther. 3:223-229 (1996), which are hereby incorporated by reference in their entirety, adeno-associated virus delivery vehicles can be constructed and used to deliver the bifunctional therapeutic agents of the present disclosure to cells.The in vivo use of these vehicles is described in Flotte et al., "Stable in Vivo Expression of the Cystic Fibrosis Transmembrane Conductance Regulator With an Adeno-Associated Virus Vector", Proc. Nat'l. Acad. Sci. 90:10613-10617 (1993), and Kaplitt et al., "Long-Term Gene Expression and Phenotypic Correction Using Adeno-Associated Virus Vectors in the Mammalian Brain", Nature Genet. 8:148-153 (1994), which are hereby incorporated by reference in their entirety. Further types of adenovirus vectors are described in U.S. Patent No. 6,057,155 to Wickham et al.; U.S. Patent No. 6,033,908 to Bout et al.; U.S. Patent No. 6,001,557 to Wilson et al.; U.S. Patent No. 5,994,132 to Chamberlain et al.; U.S. Patent No. 5,981,225 to Kochanek et al.; U.S. Patent No. 5,885,808 to Spooner et al.; and U.S. Patent No. 5,871,727 to Curiel, which are hereby incorporated by reference in their entirety.

[0116] To deliver nucleic acid molecules to target cells, retroviral vectors that have been modified to form an infectious transformation system can also be used. One such type of retroviral vector is disclosed in U.S. Patent No. 5,849,586 to Kriegler et al., which is hereby incorporated by reference in its entirety. Other nucleic acid delivery vehicles suitable for use in the present disclosure include the nucleic acid delivery vehicles disclosed in U.S. Patent Application Publication No. 20070219118 to Lu et al., which is hereby incorporated by reference in its entirety.

[0117] Regardless of the type of infectious transformation system used, the infectious transformation system must be targeted in order to deliver the nucleic acid to the desired cell type. For example, to deliver to a cluster of cells (e.g., cancer cells), a high-titer infectious transformation system can be directly injected into the site of these cells to increase the likelihood of cell infection. The infected cells then express a nucleic acid molecule that targets a tumor-associated antigen. This expression system may further contain a promoter to control or regulate the intensity and specificity of the expression of the nucleic acid molecule in the target tissue or target cells.

[0118] As noted above, the effective dose of the compositions of the present disclosure for treating metastatic disease will vary depending on many different factors including the type and stage of cancer, the means of administration, the target site, the physiological state of the patient, other drugs or therapies being administered, and the physical condition of the patient related to other medical complications. The treatment dosage needs to be adjusted to optimize safety and efficacy.

[0119] The pharmaceutical composition of the present disclosure may contain a "therapeutically effective amount" or "preventively effective amount" of the bifunctional therapeutic substance of the present disclosure. A "therapeutically effective amount" refers to the dosage necessary to achieve the desired therapeutic result and, over a period of time, an amount effective to achieve the desired therapeutic result. The therapeutically effective amount of the bifunctional therapeutic substance may vary depending on factors such as the individual's disease state, age, gender, and body weight, as well as the ability of the bifunctional therapeutic substance to induce a desired response in the individual. The therapeutically effective amount is also an amount at which the therapeutically beneficial effect is greater than any toxic or detrimental effect of the bifunctional therapeutic substance. The "therapeutically effective dosage" preferably inhibits a measurable parameter, such as the tumor growth rate, by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to untreated subjects. The ability of a compound to inhibit a measurable parameter, such as cancer, can be evaluated in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be evaluated by assays known to those skilled in the art to examine the inhibitory ability of the compound, such as in vitro inhibition.

[0120] A "preventively effective amount" refers to the dosage necessary to achieve the desired preventive result and, over a period of time, an amount effective to achieve the desired preventive result. Typically, a preventive dose is used in a subject before or at an early stage of a disease, so the preventively effective amount is less than the therapeutically effective amount.

[0121] In certain embodiments, the administering step further comprises administering a nucleotide sugar uridine diphosphate galactose (UDP-gal), uridine diphosphate-N-acetylgalactosamine (UDP-NAcGal), and / or guanosine diphosphate-fucose (GDP-fucose).

[0122] UDP-gal, UDP-NAcGal, and / or GDP-fucose can be administered by any suitable route including, but not limited to, intravenous, subcutaneous, intramuscular, intraperitoneal, oral, rectal, or any other route known in the art. Further, UDP-gal, UDP-NAcGal, and / or GDP-fucose may be co-administered simultaneously with a targeted bifunctional enzyme or administered after the targeted bifunctional enzyme. In the latter case, i.e., in the case of later administration, the interval between the targeted enzyme and the nucleotide sugar may be from 1 minute to 1 week. In a preferred embodiment, the interval is from 1 minute to 48 hours.

[0123] The bifunctional therapeutic substances described herein may be used in combination with other therapies. As used herein, "co-administered" means that two (or more) different treatments are delivered to a subject during the time the subject has a disorder. For example, two or more treatments are delivered after the subject has been diagnosed with a disorder and before the disorder is cured or eliminated or before treatment is discontinued for some other reason. In some embodiments, the delivery of one treatment is still ongoing when the delivery of the second treatment begins, and thus there is an overlap in the administration periods. This is referred to herein as "simultaneous" or "co-delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments, the effect of the treatments is enhanced for co-administration. For example, the effect of the second treatment is greater. For example, the second treatment is less but an equivalent effect is observed. Or, the second treatment reduces symptoms to a greater extent than would be observed if administered in the absence of the first treatment. Or, a situation similar to the first treatment is observed. In some embodiments, the delivery is such that the reduction in symptoms, or other parameters related to the disorder, is greater than that observed with one of the treatments delivered in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive. The delivery may also be such that the effect of the first treatment delivered is still detectable when the second delivery is made.

[0124] Exemplary therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol (see U.S. Patent No. 5,208,020, which is hereby incorporated by reference in its entirety), CC-1065 (see U.S. Patent Nos. 5,475,092, 5,585,499, and 5,846,545, which are hereby incorporated by reference in their entirety) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretharnine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0125] In other embodiments, the bifunctional therapeutic substance is administered in combination with other therapeutic modalities including surgery, radiation, cryosurgery, and / or hyperthermia. In such combination therapies, low doses of the therapeutic agent are advantageously utilized, and thus, the possible toxicities or complications associated with various monotherapies may be avoided.

[0126] In other embodiments, the bifunctional therapeutic substance is administered in combination with an immunomodulatory agent, such as IL-1, IL-24, IL-6, or IL-12, or interferon α or γ.

[0127] A further aspect of the disclosure provides nucleic acid (e.g., polynucleotide) molecules encoding the bifunctional therapeutic substances of the disclosure. The polynucleotide may be, for example, either single-stranded and / or double-stranded DNA, cDNA, PNA, RNA, or a combination thereof, or a polynucleotide in natural or synthetic form, such as a polynucleotide having a phosphorothioate backbone, and may or may not contain introns as long as it encodes the bifunctional therapeutic substance. Of course, a polynucleotide can only encode a peptide in which natural amino acid residues are joined by natural peptide bonds. A still further aspect of the disclosure provides recombinant expression vectors capable of expressing the bifunctional therapeutic substances according to the disclosure. For example, various methods have been developed for ligating polynucleotides, particularly DNA, to vectors via complementary attachment ends. For example, a complementary homopolymer region can be added to the DNA segment to be inserted into the vector DNA. Then, the vector and the DNA segment are joined by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

[0128] Synthetic linkers containing one or more restriction sites provide an alternative method of connecting DNA segments to vectors. Synthetic linkers containing various restriction endonuclease sites are commercially available from a number of suppliers, including International Biotechnologies Inc., New Haven, CN, USA. A desirable method for modifying DNA encoding the bifunctional therapeutic substance of the present disclosure uses the polymerase chain reaction as disclosed in Higuchi et al., "A General Method of In Vitro Preparation and Specific Mutagenesis of DNA Fragments: Study of Protein and DNA Interactions", Nucleic Acids Res. 16(15):7351-67 (1988), which is hereby incorporated by reference in its entirety. This method may be used, for example, to introduce DNA into an appropriate vector by manipulation at appropriate restriction sites, or may be used to modify DNA in other useful ways as known in the art.

[0129] The nucleic acids of the present disclosure may be selected to have codons that are preferred or not preferred for a particular expression system. By way of example, the nucleic acid may be one in which at least one codon, preferably at least 10% or 20% of the codons, have been changed so that the sequence is optimized for expression in E. coli, yeast, human, insect, NS0, or CHO cells.

[0130] Typically, a polynucleotide encoding a bifunctional therapeutic substance is placed under the control of a promoter that functions in the desired host cell. A wide variety of promoters are well known and can be used in the expression vectors of the present disclosure according to a particular disclosure. Usually, the promoter selected is dependent on the cell in which the promoter is active. Optionally, other expression control sequences, such as ribosome binding sites, transcription termination sites, etc. are also included. A construct containing one or more of these control sequences is called an "expression vector". Thus, the present disclosure provides an expression vector in which a nucleic acid molecule encoding a bifunctional therapeutic substance is incorporated for high-level expression in a desired host cell.

[0131] Expression control sequences suitable for use in a particular host cell are often obtained by cloning the genes expressed in that cell. Commonly used prokaryotic control sequences are defined herein as including a promoter and optionally an operator for transcription initiation, together with a ribosome binding site sequence, the β-lactamase (penicillinase) and lactose (lac) promoter systems (Change et al., Nature 198:1056 (1977), which is incorporated herein by reference in its entirety), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980), which is incorporated herein by reference in its entirety), the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A. 80:21-25 (1983), which is incorporated herein by reference in its entirety), and the λ-derived P L romoters and N-gene ribosome binding sites (Shimatake et al., Nature 292:128 (1981), which is incorporated herein by reference in its entirety) and other commonly used promoters. However, any available promoter that functions in prokaryotes can be used.

[0132] In order to express a bifunctional therapeutic substance in prokaryotic cells other than Escherichia coli, a promoter that functions in a specific prokaryotic species is required. Such promoters can be obtained from genes cloned from that species. Alternatively, heterologous promoters can be used. For example, the hybrid trp-lac promoter functions in Bacillus in addition to Escherichia coli.

[0133] The expression cassette of the present disclosure conveniently includes a ribosome binding site (RBS). The RBS in Escherichia coli consists of, for example, a nucleotide sequence 3 to 9 nucleotides in length located 3 to 11 nucleotides upstream of the start codon (Shine and Dalgarno, "Determinant of Cistron Specificity in Bacterial Ribosomes", Nature 254:34-38 (1975); Steitz, In Biological regulation and development: Gene expression (ed. R. F. Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, New York), which is incorporated herein by reference in its entirety).

[0134] In the case of mammalian cells, the control sequences include a promoter and preferably an enhancer and polyadenylation sequence derived from, for example, an immunoglobulin gene, SV40, cytomegalovirus, etc., and may include splice donor and acceptor sequences.

[0135] Either a constitutive promoter or a regulatable promoter can be used in the present disclosure. A regulatable promoter may be advantageous because the host cells can be grown to a high density before the expression of the bifunctional therapeutic substance is induced. High levels of heterologous protein expression can, in some cases, slow cell growth. An inducible promoter is a promoter that induces gene expression, where the expression level can be varied by environmental or developmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors, and chemicals. Such promoters are referred to herein as "inducible" promoters, and the use of an "inducible" promoter allows for control of the timing of the expression of the bifunctional therapeutic substance. In the case of E. coli and other bacterial host cells, inducible promoters are known to those of skill in the art. These include, for example, the lac promoter, the bacteriophage λP L promoter, the hybrid trp-lac promoter (Amann et al. Gene 25:167 (1983); de Boer et al. Proc. Nat'l. Acad. Sci. USA 80:21 (1983), which are hereby incorporated by reference in their entirety), and the bacteriophage T7 promoter (Studier et al. J. Mol. Biol (1986).; Tabor et al. Proc. Nat'l. Acad. Sci. USA 82: 1074-8 (1985), which are hereby incorporated by reference in their entirety).

[0136] Selectable markers are often incorporated into expression vectors used to express the dual-functional therapeutic substances of the present disclosure. These genes can encode gene products necessary for the survival or growth of transformed host cells growing in a selective culture medium, for example, proteins. Host cells not transformed with a vector containing a selectable gene do not survive in the culture medium. Representative selectable genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, kanamycin, chloramphenicol, or tetracycline. Alternatively, the selectable marker may encode a protein that complements an auxotrophic deficiency or supplies an essential nutrient not available from complex media. For example, in the case of Bacillus, a gene encoding D-alanine racemase. Often, the vector has one type of selectable marker that functions, for example, in Escherichia coli or in other cells in which the vector is replicated before being introduced into the host cell. A number of types of selectable markers are known to those skilled in the art.

[0137] For the construction of suitable nucleic acid constructs containing one or more of the components listed above, standard ligation methods as described in the references cited above are used. Isolated plasmids or DNA fragments are cut, adapted, and religated in a desired form to yield the required nucleic acid construct (e.g., plasmid). To confirm the correct sequence in the constructed plasmid, the plasmid can be analyzed by standard techniques such as restriction endonuclease digestion and / or by sequencing according to known methods. Molecular cloning methods for achieving these purposes are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are well known to those skilled in the art. Examples of these techniques and instructions sufficient to guide those skilled in the art through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Volume 152, Academic Press, Inc., San Diego, Calif. (Berger), and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., joint venture (1998 Supplement) (Ausubel), which are hereby incorporated by reference in their entirety.

[0138] A variety of common vectors suitable for use as starting materials for constructing the nucleic acid constructs and expression vectors of the present disclosure are well known in the art. For cloning in bacteria, common vectors include vectors derived from pBR322, such as pBLUESCRIP™, and vectors derived from λ-phage. In yeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (YRp series plasmids) and pGPD-2. Expression in mammalian cells can be achieved using a variety of commonly available plasmids including pSV2, pBC12BI, and p91023, as well as lytic virus vectors (e.g., vaccinia virus, adenovirus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retrovirus).

[0139] The nucleic acid can then be expressed in a suitable host to produce a polypeptide comprising the bifunctional therapeutic substance of the present disclosure. Accordingly, the nucleic acid encoding the bifunctional therapeutic substance of the present disclosure is appropriately modified in view of the disclosure contained herein and used according to known techniques to construct an expression vector, which is then used to transform a suitable host cell for the purpose of expressing and producing the bifunctional therapeutic substance of the present disclosure. Such techniques are described below and include, for example, the methods disclosed in U.S. Patent Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648, which are hereby incorporated by reference in their entirety.

[0140] The method for introducing an expression vector into a selected host cell is not particularly important, and such methods are known to those skilled in the art. For example, an expression vector can be introduced into prokaryotic cells including Escherichia coli by calcium chloride transformation, and can be introduced into eukaryotic cells by calcium phosphate treatment or electroporation. Other transformation methods are also suitable.

[0141] The bifunctional therapeutic substances of the present disclosure can also be linked to other bacterial proteins. This approach often results in high yields because normal prokaryotic control sequences induce transcription and translation. In Escherichia coli, lacZ fusions are often used to express heterologous proteins. Appropriate vectors such as the pUR, pEX, and pMR100 series can be easily utilized. In certain applications, it may be desirable to cleave non-enzymatic amino acids from the fusion protein after purification. This can be accomplished by any of several methods known in the art, including cleavage by cyanogen bromide, proteases, or Factor X a (see, for example, Itakura et al., Science (1977) 198: 1056; Goeddel et al., Proc. Natl. Acad. Sci. USA (1979) 76: 106; Nagai et al., Nature (1984) 309: 810; Sung et al., Proc. Natl. Acad. Sci. USA (1986) 83: 561, which are hereby incorporated by reference in their entirety). The cleavage site can be engineered within the desired cleavage point of the gene of the fusion protein.

[0142] By arranging multiple types of transcription cassettes in one expression vector or by utilizing different selection markers for each expression vector used in a cloning strategy, multiple types of bifunctional therapeutic substances can be expressed in one host cell.

[0143] The bifunctional therapeutic substance can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis, etc. (broadly, see R. Scopes, Protein Purification, Springer-Verlag, New York (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. New York (1990), which are hereby incorporated by reference in their entirety). A substantially pure composition with at least about 70-90% homogeneity is preferred, and 98-99% or greater homogeneity is most preferred. As an example, when the targeting component of the bifunctional therapeutic substance is an antibody, antibody-binding chromatography such as ion exchange chromatography can be used. Ion exchange chromatography can be anion exchange chromatography, cation exchange chromatography, or both. Types of anion exchange chromatography include, but are not limited to, Q Sepharose Fast Flow®, MacroPrep High Q Support®, DEAE Sepharose Fast Flow®, and Macro-Prep DEAE®. Types of cation exchange chromatography include, but are not limited to, SP Sepharose Fast Flow®, Source 30S®, CM Sepharose Fast Flow®, Macro-Prep CM Support®, and Macro-Prep High S Support®.

[0144] To facilitate purification of the bifunctional therapeutic substance of the present disclosure, the nucleic acid encoding the bifunctional therapeutic substance may also include an epitope or "tag" to which an affinity binding reagent is available, i.e., a coding sequence for a purification tag. Examples of suitable epitopes include the myc and V-5 reporter genes. Expression vectors useful for the recombinant production of fusion proteins having these epitopes are commercially available (e.g., Invitrogen (Carlsbad Calif.) vectors pcDNA3.1 / Myc-His and pcDNA3.1 / V5-His are suitable for expression in mammalian cells). Additional expression vectors, and corresponding detection systems, suitable for attaching tags to the bifunctional therapeutic substances of the present disclosure are known to those of skill in the art and some are commercially available (e.g., "FLAG" (Kodak, Rochester N.Y.). Another example of a suitable tag is a polyhistidine sequence that can bind to a metal chelate affinity ligand. Typically, six adjacent histidines are used, but more or fewer than six histidines can be used. Suitable metal chelate affinity ligands that can serve as a binding moiety for the polyhistidine tag include nitrilotriacetic acid (NTA) (Hochuli, E. (1990) "Purification of recombinant proteins with metal chelating adsorbents" In Genetic Engineering: Principles and Methods, J. K. Setlow, Ed., Plenum Press, New York; commercially available from Qiagen (Santa Clarita, Calif.), which is hereby incorporated by reference in its entirety).

[0145] Purification tags also include maltose binding domains and starch binding domains. The purification of maltose binding domain proteins is known to those of skill in the art. The starch binding domain is described in WO99 / 15636, which is hereby incorporated by reference in its entirety.

Examples

[0146] The following examples are intended to illustrate the implementation of aspects of the present disclosure, but in no way are they intended to limit the scope of the present disclosure.

[0147] Materials and Methods Cell Lines The human prostate cancer cell lines LNCaP and PC3 were purchased from the American Type Culture Collection (Manassas, VA). CWR22Rv1 was a gift from Thomas Pretlow, MD, Case Western Reserve University. The breast cancer cell line MDA-MB-361 was a gift from Christel Larbouret, (Institute of Cancer Research of Montpellier (France)). LNCaP, PC3, and CWR22Rv1 were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1% penicillin-streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) (all supplements were obtained from Gemini Bio-products, West Sacramento, CA). MDA-MB-361 was maintained in L-15 medium (ATCC) supplemented with 1% penicillin-streptomycin and 20% FBS.

[0148] Antibodies As described in their entirety and incorporated herein by reference, Liu et al., "Monoclonal Antibodies to the Extracellular Domain of Prostate Specific Membrane Antigen Also React With Tumor Vascular Endothelium", Cancer Res. 57:3629-3634 (1997), U.S. Patent No. 7,045,605 to Bander et al., and U.S. Patent No. 7,514,078 to Bander et al., monoclonal antibody (mAb) J591 anti-FOLH1 / PSMA, mouse and deimmunized, were generated. MAb 3E6 anti-PSMA, horseradish peroxidase-labeled polymer-conjugated goat anti-mouse Ig, and horseradish peroxidase-conjugated rabbit anti-human IgG were purchased from Dako (Carpinteria, CA). MAb 4D5 was purchased as Herceptin (Genentech / Roche). MAb anti-A antibody and anti-B antibody were purchased from Ortho Diagnostic Systems (Raritan, NJ). Ulex europaeus lectin, which recognizes α-linked fucose residues for O / H antigen detection, was purchased from Sigma-Aldrich (St. Louis, MO). Donkey anti-human IgG, horseradish peroxidase-conjugated donkey anti-human IgG, alkaline phosphatase-conjugated donkey anti-human IgG, FITC-conjugated donkey anti-mouse Ig, and FITC-conjugated donkey anti-human Ig were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-Flag M2 was obtained from Sigma-Aldrich. IRDye 800CW-goat anti-mouse secondary antibody was purchased from LI-COR Biosciences (Lincoln, Nebraska).

[0149] DNA plasmids used in this study As outlined in the following examples, a series of plasmids can be constructed for any desired targeting Ab or Ab construct or peptide, and any glycosyltransferase including, but not limited to, GTB, GTA, and fucosyltransferase (FUT1 or FUT2).

[0150] DNA plasmids and their protein products that are overexpressed in host cells after transfection or cotransfection are listed below along with a brief description. Each plasmid is described by the name of the DNA plasmid (its protein product): brief description. pMG145 (H chain): When this plasmid is introduced into host cells by transfection, the huJ591 heavy chain is produced. pMG135 (L chain): Transfection with this plasmid results in the production of the huJ591 light chain (L). pMG145 and pMG135 (huJ591 antibody): Cotransfection with these two plasmids results in the co-expression of the heavy and light chains and a functional huJ591 antibody. pMG181 (H chain-GTB): Transfection with this plasmid results in the production of a fusion protein with the huJ591 heavy chain (H) at the N-terminus and GTB at the C-terminus (see below). pMG181 and pMG135 (huJ591-GTB fusion antibody): Cotransfection with these two plasmids results in the production of heavy and light chains containing GTB.

[0151] Construction of GTB and huJ591 or 4D5 heavy chain-GTB (H-GTB) fusion expression plasmids The region of α1,3-galactosyltransferase (GTB) containing the catalytic domain (amino acids 57 - 354) was subcloned by PCR using the GTB coding plasmid pBBBB as a template. For tracking the fusion protein or to assist in the purification of the fusion protein, Flag and His tags may be added to the 3' end if desired. For example, to construct an antibody - GTB fusion protein, the DNA sequence encoding the huJ591 heavy chain (H) was ligated to the GTB catalytic domain DNA sequence to obtain plasmid pMG181. The same procedure is followed to generate 4D5 - GTB or any Ab (or Ab derivative or peptide) - GTB (or the catalytic domain of GTA for A antigen). Alternatively, if it is desired to target the synthesis of the H antigen, the catalytic domain of FUT1 or FUT2 can be incorporated. The glycosyltransferase enzyme is preferentially ligated to the C - terminus of either the heavy or light chain of the Ab construct.

[0152] A (G4S)3 spacer sequence was inserted between the Ab sequence and the enzyme sequence. Alternatively, various fusion protein linkers or spacers as described in Chen et al., 「Fusion Protein Linkers: Property, Design and Functionality」, Adv. Drug Deliv. Rev. 65(10):1357 - 69 (2013), which is hereby incorporated by reference in its entirety, can be used.

[0153] DNA Transfection and Fusion Protein Expression For the production of huJ591 - GTB (similarly 4D5 - GTB) fusion antibodies, CHO cells were co - transfected with pMG181 (H - chain - GTB) and pMG135 (L - chain) using FreeStyle MAX (ThermoFisher scientific) according to the manufacturer's instructions. Five days after transfection, the supernatant containing the fusion antibody was collected and concentrated using an Amicon Ultra 10K centrifugal filter (Merck Millipore).

[0154] Purification of the Overexpressed Fusion Protein huJ591 was purified using Protein G-Sepharose (GE healthcare) according to the manufacturer's instructions. J591-GTB was purified using anti-FLAG M2 affinity gel (Sigma-Aldrich) according to the manufacturer's instructions. Briefly, the supernatant containing the fusion protein was incubated with M2 affinity gel for 2 hours, washed, eluted with 3xFLAG peptide (Sigma-Aldrich), and dialyzed against PBS.

[0155] Western Blot Analysis The supernatant or purified fraction containing the fusion protein was separated on a 4-20% SDS-PAGE gel (Life Technology) under reducing and non-reducing conditions and transferred to a polyvinylidene fluoride membrane (PVDF) (Millipore, Billerica, MA). The membrane was blocked with 5% dry milk / PBST for 60 minutes. Anti-flag M2 was incubated with the membrane for 60 minutes. After washing, the IRDye 800CW-goat anti-mouse secondary antibody was incubated with the membrane for 60 minutes. After washing, the membrane was analyzed using an Odyssey Infrared Imaging System (LI-COR Biosciences).

[0156] Immunostaining Cells (2x10 5 / Well) was grown on a cover glass in a 12-well plate. Cells were fixed with 4% paraformaldehyde (PFA) dissolved in PBS and then washed three times with PBS. To detect human tissue blood group antigens, mouse monoclonal anti-A antibody or anti-B antibody was added at RT for 60 minutes. After washing with PBS, cells were incubated with FITC-conjugated donkey anti-mouse immunoglobulin for 60 minutes and washed with PBS. Expression of HBGA O was detected by incubating cells with FITC-conjugated wheat germ agglutinin at RT for 60 minutes and then visualizing under a UV microscope. HuJ591 was added at RT for 60 minutes for PSMA detection. After washing with PBS, cells were stained with FITC-conjugated anti-human Ig for 60 minutes and washed with PBS. The cover glass was mounted and examined under a UV microscope.

[0157] To immunostain tissue sections derived from xenograft tumors, 3E6 was used for the detection of PSMA expression in paraffin sections and huJ591 was used for frozen sections. Antibodies against blood group antigens were the same as above. Paraffin sections were deparaffinized by placing the slides in Histo-Clear and then rehydrated by sequential alcohol and washing with Tris-buffered saline-Tween 20 (TBST). The deparaffinized and rehydrated sections were placed in Target Retrieval Solution pH9.0 (Dako) and heated in a water bath (95 - 99 °C) for 30 minutes. The sections were washed with TBST. Peroxidase block was added for 5 minutes. After washing with TBST, mAb was added for 60 minutes at RT. Antibody binding was detected using peroxidase-labeled polymer-conjugated goat anti-mouse Ig and 3,3'-diaminobenzidine (DAB) substrate. Sections were visualized after counterstaining with 10% hematoxylin. Frozen sections were used to detect J591-GTB fusion antibody bound to cell surface PSMA in vivo. Frozen sections were fixed with pre-cooled acetone for 10 minutes and then washed with PBS. Peroxidase block was added for 5 minutes. After washing with PBS, J591-GTB was detected by horseradish peroxidase-conjugated rabbit anti-human IgG followed by DAB and counterstaining as described above. Sections incubated directly with huJ591 were used as positive controls.

[0158] Competitive ELISA Plates were coated overnight at 4°C with 15 μg / ml of 7E11 antibody (an antibody that binds to the N-terminal / cytoplasmic domain of PSMA) dissolved in 0.05 M carbonate buffer (pH 9.5). Wells were blocked with 2% HSA dissolved in PBS for 30 minutes at RT and washed. Lysates of LNCaP cells (containing PSMA), diluted 1:8, were added for 60 minutes at RT. After washing with PBS, serial dilutions of mouse J591 antibody (30 μl) were added for 60 minutes, followed by overnight co-incubation at 4°C with supernatants containing J591-GTB or huJ591 (1.6 μg Ig / ml; 30 μl). After washing, donkey anti-human IgG-alkaline phosphatase (1:1,000) was added for 60 minutes at RT. After washing, plates were incubated with pNPP (Sigma) and read at 405 nm.

[0159] In vitro blood group antigen conversion Cells (2x10 5 ) were grown for 24 hours on coverslips in 12-well plates. Coverslips were washed with PBS and transferred to a humid chamber. Cells were then incubated with huJ591-GTB (or 4D5-GTB) fusion antibody + UDP-galactose for 30 minutes at 37°C. After washing with PBS, cells were fixed with PFA. B antigen conversion on the cell surface was detected by immunostaining as described above.

[0160] Lysis activity of normal human O or A serum after in vitro conversion of cancer cells to HBGA B LNCaP cells were grown on 60-well microtiter plates. Cells were incubated with either native J591 or J591-GTB fusion protein, or were not incubated with either agent. UDP-gal was added to all wells. Then, sera from type A or type O patients were added as a source of natural anti-B Ab and complement. Control wells contained J591 without GTB or without sera. After 3 hours, the wells were washed, fixed with methanol, incubated with 2% Giemsa dye for 25 minutes, washed, and read. Using a similar method, a larger panel of suspended prostate cancer cell lines and breast cancer cell lines was tested. Lytic activity was evaluated by both trypan blue exclusion and propidium iodide uptake measured by FACS.

[0161] Blood group antigen conversion in vivo Under a protocol approved by the Institutional Animal Care and Use Committee (IUCUC), 6- to 8-week-old NOD SCID mice (Charles River, Wilmington, MA) were subcutaneously injected with 5 x 10 6 cells suspended in 200 μl Matrigel (Corning Life Sciences, Bedford, MD). Cell lines LNCaP, CWR22Rv1, PC3, and MDA-MB-361 were used in the animal experiments. After 14-21 days, the established tumors reached 8-10 mm in diameter. HuJ591, huJ591-GTB, 4D5, or 4D5-GTB was injected intravenously (IV) or intratumorally. UDP-gal was injected IV, intraperitoneally (IP), or subcutaneously (SQ). On day 1, 2, or 3, the mice were euthanized and the tumors and other organs were collected. Half of each tumor was prepared for frozen sections using OCT compound. The other half was placed in phosphate-buffered formalin for preparation of paraffin sections. Immunostaining was as described above.

[0162] An intraperitoneal xenograft model in NOD / SCID mice was also developed using the castration-resistant human PC cell line C4-2-luciferase. In this model, human plasma can be IP injected to supply natural Ab and complement without causing fluid overload when using the IV route. 10x10 6 After IP injection of 10x10 6 C4-2-luc cells and confirmation of tumor uptake by bioluminescence imaging several days later, two groups of five animals each with equivalent median / range bioluminescence photon fluxes were given a single IP treatment with J591-GTB, UDP-gal, and human type O serum. For the control group, type O serum was heat inactivated prior to injection. The total flux of each animal was measured every 3 - 4 days over a period of approximately 2 weeks.

[0163] Example 1 - Preparation of Antibody-Glycosyltransferase Fusion Protein First, a chimeric protein composed of an Ab targeting tumors and a glycosyltransferase, which is a prototype construct for providing a highly versatile modular system with multiple functionalities, was prepared. (1) The Ab specificity is exchangeable so that various tumor-related antigens can be targeted. Examples of such tumor antigen targets include, but are not limited to, FOLH1 / PSMA, VEGFr, CD19, CD20, CD25, CD30, CD33, CD38, CD52, CD79, B cell maturation antigen (BCMA), somatostatin receptors (e.g., SSTR1-5), 5T4, gp100, CEA, mammaglobin A, Melan A / MART-1, PSA, tyrosinase, HER-2 / neu, EGFr, hTERT, MUC1, mesothelin, nectin-4, TROP-2, and many other tumor antigen targets known in the art. The structure of the targeting moiety may vary from intact (full-length dimer) to monomeric single-chain Ab structure, Fab, Fab’2, scFv, or other Ab fragment derivatives, e.g., minibody, diabody, triabody, etc. These may maintain or lack the FcRn binding domain. Alternatively, the targeting moiety may be a peptide that binds to the target antigen. Examples include, but are not limited to, glutamate-urea-lysine derivatives, e.g., ACUPA (2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid) that binds to FOLH1 / PSMA, somatostatin derivatives that bind to SSTR2, Arg-Gly-Asp (RGD) peptides that bind to α-v / β-3 integrin expressed on proliferating endothelial cells, and other targeting peptides known in the art. These types of targeting agents, due to their different physical properties, enable adjustment of different pharmacokinetics and biodistributions. For example, large molecular constructs, e.g., intact full-length Abs containing the FcRn (neonatal receptor) binding site, have a long plasma half-life and whole-body half-life and tend to remain in circulation. Large molecular constructs are likely to be excreted through the liver rather than the kidney and have a low potential to penetrate normal tissues due to intervening normal cell layers and tight junctions.Conversely, constructs made using small, FcRn-binding-free targeting peptides rather than antibodies have a short half-life, are likely to be excreted through the kidney / urinary tract, and tend to rapidly penetrate normal tissues and tumors. In addition to the specificity of target binding, these different physical properties, PK, and biodistribution affect the adverse event profile of the constructed agent. (2) The glycosyltransferase component can be altered based on a substantial body of knowledge of the natural allelic variants and their respective properties that can be utilized to modulate its functionality. The glycosyltransferase component may also include α-gal-transferase, which generates highly immunogenic α-gal epitopes that are not naturally present in humans. The use of any enzyme involved in post-translational modification is possible. Examples in addition to glycosylation are phosphorylation and lipid modification.

[0164] As a further alternative to the production of genetically engineered fusion proteins, targeting agents and post-translational enzymes can be linked by using chemical bonds between two individual moieties. Such chemical bonds are known to those skilled in the art.

[0165] For the initial proof-of-concept effort, three well-characterized and clinically validated Abs, namely J591 (anti-FOLH1 / PSMA (folate hydrolase-1 / prostate-specific membrane antigen)), 4D5 (trastuzumab; anti-her2), and obexelimab (anti-CD19); four Ab structures, namely intact dimer, intact monomer, Fab, and scFv, and four glycosyltransferase variants, namely α1,3-galactosyltransferase (GTB; AF134414), α1-3-N-acetylgalactosaminyltransferase (GTA; AF134415), α-1,3-galactosyltransferase (α-1,3-GalT or α-GalT; EC2.4.1.87), and the following entirely novel structures were selected. GTB transfers the galactose moiety from the nucleotide-donor UDP-gal in an α1,3 linkage to the acceptor H antigen to form Galα(1-3)[Fucα(1-2)]Galβ1,4 GlcNAc-R (HBGA B). GTB requires the H antigen to be modified with an α1-2 linked fucose for activity. This is because B-transferase does not add to the unmodified type 2 precursor. α-1,3-GalT transfers the galactose moiety from the nucleotide-donor UDP-gal in an α1,3 linkage to Galβ1,4 GlcNAc-R. This enzyme does not require the H antigen to be modified with an α1-2 linked fucose for activity. GTB was selected because HBGA O-type and A-type individuals constitute 85-90% of the population (Galili et al., 「A Unique Natural Human IgG Antibody With Anti-Alpha-Galactosyl Specificity」, J. Exp. Med. 160:1519-1531 (1984), which is hereby incorporated by reference in its entirety). As previously described, these individuals have high levels of anti-HBGA B antibodies. α-1,3-GalT was selected because it can add terminal Gal to cells that do not form the H antigen, such as cells derived from hematopoietic or mesenchymal cells.The selection of GTB results in a high level of polyclonal anti-gal activity that cross-reacts with HBGA B, and as a result of the substantially identical structure, further benefits are obtained as a result of the high level of polyclonal anti-gal activity (responsible for the hyperacute rejection of xenografts), which is incorporated herein by reference in its entirety (Macher et al., "The Gal Alpha1,3Gal beta1,4GlcNAc-R (Alpha-Gal) Epitope: a Carbohydrate of Unique Evolution and Clinical Relevance", Biochim. Biophys. 1780:75-88 (2008)). From the GTB (or GTA, α-GalT, or FUT) sequence, short cytoplasmic regions, transmembrane regions, and stem regions unnecessary for enzymatic activity were excised and replaced with respective antibody (or derivative) or peptide sequences to create chimeric proteins. The membrane binding is reconstituted by the binding of the antibody or peptide domain to the cognate antigen located on the plasma membrane. ELISA assays of the chimeric proteins confirmed that the respective Ab binding specificities and immunoreactivities remained intact regardless of whether intact or antibody fragments were used (Figures 2A-2B). Incorporation of 14C-gal from UDP-14C-gal into the synthetic substrate (fucosyl-lactose (FL)) was also measured as described in Yamamoto et al., "Amino Acid Residue at Codon 268 Determines Both Activity and Nucleotide-Sugar Donor Substrate Specificity of Human Histo-Blood Group A and B Transferases. In Vitro Mutagenesis Study", J. Biol.Chem. 271:10515-10520 (1996), which is incorporated herein by reference in its entirety. High GTB activity was confirmed to be maintained from (Figures 2A-2B).

[0166] In addition to creating such fusion proteins by genetic engineering, using one of the knowledge in the art, targeting proteins, peptides, or other biopharmaceuticals can be chemically linked to effector enzymes (e.g., glycosyltransferases) that can post-translationally modify cellular proteins.

[0167] Example 2 - Modulation of the functionality of glycosyltransferase activity In-depth knowledge of the A, B, and O alleles provides ample opportunity to further refine the functionality of this component. For example, among the alternative allele variants that could be selected, there is a so-called "cisA,B" sequence that exchanges the two most important amino acid residues (aa266 and 268 (leu and gly) of GTA and aa266 and 268 (meth and ala) of GTB) to create a hybrid sequence (meth and gly) (Yazer et al., "The Cis-AB Blood Group Phenotype: Fundamental Lessons in Glycobiology", Transfus. Med. Rev. 20:207-217 (2006), which is hereby incorporated by reference in its entirety). This cis A,B enzyme sequence synthesizes both the HBGA A and B specificities.

[0168] Other sequences are known that allow the regulation of enzyme activity to adjust the potency of the enzyme. For example, a completely new version of GTB was developed based on two natural mutant alleles of GTA (referred to as Ae101 and A201). Ae101 has a single base insertion, and A201 has a single base deletion. Each causes a frameshift. Due to the frameshift, transferases with 37 amino acid extensions and 21 amino acid extensions at the C-terminus are produced, respectively. The resulting extended transferases have enzyme activity that is reduced to 1 / 30 to 1 / 50 or less (Yip, "Sequence Variation at the Human ABO Locus", Annals of Human Genetics 66:1-27 (2002), which is hereby incorporated by reference in its entirety). These two mutant alleles were defined with respect to GTA, but such mutant alleles have not been described for GTB. Nevertheless, a completely new sequence was created by directly incorporating a C-terminal sequence extension into GTB by inserting various amino acid sequences of various lengths before the stop codon. When four versions of GTB incorporating extensions of 2, 7, 14, and 54 amino acids were tested, the GTB activity gradually decreased to 93% (Figure 3). Thus, by incorporating a cleavable sequence that cleaves the extension in the presence of tumor- or tissue-associated endoproteases or endopeptidases, such as PSA, metalloprotease, etc., a mechanism can be obtained to adjust the desired activity level and on-off switch as described above. Any sequence selection for the extension is at the discretion of the practitioner, and the only requirements are that the sequence is selected to achieve the desired level of enzyme activity that can be measured as described below and that it is non-immunogenic. Non-immunogenicity may be achieved using the sequence information of natural non-immunogenic proteins (e.g., albumin), or may be achieved by methods known in the art that elicit or determine immunogenicity, e.g., by eliminating T cell binding motifs.

[0169] Example 3 - Inducible expression of HBGA B in vitro To demonstrate the functionality of the construct, human prostate cancer cell lines LNCaP (PSMA-high), CWR22Rv1 (PSMA heterologous and low), and PC-3 (PSMA-neg), all of which are naturally HBGA O, were incubated in vitro and on tissue sections of xenografts from SCID mice with both chimeric J591 (anti-FOLH1 / PSMA)-GTB or J591 (without GTB) and UDP-gal. Cell lines and tissue sections incubated with chimeric J591-GTB + UDP-gal were converted to HBGA B, whereas cell lines and tissue sections incubated with J591 (without GTB) + UDP-gal were not converted to HBGA B. This proves that GTB was required for the conversion (Figure 4).

[0170] In vitro, J591-GTB converted LNCaP (PSMA-high) from HBGA O to HBGA B, but did not convert PC3 (PSMA-neg) (Figure 5). Testing PC3 cells transfected with PSMA (PC3-PSMA) confirmed the high specificity of HBGA conversion. In these PSMA heterologous expressing cells, only PSMA-pos cells were converted. Adjacent PSMA-neg cells were not converted (Figure 6).

[0171] HBGA O LNCaP cells were co-incubated in O-type whole blood + UDP-Gal with J591 or J591-GTB or J591-GTB-54 amino acid extension. As shown in Figure 7, J591 did not convert the cells at all, whereas J591-GTB converted LNCaP cells from O-type to HBGA B-type with or without the extension and did not convert RBCs.

[0172] Example 4 - Lytic activity of normal human O or A serum after cancer cells are converted to HBGA B Using in vitro assays, the lysis ability of prostate cancer cells or breast cancer cells after conversion to HBGA B expression to be lysed by normal human O and A sera was tested. Figures 8A - 8D show that LNCaP cells (HBGA O) are lysed when incubated with J591 - GTB+UDP - gal+human A (or O serum) (as a source of anti - B component and complement components). Removal of human A or O serum and / or replacement of J591 - GTB with J591 without GTB resulted in no lysis.

[0173] A larger panel of prostate cancer cell lines, all of which are HBGA O, was assayed by trypan blue exclusion (Figure 9) and propidium iodide uptake by FACS analysis (Figure 10). Four of these lines (LNCaP, VCaP, MDA - PCa - 2b, and CWR22Rv1) express PSMA at various levels from high to low and were all lysed when incubated with human O or A serum containing J591 - GTB+natural anti - B Ab+endogenous complement. The fifth cell line, PC3, is PSMA - neg, not converted, and did not lyse (Figures 11A - 11B). Similar results were obtained using the breast cancer cell line MDA - MB - 361 after conversion with the chimeric agent mAb 4D5 - GTB.

[0174] Example 5 - Conversion of HBGA Expression in Vivo The rapid rejection and destruction of HBGA-incompatible solid organ transplants in humans has been well documented and established since the early days of kidney transplantation (T. Starzl, Experience In Renal Transplantation. (WB Saunders Company, Philadelphia, PA, chapter 6 (1964); L. Altman, Doctors Discuss Transplant Mistake. New York Times. (2003), which are hereby incorporated by reference in their entirety). The in vivo experiments of great significance were to prove that the HBGA of established human cancers can be converted to that of highly immunogenic HBGA by "molecular transplanting" the allogeneic glycosyltransferase that normally functions inside the Golgi / ER to the plasma membrane of tumor cells using a systemic tumor-targeting approach. For proof of concept, two clinically well-established tumor-associated antigens (FOLH1 / PSMA and HER2) obtained from two of the most common types of solid tumors, prostate cancer and breast cancer, were selected. Multiple tumor lines expressing a wide range in terms of target expression levels were tested. PSMA-pos prostate cancers LNCaP, C4-2, and CWR22Rv1, as well as her2-pos breast cancer, MDA-MB361 were established in the subcutaneous sites of NOD SCID mice. J591-GTB or 4D5 / trastuzumab-GTB was administered IV. UDP-gal was administered via the IV, IP, or subcutaneous route. J591-GTB and 4D5 / trastuzumab-GTB each converted PSMA-pos prostate cancer and her2-pos breast cancer (see Figures 10A - 10H. See also Figures 12A - 12E).

[0175] HBGA B conversion was insufficient after IP administration of UDP-gal compared to IV or SQ administration. HBGA expression was clearly present on the plasma membrane. As expected, no HBGA B expression occurred when Ab-GTB was replaced with each Ab alone. No conversion could be detected in any other tissue, and the animals showed no evidence of toxicity.

[0176] Example 6 - Antitumor Activity In Vivo Testing for antitumor activity resulting from Ab-GTB-induced HBGA expression conversion in animal models has presented several difficulties. This is because both mice and rats express the cisA,B alleles as well as the α1,3GalT alleles. As a result, these rodent models are HBGA A positive, B positive, and α1,3gal positive and are thus tolerant to all of these sugar structures. Additionally, mice have a very weak or inactive complement system (Bergman et al., Cancer Immunol. Immunother. (2000) 49:259-266, which is incorporated herein by reference in its entirety; Drake et al., 「Passive Administration of Antiserum and Complement in Producing Anti-EL4 Cytotoxic Activity in the Serum of C57BL / 6 Mice」, J Natl. Cancer Inst. 50:909-14 (1973); Ong et al., 「Mouse Strains With Typical Mammalian Levels of Complement Activity」, J. Immunol. Methods 125:147-158 (1989)), and in some cases, even inhibit the function of complement activity in other species, including humans (Ratelade et al., 「Inhibitor(s) of the Classical Complement Pathway in Mouse Serum Limit the Utility of Mice as Experimental Models of Neuromyelitisoptica」, Mol. Immunol. 62:104-113 (2014), which is incorporated herein by reference in its entirety). Indeed, the complement activity of normal human plasma was assayed in the presence of C57BL / 6 plasma. A decrease of approximately 33% in human complement lysis activity was found.In order to supply the necessary natural Abs and functional complement proteins to these animal models, which lack natural Abs and have a weak or even inhibitory complement system, it would be necessary to almost completely replace the animal's plasma with human type O or type A plasma. This plasma exchange is physically impossible, would result in fluid overload, would not provide an appropriate immunoglobulin in vivo distribution balance reflecting the human steady state, and would be impaired by the inhibitory effects of natural mouse plasma. These problems were overcome by developing an intraperitoneal xenograft model in NOD / SCID mice using the castration-resistant human PC cell line C4-2-luciferase. In this model, human plasma could be injected IP to supply natural Abs and complement without causing fluid overload. 10x10. 6 After injecting 10x106 C4-2-luc cells IP and confirming tumor uptake by bioluminescence imaging several days later, two groups of five animals each with equivalent median / range bioluminescence photon fluxes were given a single IP treatment with J591-GTB, UDP-gal, and human type O serum. For the control group, type O serum was heat-inactivated prior to injection. The total flux of each animal was measured every 3 - 4 days over approximately 2 weeks. Animals given heat-inactivated serum experienced significant tumor progression by day 13. In contrast, animals treated with serum containing active complement regressed by 80% compared to the flux of the control group (p = 0.0032; Figures 13A - 13B). Consistent results were obtained from the experiment repeated twice (Figures 14A - 14B).

[0177] Discussion of Examples 1 - 6 The immune response to cancer is quite different from the response to mismatched allografts. As described herein, a strategy is presented to selectively modify tumor cells to express mismatched HBGA expression, a non-self, highly immunogenic phenotype. The rapid and destructive outcome of HBGA mismatched allografts in humans was reported early in renal transplantation by Starzl (T. Starzl, Experience In Renal Transplantation. (WB Saunders Company, Philadelphia, PA, chapter 6 (1964)), which is hereby incorporated by reference in its entirety), leading to the HBGA compatibility test, an essential and important part of donor-recipient matching. This caution is repeated and reinforced only in the rare case where an error occurs and the requirement for HBGA compatibility is breached (L. Altman, Doctors Discuss Transplant Mistake. New York Times (2003), which is hereby incorporated by reference in its entirety).

[0178] To implement this strategy, clinically validated tumor-restricted antibodies (exemplified by anti-FOLH1 / PSMA and anti-HER2) were fused to GTB to generate a single bifunctional protein. Similarly constructed glycosyltransferases (GTs) targeted with various antibody fragments, peptides / ligands, and constructs. Although the outcome of mismatched ABO allografts in humans is well documented, the challenge in this endeavor was to molecularly "transplant" the post-translational glycosyltransferase function, normally found in the Golgi, to the tumor (or neovasculature endothelial) cell surface, and to do so in a tumor-targeted manner by systemic administration.

[0179] These chimeric proteins successfully altered the HBGA of various cancer cell lines both in vitro and in vivo. No off-target HBGA conversion or toxicity was observed in animal experiments. HBGA-incompatible cells, as shown herein, induce complement-mediated lysis, a response expected to occur in cancer patients, as has been demonstrated numerous times in the context of clinical transplantation (the entirety of which is incorporated herein by reference, L. Altman, Doctors Discuss Transplant Mistake. New York Times (2003); T. Starzl, Experience In Renal Transplantation. (WB Saunders Company, Philadelphia, PA, chapter 6 (1964))).

[0180] For the biosynthesis of the new HBGA, both a GT and the (fucosylated) H antigen “acceptor structure” on glycoproteins and glycolipids of the target cells are required for the addition of the HBGA (Milland et al., “ABO Blood Group and Related Antigens, Natural Antibodies and Transplantation”, Tissue Antigens 68:459 - 466 (2006), which is hereby incorporated by reference in its entirety). Since a variety of cancers, including lung, stomach, colorectal, breast, prostate, ovarian, bladder, and pancreatic cancers, express the H antigen, these tumor types would be candidates for this strategy. (1) Normal non - target cells do not undergo HBGA conversion because (2) the required HAg is absent in many normal cell types (e.g., bone marrow, liver, spleen, kidney, myocardium, central nervous system, and peripheral nervous system, etc.), and the GTB (or GTA) transferase activity at these sites is inhibited, so the targeted GTB (or GTA) enzyme does not bind. FOLH1 / PSMA expression has been reported in the tumor neovessels of a wide variety of tumors but is absent in the vasculature of normal tissues. Examples of tumor types with FOLH1 / PSMA - positive neovessels include kidney tumors, lung tumors, colon tumors, stomach tumors, breast tumors, brain tumors, pancreatic tumors, liver tumors, bladder tumors, esophageal tumors, adrenal tumors, head and neck tumors, melanoma, and brain tumors, etc. Testis, lymphatic system, and sarcomas are not common but may sometimes be FOLH1 - positive. Targeting FOLH1 / PSMA expression in tumor neovessels provides a means to alter HBGA expression within the vasculature of a wide variety of tumors.And next, this would lead to the same kind of hyperacute rejection phenomenon seen in the solid tissue allografts of the wrong HBGA (L. Altman, Doctors Discuss Transplant Mistake. New York Times (2003); T. Starzl, Experience In Renal Transplantation. (WB Saunders Company, Philadelphia, PA, chapter 6 (1964)), which is incorporated herein by reference in its entirety).

[0181] The enzymatic nature of the reaction results in an amplification effect. This is because each targeted enzyme molecule converts a very large number of acceptor molecules. Furthermore, not only is the tumor - associated antigen itself targeted by the Ab enzymatically converted, but all adjacent molecules within the range of the enzyme are also enzymatically converted. And since most cell - surface molecules have multiple glycosylation sites, and FOLH1 / PSMA has, for example, 10 (20 if considering that FOLH1 / PSMA is usually expressed as a homodimer) glycosylation sites, the amount of non - self HBGA sites that can be created by this approach is quite large. Additionally, glycoproteins secreted by the targeted neovascular cells or tumor cells are also HBGA - converted, complement activation occurs in the tumor microenvironment, thereby enhancing the immune environment around the tumor.

[0182] These above-mentioned factors, namely, enzyme amplification, conversion of directly targeted molecules and adjacent and secreted glycoproteins, and amplification factors of abundant glycosylation sites, should result in unprecedentedly high immunogenic antigen expression levels by tumor cells and within the tumor microenvironment, even in the case of weakly expressed tumor-associated antigen targets. Since HBGA O and A patients constitute approximately 85% of the population, GTB was utilized in the proof-of-concept endeavor. Additionally, since polyclonal anti-gal activity that impedes xenotransplantation cross-reacts with HBGA B (Macher et al., "The Gal alpha1,3Gal beta1,4GlcNAc-R (alpha-Gal) Epitope: a Carbohydrate of Unique Evolution and Clinical Relevance", Biochim. Biophys. 1780:75-88 (2008), which is hereby incorporated by reference in its entirety), HBGA B becomes a target for an unprecedent level of attack by complement-binding antibodies that can mediate high levels of inflammation and hyperacute rejection due to the inducible expression of HBGA B by the tumor and / or its blood supply. This strategy might be expandable to cover HBGA O, A, and B patients (approximately 95% of the population) by using GTs with both A and B activities. This is a mutation that naturally occurs in so-called cisAB GT and is achievable by a single nucleotide / amino acid change 803G>C (Gly268Ala) in GTA that results in both HBGA A and B. This approach would be applicable to all patients except AB patients (approximately 5% of the population) who have neither natural anti-A nor anti-B antibodies.

[0183] The methods described herein share many features with and are complementary to recent successful immunotherapy approaches. Similar to CAR-T and bispecific Ab approaches that utilize T cell lysis mechanisms, this approach uses the lysis mechanism of the complement cascade. Beyond direct lysis, induction of the complement cascade within the tumor microenvironment serves as a bridge to enhance the efficacy of the cellular immune response to promote initial antigen stimulation of T cells (Kopf et al., “Complement Component C3 Promotes T-cell Priming and Lung Migration to Control Acute Influenza Virus Infection”, Nature Med. 8:373-378 (2002), which is incorporated herein by reference in its entirety), when C3 activates APCs (Baudino et al., “C3 Opsonization Regulates Endocytic Handling of Apoptotic Cells Resulting in Enhanced T-cell Responses to Cargo-Derived Antigens”, Proc. Natl. Acad. Sci. USA 111:1503-1508 (2014); Surace et al., “Complement is a Central Mediator of Radiotherapy-Induced Tumor-Specific Immunity and Clinical Response”, Immunity 42:767-777 (2015), both of which are incorporated herein by reference in their entirety).Furthermore, recently, it has been shown that the release of free C3d, a fragment of C3, depletes Tregs (via apoptosis), increases the infiltration of CD8+ T cells that produce perforin, TNF-α, and IFN-γ, and decreases PD-1 expression by T cells (Platt et al., “C3d Regulates Immune Checkpoint Blockade and Enhances Antitumor Immunity”, JCI Insight. 2:e90201 (2017), which is hereby incorporated by reference in its entirety). Additionally, activation of the complement system generates chemotactic factors such as C3a and C5a that induce inflammation and recruit inflammatory cells. This would convert the “cold” tumor microenvironment into a “hot” microenvironment that is more supportive of the immune response. In short, the approach described herein offers the potential to broaden the breadth and intensity of the immune attack against cancer by directly using the humoral immune system and the complement cascade, as well as by its role in enhancing the cellular immune response.

[0184] Example 7 - B conversion of MM1-S using anti-CD19-GTB Figures 15 to 17 show the ability to convert CD19-positive / HBGA O-positive myeloma cells to express HBGA B. In this case, MM1-S myeloma cells that were passaged in tissue culture were tested by fluorescence-activated cell sorting (FACS) using mouse monoclonal antibodies against CD19, CD20, CD38 (Figure 15), HBGA A, HBGA B (Fisher Scientific (Ortho)), and Ulex-FITC or Ulex-Dylight (Vector Labs) to detect HBGA O (Figures 16A to 16B). MM1-S cells were incubated with each antibody for 1 hour, the cells were washed, and then incubated with an appropriate secondary antibody such as anti-mouse IgM-Alexa488 or 647 (Jackson ImmunoResearch). In this case, the primary antibody was IgM or tagged anti-mouse IgG when the primary antibody was IgG. After washing once more, the cells were analyzed by FACS. As shown in Figure 17, MM1-S cells were incubated with anti-CD19-GTB fusion protein + UDP-gal, and HBGA B expression was compared to untreated cells by FACS.

[0185] Figure 15 shows that MM1-S myeloma cells are CD20-negative, CD19 + , and CD38 + . Figures 16A to 16B show that MM1-S cells are HBGA A-negative and B-negative (Figure 16A), but HBGA O-positive (Figure 16B). Figure 17 shows that MM1-S cells incubated with anti-CD19-GTB fusion protein + UDP-gal are converted to high-level HBGA B expression compared to untreated cells.

[0186] These experiments show another example of tumor-targeted conversion to express the foreign antigen: HBGA B. In this case, the target is CD19, a B-cell marker that is also present in B-cell neoplasms. This is a conversion of a hematopoietic tumor type, whereas prostate (PSMA) and breast (HER2) shown in other examples are examples of solid tumors.

[0187] Example 8 - Targeting glycosyltransferase via a small molecule ligand instead of an antibody or antibody derivative Figure 18 demonstrates that targeting of GTA, GTB, or α-gal can be achieved not only with antibody-based constructs but also with peptide / small molecule ligand-based targeting agents. In this case, the GTB enzyme was conjugated with 2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid (ACUPA), a galactose-urea-lysine-based ligand that binds to PSMA. To achieve high-level binding, a PEG1500 spacer was used between ACUPA and the GTB moiety. This provided sufficient steric freedom for ACUPA to bind to the PSMA enzyme pocket without steric hindrance from the much larger GTA / GTB enzyme than ACUPA.

[0188] Figure 18 shows that ACUPA-PEG1500-GTB can convert LNCaP cells from HBGA O to HBGA B (left panel). No conversion occurred when using pure GTB without the ACUPA moiety for targeting (right panel).

[0189] Having the flexibility to use various targeting moieties, from a large 150 kd antibody to small antibody-derived formats such as monomer (75Kd), Fab’2 (100kd), Fab (50kd), scFv (25kd), and short peptides such as ACUPA (1.0kd), enables the construction of fusion proteins with various pharmacokinetic and biodistribution properties. Larger fusion proteins tend to have a longer circulation, stay in the blood compartment for a longer time, and are excreted through the liver, while smaller constructs tend to have a shorter serum half-life, reach / contact the tumor target quickly, and are excreted by the kidneys. These various options can be utilized to tailor the therapy according to the requirements of various tumor types (e.g., hematological tumors vs. solid tumors).

[0190] Example 9 - Specificity and accuracy of conversion; absence of the bystander effect As shown in FIGS. 19 and 20, the breast cancer cell line SK-BR5 (PSMA negative) and the LNCaP (PSMA positive) prostate cancer cell line were co-cultured. These cells can be distinguished because they have characteristic morphologies. That is, SK-BR5 is spherical, and two clusters can be seen near the center and upper part (red circles) of the field of view in FIG. 19. LNCaP is more elongated, and these cells also express GFP as a discriminant marker.

[0191] When the culture was treated with J591-GTB + UDP-gal, HBGA B (stained with Cy5 (purple)) appeared only on PSMA-positive cells. The adjacent PSMA-negative SKBR5 cell cluster (highlighted in the red circle), even in the immediate vicinity of the converted cells, was not converted to HBGA B. Similarly, FIG. 20 shows the same, distinguishable cell types. The left panel shows all of the cell nuclei stained with DAPI. The central panel shows spindle-shaped LNCaP cells with green fluorescence due to GFP expression. The right panel, after treatment with J591-GTB + UDP-gal, shows that HBGA B is expressed only in PSMA-positive LNCaP cells, while the PSMA-negative SK-BR5 cells remain HBGA B-negative.

[0192] This demonstrates a high degree of specificity and the absence of a bystander effect. That is, even adjacent cells are not converted unless they are directly targeted and bind to the fusion protein.

[0193] Example 10 - Quantification of Specificity Index Figures 21A - 21B and Figure 22 quantify the specificity index for the same two types of PSMA - positive cell lines and PSMA - negative cell lines. Anti - PSMA - GTB at various concentrations from 100 μg / mL to 0.003 μg / mL was incubated one by one with each cell line in the presence of the nucleotide donor UDP - gal. The specificity of the conversion was quantified by using FACS to compare the concentration of J591 (anti - PSMA) - GTB required to convert LNCaP (PSMA+) to HBGA B with that in SK - BR5 (PSMA - negative) cells. Both cell lines are O+. FACS histograms are shown in Figures 21A - 21B. Note that concentrations above 12.5 μg / mL overlap the 12.5 μg / mL curve and have been removed from the FACS histogram for ease of viewing.

[0194] At J591 - GTB concentrations up to 100 μg / mL, B - conversion of SK - BR5 does not occur. In comparison, anti - PSMA - GTB at concentrations as low as 0.012 μg / mL begins to induce conversion of PSMA - positive cells. This data is shown in histogram form in Figure 22. From this, it can be seen that the fusion protein is at least 8,196 - fold more specific for PSMA - positive cells than for PSMA - negative cells. This is due to the ability of the fusion protein to directly bind to PSMA - positive cells, accumulate on the cell surface of PSMA - positive cells, and perform its enzymatic function. If the enzyme does not bind to the target cell surface, the enzymatic reaction is either very weak or non - existent. For practical reasons, concentrations above 100 μg / mL were not tested. The calculated specificity index may actually be well above 8,000 - fold.

[0195] Figures 19 - 22 demonstrate that the very excellent conversion reaction specificity is limited only to target - positive cells and the lack of a bystander effect where cells that are target - negative and do not bind to the fusion protein are not converted, even if adjacent to the target - positive cells.

[0196] Example 11 - Both cell - surface glycoproteins and secreted glycoproteins are glycosylated by this method Since both cell surface glycoproteins and secreted glycoproteins are glycosylated by the same cellular processes in the Golgi / apparatus, in addition to the conversion of HBGA of cell surface molecules, the glycoproteins secreted by target cells are also HBGA-converted. In this exemplary case, the secreted glycoprotein is converted to HBGA B positive. LNCaP cells were treated with J591-GTB+UDP-gal for 5 hours (10 μg / ml anti-PSMA-GTB+100 μM UDP-gal). As a negative control, another set of LNCaP cells was incubated with 10 μg / ml anti-PSMA-GTB without UDP-gal. As a positive control, the measurement of cell surface conversion was performed together with unconverted cells that served as a background control. After 5 hours of incubation, the spent medium containing secreted glycoproteins was collected from each set of cells and concentrated 10-fold using an Amicon 3,000 Dalton cut-off. The spent medium was adsorbed to the wells of the plate and analyzed by ELISA for the presence of HBGA B using IgM anti-HBGA B followed by anti-mouse IgM-alkaline phosphatase.

[0197] Figure 23 shows that for the presence of HBGA B on the secreted protein, the converted medium was positive compared to the negative control (unconverted spent medium).

[0198] This demonstrates that HBGA B conversion is not limited to the cell surface and also includes glycoproteins secreted by target cells. In vivo, this suggests that these converted secreted glycoproteins penetrate the tumor extracellular space, bind to natural anti-B antibodies, induce complement, create an inflammatory microenvironment, recruit inflammatory and immune cells via chemotaxis, and further convert the tumor microenvironment into a "hot" microenvironment.

[0199] Analysis of the utilization of example 12 - α1,3 galactosyltransferase (aGalT) In addition to using human glycosyltransferase A or glycosyltransferase B enzymes, another approach utilizes the enzyme α1,3 galactosyltransferase (aGalT, EC 2.4.1.87) that functions in all mammals but is inactive in humans and Old World monkeys due to evolutionary mutations. GTA, GTB, and α1,3GalT are highly homologous and are thought to be derived from the same ancestral gene. Similar to GTB, αGalT adds terminal α1,3 galactose to the carbohydrate chains of cell surface - type and secreted glycoproteins and glycolipids, but unlike GTB, αGalT can add its Gal in the absence of the H - antigen fucose acceptor structure. All humans lack functional αGalT and thus do not express this terminal α1,3Gal epitope, and have high levels of anti - αGal antibodies of the IgM, IgG, IgA, and IgE classes, which are estimated to make up about 1% of all circulating immunoglobulins. What prevents xenotransplantation from other mammals that have functional aGalT and express terminal α1,3Gal on the surface of tissues including blood vessels is the immunogenicity of this α1,3Gal epitope.

[0200] Using αGalT eliminates the need to select GTA or GTB depending on the blood type of the subject. Thus, it becomes possible to use this treatment approach in AB - blood - type patients who have no natural antibodies against HBGA A or B but do have antibodies against α1,3Gal. In the case of αGalT, since it does not require the H - antigen fucose as an acceptor, the range of tissue types that can be addressed also expands. For example, hematopoietic cells and mesenchymal - derived cells (and tumors derived from these cell types) as well as other tissues do not express the H - antigen acceptor. These tissues / tumors cannot be addressed by GTA or GTB but may be addressed by α1,3GalT.

[0201] One concern regarding the use of αGal transferase is whether the enzyme is immunogenic in humans if humans do not express a functional version of the enzyme. If this were true, the enzyme would need to be humanized or deimmunized for repeated dosing. This concern was evaluated by assaying sera from 50 randomly selected patients of different blood types to see if anti-αGalT antibodies were present.

[0202] An ELISA assay was performed by coating α1,3GalT (500 ng / ml) overnight at 37 °C in 96-well Half Area High Bind microplates. The negative control wells were not coated with the enzyme. After washing and blocking with PBS-HSA (5%), sera from 50 different donors were added to the plates at room temperature (RT) for 2 hours. This included sera from 21 O-type patients, 20 A-type patients, 3 B-type patients, and 6 AB-type patients. After washing, an anti-human IgG+IgM-Alk Phos antibody solution was added, followed by addition of PNPP, and the plates were read at 405 nm to detect anti-α1,3GT antibodies. To ensure that α1,3GT was correctly coated, since this protein was labeled with a His-tag, this protein was detected using an anti-His-tag antibody (positive control). As another control, to ensure that AP anti-human IgG+IgM functioned, 500 ng / ml of BSA was coated and anti-BSA antibodies derived from human serum (HBGA A) were measured using the same method.

[0203] None of the 50 sera were found to contain antibodies against α1,3GalT (Figures 24A - 24B). This is presumably due to the homology of a wide variety of glycosyltransferases including, but not limited to, GTA and GTB. From this result, it can be seen that the αGalT enzyme can be used in the fusion protein without concern that the fusion protein will be immunogenic to generate the αGal epitope. Therefore, the likelihood of requiring deimmunization or humanization is low.

[0204] Example 13 - Expression and Purification of Recombinant α1,3 Galactosyltransferase (aGalT) Similar to the approach using GTA and GTB, an anti - CD19 scFv fused to a part of the α1,3GalT sequence (aa90 - 376) was constructed. The scFv sequences used were derived from Seattle Genetics' Denintuzumab (Den) and Xencor's Obexelumab (Obx), which recognize and bind to both cynomolgus CD19 and human CD19. The same (G4S)3 spacer / linker previously described in the Ab - GTB construct was used. For αGalT, a marmoset sequence with 376 amino acid residues, corresponding to a general glycosyltransferase topology: a 6aa cytoplasmic domain, a 16aa transmembrane domain, and a 354aa lumen domain containing the enzyme activity, was selected.

[0205] The stem region of marmoset α1,3GT consists of 67 amino acids and extends from amino acids 23 - 89 of the lumen part of the enzyme. This can be removed without affecting enzyme activity. The truncated 90 - 376α1,3GT was functional and was selected for the fusion protein. A His - tag was added to this enzyme. The construct was expressed in Expi293F cells and purified using a metal affinity column.

[0206] From SDS - PAGE electrophoresis probed with anti - his, a high - purity preparation of the desired construct was revealed at the appropriate predicted molecular weight (Figure 25).

[0207] Example 14 - Investigation of the Functionality and Specificity of the Anti - CD19 scFv - αGalT Construct To demonstrate the functionality and specificity of the anti-CD19 scFv-αGalT construct, hematopoietic target Raji B lymphoma cells that express neither the H antigen acceptor structure nor HBGA A or B were selected for the presence of CD19. CD19 is also a validated tumor target. In addition to functionality, specificity was evaluated by comparing αGal addition to CD19+ Raji cells co-incubated with CD19-neg MM1.S cells.

[0208] The CD19-positive cancer cell line Raji-GFP was mixed with CD19-negative cancer cells (MM1.S) at different ratios and incubated with the scfv-αGalT construct (10 μg / ml) and UDP-Gal (5 mM) at 37 °C for 1 hour. Subsequently, the presence of the α1,3Gal epitope was evaluated by flow cytometry using an anti-α1,3Gal antibody. Raji-GFP cells were used to distinguish between Raji-GFP cells and MM1.S cells.

[0209] The fusion protein binds to CD19-positive Raji cells and saturates at 1 - 10 μg / mL, but does not bind to CD19-negative MM1.S cells (Figures 26A - 26B). The anti-CD19 scFv-aGalT construct added terminal α1,3Gal to CD19-pos Raji cells, but did not add terminal α1,3Gal to CD10-neg MM1.S cells even when CD10-neg MM1.S cells were present at 30-fold excess of CD19-pos Raji cells (Figure 27). MM1.S never became αGal-positive in the presence of scfv-αGalT fusion protein + UDP-Gal, even at a high ratio to Raji cells.

[0210] α1,3GalT itself does not have an scFv binding domain and does not add a Gal moiety. From this, it is proven that for the addition of α1,3Gal, binding via the antibody (or fragment) moiety of the fusion protein is required (Figure 28). Furthermore, when UDP-gal was not added, Gal was not added to the target cells. In the presence of UDP-gal, the α1,3Gal moiety was added, but no HBGA B epitope was generated as proven by the non-binding of the antibody to HBGA B (Figure 28).

[0211] Example 15 - Ability of anti-CD19 scFv-aGalT to convert fresh human lymphocytes Similarly, the ability of anti-CD19 scFv-aGalT to convert fresh human lymphocytes was tested. To avoid distorting the results due to the presence of the anti-CD19 construct, anti-CD20 was used to identify B cells in this experiment. Cells were incubated with only the indicated concentration of UDP-gal, only Obx CD19-αGalT, or both ObxCD19-αGalT + UDP-gal. Using two-channel FACS, the binding of CD19-αGalT (x-axis) and the expression of the αGal epitope (y-axis) were measured (Figure 29). An untreated negative control was also performed.

[0212] CD20-negative cells did not bind the fusion protein and were not converted to express α1,3Gal (Figure 29, upper panel). CD20-positive cells (Figure 29, lower panel) showed binding of the fusion protein and were converted to express α1,3Gal only when UDP-gal was also added.

[0213] Example 16 - Lytic functionality of human sera from different blood type donors against CD19-positive Raji-GFP cells The lytic functionality of human sera from different blood type donors against CD19-positive Raji-GFP cells was tested. CD19+ Raji-GFP cells were incubated with Obx-αGT (10 μg / ml) at 37 °C for 1 hour with or without UDP-Gal (5 mM). Subsequently, sera from different donors were added to the cells and incubated at 37 °C for 4 hours. The viability of the cells was evaluated by measuring GFP expression by flow cytometry.

[0214] When UDP-gal, a nucleoside donor for completing anti-CD19 scFv-aGalT conversion and expressing terminal α1,3Gal, was not provided, only background lysis was observed (Figure 30). However, when UDP-gal was included and αgal was converted, the human sera lysed the converted cells. In this experiment, it was found that O-type serum and A-type serum caused greater lysis than B-type serum or AB-type serum.

[0215] Example 17 - Conversion and lysis of fresh human B cells using autologous serum The above was extended to examine the conversion and lysis of fresh human B cells using autologous serum (from the same donor). Here, the anti-α1,3Gal IgG and IgM levels of individual donors were also measured. Human PBMCs from donors of different blood types were incubated with Obx-αGT (10 μg / ml) and UDP-Gal (5 mM) at 37 °C for 1 hour. Cell aliquots were analyzed by FACS using anti-γ chain specific antibody or anti-μ chain specific antibody to determine whether human IgG and human IgM bound. Subsequently, serum from the same donor was added to another cell aliquot and incubated at 37 °C for 4 hours. B cell depletion was measured by flow cytometry using anti-CD20.

[0216] The greatest degree of lysis of the transformed cells was found in A-type and O-type patients. This was consistent with the anti-α1,3 Gal levels of individual patients (Figures 31A - 31B). This suggests that measuring anti-αgal before treatment can predict whether a patient is likely to respond well or poorly to this treatment approach. Furthermore, it is also suggested that some patients, particularly type B or AB, may benefit from a first antigenic stimulation by exposure to the α1,3gal antigen in order to stimulate higher levels of anti-αgal antibodies. This could occur by subcutaneous administration of polysaccharides or glycoproteins containing αgal at least one week before this therapeutic approach. Or, the αgal epitope induction in the first treatment cycle may help to stimulate anti-αgal antibody production such that it is present for subsequent cycles. One piece of knowledge in the art is that a series of patients can be evaluated for pre-treatment anti-αgal levels and the relationship between pre-treatment anti-αgal levels and response to determine a threshold. Below the threshold, the likelihood of a response without a first antigenic stimulation is low.

[0217] Example 18 - Determination of Optimal Concentrations of Anti-CD19 scFv-aGalT and UDP-gal for Generating Human Donor CD19 Cell Lysis Using Autologous Serum The optimal concentrations of anti-CD19 scFv-aGalT and UDP-gal for generating human donor CD19 cell lysis using autologous serum were determined. Human PBMCs from HBGA A-type donors, which are known to induce serum-mediated lysis of αGal-converted cells, were incubated with various concentrations of Obx-αGT and UDP-Gal at 37°C for 1 hour. Serum from the same donor was then added to the cells and incubated at 37°C for 4 hours. Binding, α1,3Gal transfer, and B cell depletion were evaluated by flow cytometry.

[0218] Anti-CD19 scFv-aGalT saturated CD19 cells at approximately 10 ug / mL (Figures 32A - 32C). The expression of α1,3gal was best at a UDP-gal concentration of approximately 10 mM. B cell lysis was maximal using 10 ug / mL of anti-CD19 scFv-aGalT and UDP-gal in the range of 5 - 20 mM. B cell lysis decreased progressively at anti-CD19 scFv-aGalT concentrations >10 ug / mL, particularly >=25 ug / mL which is above the saturation point of CD19. This is likely because unbound anti-CD19 scFv-aGalT competes for UDP-gal, thereby reducing the UDP-gal available to anti-CD19 scFv-aGalT bound to the cells.

[0219] The concentrations of scFv-aGalT and UDP-gal may vary depending on the cancer target antigen, and their density and lysis efficacy on the tumor cell membrane may vary depending on the levels of anti-α1,3Gal antibodies (IgM and / or IgG and / or IgA and / or IgE). All of these parameters can be measured prior to treatment, and one of ordinary skill in the art can determine the optimal concentrations of the various components for treating each individual patient.

[0220] Example 19 - Manipulation of Anti-CD19 scFv-αGal Transferase Obexelimab-scFv-α-1,3 Gal (SEQ ID NO:63, Table 1) was constructed by fusing the obexelimab single-chain variable fragment (scFv) to the N-terminus of cynomolgus-derived α-1,3 galactosyltransferase (aa90 - 376) in the vH-vL orientation via a (G4S)3 linker. A 6His tag was added to the C-terminus of the fusion protein to enable affinity chromatography purification.

[0221] The above-mentioned protein was produced by WuXi Biologics. Briefly, the target DNA sequence (SEQ ID NO: 63) encoding obexelimab-scFv-α-1,3 Gal was codon-optimized, synthesized, and subcloned into an expression vector protected by the intellectual property rights of WuXi Biologics. The fusion protein was expressed by transient transfection in CHO cells scaled up to 2L. Obexelimab-scFv-α-1,3Gal was purified from the cell culture supernatant by a three-step column purification process. Nickel affinity chromatography was used in the first capture step, followed by anion exchange chromatography and then size exclusion chromatography to obtain a protein purity of 95% with an endotoxin level of <1 EU / mg. The purified protein was formulated by dissolving it in 20 mg / ml histidine buffer pH 6.0. Protein purity was evaluated by SDS-PAGE and SEC-HPLC, and endotoxin levels were tested.

[0222] (Table 1) Protein sequence of obexelimab bifunctional therapy TIFF2025518735000007.tif153163

[0223] Example 20 - In vivo treatment of non-human primates with anti-CD19 scFv-αGal transferase fusion protein + UDP-Gal Next, non-human primates were treated in vivo with anti-CD19 scFv-αGal transferase fusion protein + UDP-Gal to examine whether the number of CD19 + B cells or toxicity would decrease.

[0224] To confirm the acceptable test values and to measure the baseline B cell and T cell counts, two cynomolgus monkeys (each weighing 5 kg) received baseline blood tests. Next, at time 0, the cynomolgus monkeys were injected intravenously with anti-CD19 scFv-αGal transferase (SEQ ID NO: 63), and 1 hour later, UDP-gal was injected.

[0225] One hour, four hours, and 24 hours after treatment, and on days 7, 14, 30, and 60, complete blood counts, serum chemistry, liver function tests, and total lymphocytes, B cell counts, and T cell counts were measured (Figure 33). CD20 + / CD3 - B cell counts were determined by examining fluorescence. CD20 was used to avoid misinterpreting B cell counts due to the presence of anti-CD19 scFv.

[0226] When the first subject monkey was treated with anti-CD19 scFv-α-Gal transferase + UDP-Gal, CD19 / CD20 + B cells decreased by 70% seven days after treatment. This persisted for eight weeks before returning to baseline levels. More recently, a second monkey given a higher dose of anti-CD19 scFv-α-Gal transferase (3 mg / kg) had an 80% reduction in B cells at the first measurement taken at four hours. There were no visible signs of toxicity observed by the veterinarian in this subject monkey. The body weight of the subject monkey did not change. There were no measurable signs of toxicity in the subject monkey during blood tests. In the first monkey, blood test values other than CD19 / CD20 counts remained stable over the two-month follow-up period. In the second monkey, there were also no observable signs of toxicity or measured signs of toxicity in the experiment.

[0227] From these results, it is demonstrated that non-human primates can be safely treated by the methods disclosed herein, and treatment results in a significant reduction in target cells.

[0228] Example 21 - Engineering of a Bifunctional Therapeutic Protein Targeting Tumors A fusion protein targeting a tumor was constructed by gene fusion of the H chain of an antibody targeting the tumor with a glycosyltransferase enzyme. In the examples described herein, GTA (SEQ ID NO:64) and GTB (SEQ ID NO:65) are derived from known human sequences, while αGalT is derived from a marmoset sequence (SEQ ID NO:66). All of the translated enzymes used in the examples presented herein were naturally expressed in the vesicular membranes of the Golgi and / or endoplasmic reticulum. To construct the bifunctional fusion proteins described herein, enzyme portions unnecessary for enzyme function (e.g., extracellular regions, transmembrane regions, and stem regions) have been removed.

[0229] As previously described herein, the tumor-targeting portion of the fusion protein may be a full-length Ab, Fab’2, Fab, scFv, monomeric Ab, or any Ab / immunoglobulin derivative thereof. The enzyme portion of the fusion protein may be any post-translational modification enzyme. Its sequence is generally human, humanized, primatized (from non-human primates), or otherwise deimmunized. Attachment of the targeting moiety and the enzyme may or may not use a linker / spacer. In the examples provided herein, the (G4S)3 (SEQ ID NO:67) linker / spacer is used, but any linker / spacer known to those skilled in the art can be used.

[0230] The preparation of these fusion proteins is modular, and thus, according to some of the examples provided herein, any tumor / tissue-targeting moiety can be fused with any post-translational enzyme. The sequences used in the manipulation and production of the huJ591 and 4D5 bifunctional therapeutic substances are shown in Table 2. The bifunctional therapeutic substances are shown in Tables 3 and 4.

[0231] (Table 2) Sequences TIFF2025518735000008.tif81161TIFF2025518735000009.tif246161TIFF2025518735000010.tif246161TIFF2025518735000011.tif133161

[0232] (Table 3) Protein sequences for dual-functional treatment of J591 TIFF2025518735000012.tif103163TIFF2025518735000013.tif202163TIFF2025518735000014.tif184163TIFF2025518735000015.tif249163TIFF2025518735000016.tif242163TIFF2025518735000017.tif249163TIFF2025518735000018.tif249163TIFF2025518735000019.tif249163TIFF2025518735000020.tif234163

[0233] (Table 4) Protein sequences for dual-functional treatment of 4D5 TIFF2025518735000021.tif165163TIFF2025518735000022.tif213163TIFF2025518735000023.tif249163TIFF2025518735000024.tif242163TIFF2025518735000025.tif246163TIFF2025518735000026.tif235163TIFF2025518735000027.tif136163

[0234] huJ591-GTB huJ591-GTB (heavy chain) (SEQ ID NO:34) was constructed by linking the huJ591 heavy chain (SEQ ID NO:68) to the N-terminus of human GTB (aa57 - 354) (SEQ ID NO:69). huJ591-LC (light chain) (SEQ ID NO:36) was constructed by adding a 6His-tag (SEQ ID NO:70) to the C-terminus of huJ591-LC (SEQ ID NO:70) to facilitate affinity chromatography purification.

[0235] The DNA sequences encoding the heavy and light chains were subcloned into the pcDNA3.1 expression vector. Protein production was carried out using a transient expression method by co-transfection of the heavy and light chains into CHO cells. The huJ591-GTB fusion protein was purified from the cell culture supernatant by nickel affinity chromatography and evaluated by SDS-PAGE.

[0236] huJ591Fab-GTB huJ591Fab-GTB (heavy chain) (SEQ ID NO:37) was constructed by linking the truncated fragment of the huJ591 heavy chain (VH-CH1-partial hinge sequence) (SEQ ID NO:72) to the N-terminus of human GTB (aa57 - 354) (SEQ ID NO:69). A Myc / his-tag (SEQ ID NO:73) was added to the C-terminus to facilitate expression monitoring and affinity chromatography purification. huJ591-LC (light chain) (SEQ ID NO:39) encodes the huJ591 light chain sequence (SEQ ID NO:71).

[0237] The DNA sequences encoding the heavy and light chains were subcloned into the pcDNA3.1 expression vector. Protein production was carried out using a transient expression method by co-transfection of the heavy and light chains into CHO cells. The Fab-GTB fusion protein was purified from the cell culture supernatant by nickel affinity chromatography and evaluated by SDS-PAGE.

[0238] huJ591-HC67-GTB huJ591-HC67-GTB (heavy chain) (SEQ ID NO:40) was constructed by linking the huJ591 heavy chain (SEQ ID NO:74) to the N-terminus of human GTB (aa57-354) (SEQ ID NO:69) via a (G4S)3 (SEQ ID NO:67) linker. The altered aa are shown in bold double underline in Table 3 and in bold letters in Table 2. J591-LC (light chain) (SEQ ID NO:42) was constructed by adding a 6His-tag (SEQ ID NO:70) to the C-terminus of J591-LC (SEQ ID NO:71) to facilitate affinity chromatography purification.

[0239] The production of the monomeric Fc fusion protein was performed as follows. The DNA sequences encoding J591HC67-GTB and the light chain were synthesized, subcloned into an expression vector, and introduced into CHO cells by co-transfection. The cell culture supernatant was collected. The J591HC67-GTB fusion protein was purified using nickel affinity chromatography and evaluated by SDS-PAGE.

[0240] huJ591-HC67-GTB54aa huJ591-HC67-GTB54aa (heavy chain) (SEQ ID NO:43) was modified from huJ591-HC67-GTB (heavy chain) (SEQ ID NO:74) by adding a 54aa tail (SEQ ID NO:75) to the C-terminus of GTB. huJ591-LC (light chain) (SEQ ID NO:45) was constructed by adding a 6His-tag (SEQ ID NO:70) to the C-terminus to facilitate affinity chromatography purification.

[0241] Protein production was performed by introducing the heavy and light chains into CHO cells by co-transfection using a transient expression method. The huJ591-GTB fusion protein was purified from the cell culture supernatant by nickel affinity chromatography and evaluated by SDS-PAGE.

[0242] huJ591scFv-Fc67-GTB huJ591scFv-Fc67-GTB (SEQ ID NO:46) encodes (from the N-terminus to the C-terminus) the huJ591 single-chain variable fragment (scFv) / J591 Fc fragment (SEQ ID NO:76), human GTB (aa57 - 354) (SEQ ID NO:69). A (G4S)3 (SEQ ID NO:67) linker was added between Fc (SEQ ID NO:76) and GTB (SEQ ID NO:69).

[0243] The DNA sequence encoding huJ591scFv-Fc67-GTB (SEQ ID NO:46) was synthesized, subcloned into an expression vector, and introduced into CHO cells by transfection. The cell culture supernatant was collected. The huJ591scFv-Fc67-GTB (SEQ ID NO:46) fusion protein was purified using nickel affinity chromatography and evaluated by SDS-PAGE.

[0244] huJ591scFv-GTB huJ591scFv-GTB (SEQ ID NO:48) encodes (from the N-terminus to the C-terminus) the deimmunized version of the huJ591 single-chain variable fragment (scFv) (SEQ ID NO:77), human GTB (aa57 - 354) (SEQ ID NO:69). A (G4S)3 (SEQ ID NO:67) linker was added between scFv (SEQ ID NO:77) and GTB (SEQ ID NO:69). A Myc / his tag (SEQ ID NO:73) was added to the C-terminus to facilitate expression monitoring and affinity chromatography purification.

[0245] The DNA sequence encoding huJ591scFv-GTB (SEQ ID NO:48) was subcloned into the pcDNA3.1 expression vector. Protein production was performed by introducing the plasmid into CHO cells by transfection using a transient expression method. The huJ591scFv-GTB fusion protein was purified from the cell culture supernatant by nickel affinity chromatography and evaluated by SDS-PAGE.

[0246] GTA construct Using the human GTA (aa57-354) sequence (SEQ ID NO:78) instead of the GTB sequence (SEQ ID NO:69) in the DNA construct described above, the following recombinant protein: TIFF2025518735000028.tif19164 was also prepared.

[0247] Trastuzumab (4D5) construct To target HER2 on the surface of breast cancer and other cancers, using the trastuzumab (4D5) sequence instead of the huJ591 sequence in the DNA construct described herein, the following recombinant protein: TIFF2025518735000029.tif26170 was prepared.

[0248] Preferred embodiments have been described and explained in detail herein, but various modifications, additions, substitutions, etc. can be made without departing from the spirit of the present disclosure, and thus it will be apparent to those skilled in the relevant art that these are considered to be within the scope of the present disclosure as defined in the following claims.

Claims

1. Targeting components that target tumor-associated antigens, and An enzyme that, when delivered to a tumor by the targeting component, enzymatically converts the tumor phenotype to the phenotype of an incompatible allograft or xenograft, the enzyme being connected to the targeting component. A bifunctional therapeutic substance for treating cancer, which includes, The targeting component is TCR, a single-domain antibody, or Anticalin®. Said bifunctional therapeutic substance.

2. The aforementioned tumor-associated antigens include FOLH1 / PSMA, VEGFR, CD19, CD20, CD25, CD30, CD33, CD38, CD52, B-cell maturation antigen (BCMA), CD79, somatostatin receptor, 5T4, gp100, CEA, Mellan A / MART-1, MAGE, NY-ESO-1, PSA, tyrosinase, HER-2, HER-3, EGFR, hTERT, MUC1, mesothelin, nectin-4, TROP-2, tissue factor, CA-125, ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) Family, IGF-binding protein (IGFBP), IGFR ligand family (IGF-1R); platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, VEGF family; HGF receptor family: TRK receptor family; ephrin (EPH) receptor family: AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1, 2; receptor tyrosine kinase-like enzymes Fan receptor (ROR) receptor family; Discoidine domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; Transforming growth factor α (TGF-α), TGF-α receptor; Transforming growth factor-β (TGF-β), TGF-β receptor; Interleukin β receptor α2 chain (IL13Rα2), Interleukin-6 (IL-6), IL-6 receptor, Interleukin-4, IL-4 receptor, Cytokine receptor, Class I (Hematopoietin) Family) and Class II (Interferon / 1L-10 family) receptors, Tumor Necrosis Factor (TNF) family, TNF-α, Tumor Necrosis Factor (TNF) receptor superfamily (TNTRSF), Death Receptor family, TRAIL receptor; Cancer Testis (CT) antigen, Lineage-Specific Antigen, Differentiation Antigen, α-Actinin-4, ARTC1, B-RAF, Caspase-5 (CASP-5), Caspase-8 (CASP-8), β-Catenin (CTNNB1), Cell Division Cycle 27 (CDC27), Cyclin-Dependent Kinase 4 (CDK4), CDKN2A,COA-1, dek-can fusion protein, EFTUD-2, extension factor 2 (ELF2), Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low-density lipid receptor / GDP-L-fucose:β-D-galactose 2-α-L-fucosyltransferase (LDLR / FUT) fusion protein, HLA-A2, MLA-A11, heat shock protein 70-2 variant (HSP70-2M), KIAA0205, MART2, melanoma ubiquitous variants 1, 2, 3 (MUM-1, 2, 3), neoPAP, myosin class 1, NFYC, OGT, OS-9, pml-RARα fusion protein, P RDXS, PTPRK, N-ras (NRAS), HRAS, RBAF600, SIRT12, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, triose phosphate isomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8, GnT-V (abnormal N-acetylglucosaminyltransferase V, MGATS), HERV-K MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognizing antigen on melanoma (CAMEL), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, gp100 / Pme117(S1LV), TRP-1, HAGE, NA-88, NY-ESO-1 / LAGE-2, SAGE, Sp17, SSX-1, 2, 3, 4, TRP2-1NT2, kallikrein 4, Mammaglobin-A, OA1, TRP-1 / , 75, TRP-2, Adipophyllin, Interferon-inducible protein 2 (AIM-2) not present in melanoma, BING-4, CPSF, Cyclin D1, Epithelial cell adhesion molecule (Ep-CAM), EpbA3, Fibroblast growth factor-5 (FGF-5), Glycoprotein 250 (gp250), Intestinal carboxylesterase (iCE), M-CSF, mdm-2, MUCI, PBF, PRAME, RAGE-1, RNF43, RU2AS, SOX10, STEAP1,SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, C SAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE, TPTE, immunoglobulin idiotype, Bence Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD4, CD3, cancer antigen 72-4 (CA 72-4), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96, GM2, salglamostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen 4 recognized by T cells (ART-4), carcinoembryonic antigen peptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2), cyclophyllin B (Cyp-B), human signet ring tumor-2 (HST-2), PR1, claudin (e.g., claudin 1, claudin 3, claudin The bifunctional therapeutic substance according to claim 1, which is one of the following: Caudin 4, Claudin 6, Claudin 7, Claudin 18.2), GPC3, GD2, EpCam, CD70, CD123, prostate stem cell antigen (PSCA), CD133, ROR1, FAP, GD2, EGFRVIII, CA9, ML-IAP, ERG, NA17, PAX3, ALK, MYCN, RhoC, GD3, PLAC1, GM3, CD166, LIV1A, CD71, CD228, P-cadherin, LAMP1, Napi2b, or an MHC / neoantigen complex.

3. Targeting components that target tumor-associated antigens, and An enzyme that, when delivered to a tumor by the targeting component, enzymatically converts the tumor phenotype to the phenotype of an incompatible allograft or xenograft, the enzyme being connected to the targeting component. A bifunctional therapeutic substance for treating cancer, which includes, The tumor-associated antigens include ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF-binding protein (IGFBP), IGFR ligand family (IGF-1R); platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, VEGF family; HGF receptor family: TRK receptor family; ephrin (EPH) receptor family: AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR) receptor family; discoidin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; transforming growth factor α (TGF-α), TGF-α receptor; transforming growth factor-β (TGF-β), TGF-β receptor; and - Leukin-β receptor α2 chain (IL13Rα2), interleukin-6 (IL-6), 1L-6 receptor, interleukin-4, IL-4 receptor, cytokine receptor, class I (hematopoietin family) and class II (interferon / 1L-10 family) receptors, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily (TNTRSF), death receptor family, TRAIL receptor; cancer testis (CT) antigen, lineage-specific antigen, differentiation antigen, α-activator Nin-4, ARTC1, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, prolongation factor 2 (ELF2), Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low-density lipid receptor / GDP-L-fucose:β-D-galactose 2-α-L-fucosyltransferase (LDLR / FUT) fusion protein, HLA-A2, MLA-A11, heat shock protein 70-2 mutant (HSP70-2M), KIAA0205, MART2, melanoma ubiquitous mutant 1, 2, 3 (MUM-1, 2, 3), neoPAP, myosin class 1, NFYC, OGT, OS-9, pml-RARα fusion protein, P RDXS, PTPRK, N-ras (NRAS), HRAS, RBAF600, SIRT12, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, triose phosphate isomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8, GnT-V (abnormal N-acetylglucosaminyltransferase V, MGATS), HERV-K MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognizing antigen on melanoma (CAMEL), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, gp100 / Pme117(S1LV), TRP-1, HAGE, NA-88, NY-ESO-1 / LAGE-2, SAGE, Sp17, SSX-1, 2, 3, 4, TRP2-1NT2, Kallikrein 4, Manmaguro Bing-A, OA1, TRP-1 / , 75, TRP-2, adipophyllin, interferon-inducible protein 2 (AIM-2) not present in melanoma, BING-4, CPSF, cyclin D1, cyclin B1, epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250), intestinal carboxylesterase (iCE), M-CSF, mdm-2, MUCI, PBF, PRAME, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE, TPTE, immunoglobulin idiotype, Bence Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD4, CD3, cancer antigen 72-4 (CA 72-4), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96, GM2, salglamostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen 4 recognized by T cells (ART-4), carcinoembryonic antigen peptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2), cyclophyllin B (Cyp-B), human signet ring tumor-2 (HST-2), PR1, claudin (e.g., claudin 1 Din-3, Claudin-4, Claudin-6, Claudin-7, Claudin-18.2), GPC3, GD2, EpCam, CD70, CD123, Prostate Stem Cell Antigen (PSCA), CD133, ROR1, FAP, EGFRVIII, CA9, ML-IAP, ERG, NA17, PAX3, ALK, MYCN, RhoC, GD3, PLAC1, GM3, CD166, LIV1A, CD71, CD228, P-cadherin, LAMP1, Napi2b, or MHC / neoantigen complexes. Said bifunctional therapeutic substance.

4. The bifunctional therapeutic substance according to claim 3, wherein the targeting component is selected from an antibody or its antigen-binding fragment, a TCR, a single-domain antibody, anticalin, a protein, a peptide, an aptamer, and a low molecular weight ligand.

5. A pharmaceutical product for use in a method of treating cancer, comprising a dual-function therapeutic substance according to any one of claims 1 to 4, wherein the method comprises the step of administering the dual-function therapeutic substance to a subject having cancer.

6. The pharmaceutical product according to claim 5, wherein the subject is a human.

7. The pharmaceutical product according to claim 5, wherein the cancer is lung cancer, stomach cancer, colorectal cancer, breast cancer, prostate cancer, blood cancer, cervical cancer, endometrial cancer, ovarian cancer, bladder cancer, kidney cancer, brain cancer, liver cancer, esophageal cancer, adrenal cancer, head and neck cancer, melanoma, or pancreatic cancer.

8. The aforementioned administration step is Administer uridine diphosphate-galactose (UDP-gal), uridine diphosphate-N-acetylgalactosamine (UDP-NAcGal), and / or guanosine diphosphate-fucose (GDP-fucose). The pharmaceutical product according to claim 5, further comprising:

9. A dual-function therapeutic substance according to any one of claims 1 to 4, and Pharmacologically acceptable carriers A pharmaceutical composition containing [the specified substance].

10. A nucleic acid molecule encoding a bifunctional therapeutic substance according to any one of claims 1 to 4.

11. An expression vector comprising the nucleic acid molecule described in claim 10.

12. Recombinant host cells transformed with the nucleic acid molecule described in claim 10.

13. Recombinant host cells transformed with the expression vector according to claim 11.

14. A host cell expressing a bifunctional therapeutic substance according to any one of claims 1 to 4.

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