Cancer immunotherapy for promoting super-acute rejection

Abstract

The present application relates to a bifunctional therapeutic agent for the treatment of cancer, the bifunctional therapeutic agent comprising: a targeting component that targets a tumor-associated antigen; and an enzyme that, when delivered to a tumor by the targeting component, converts a tumor phenotype to a phenotype of an incompatible allograft or xenograft. The enzyme is coupled to the targeting component. Also disclosed is a method for treating cancer comprising administering the bifunctional therapeutic agent.

Description

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

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

[0003] Combination therapy is a universally accepted treatment for nearly all types of cancer and has been the standard of care for decades. The basis for the use of combination therapy is the early experience with chemotherapy, where it was found that due to the high mutation rate of cancer, resistant strains of tumor cells would quickly develop when only a single agent was used. The goal of combination therapy is to improve the efficacy and minimize the occurrence of tumor resistance or escape. This is usually achieved by using two or more anticancer agents, each with a different mechanism of action, making it difficult and less likely for resistant tumor cells to develop. The additive or synergistic effects of the combination of two or more agents may be the key to determining the success of a patient's treatment.

[0004] Many combination treatments are well known in the field of oncology. As an example, MOPP (an acronym for mechlorethamine, vincristine, procarbazine, prednisone) is a curative treatment for Hodgkin's disease. Several different combination regimens (all including cisplatin, vinblastine, and bleomycin) have been used to treat testicular cancer, with cure rates as high as 98% in confirmed cases. In total, more than 300 different combination regimens have been used.

[0005] The major drawback of combination therapy is often that it also results in increased toxicity. For example, most nonsurgical forms of cancer treatment, such as external radiation and chemotherapy, have limited efficacy due to toxic side effects on normal tissues and cells and the limited specificity of these treatment modalities for cancer cells. This limitation is also important when anticancer antibodies are used to target toxic agents such as isotopes, drugs, and toxins to cancer sites because, as systemic agents, they also circulate to sensitive cellular compartments such as the bone marrow. In acute radiation injury, destruction of lymphoid and hematopoietic compartments is a major factor in the development of sepsis and subsequent mortality. Therefore, methods that can reduce the toxic effects of cancer therapies while maintaining or even improving their efficacy are urgently needed.

[0006] As an alternative to combination therapy, recent advances in immunotherapy have clearly demonstrated that the immune system can mount responses against cancer, and that these responses are highly potent and durable. Extensive experience with immune checkpoint inhibition suggests that its greatest benefit is in cancers with relatively high mutational loads. Even in these settings, however, only a minority of patients respond. Some cancers, such as prostate cancer, lack immune cells in the tumor microenvironment. This lack of immune cells, sometimes referred to as a ‘cold’ microenvironment or immune ‘desert’, severely limits the ability to activate the immune system. Chimeric antigen receptor T (CAR-T) cells and bispecific T cell engagers (BiTEs) use antibodies targeting tumor-associated antigens to direct the lytic machinery of T cells to lyse cancer cells. However, to date, the antitumor activity of CAR-T and BiTEs has been limited to blood-borne cancers, rather than the more common solid tumors. Clearly, additional approaches are still needed to treat a variety of cancers.

[0007] The present disclosure is directed to overcoming these and other deficiencies in the art. Summary of the invention

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

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

[0010] A novel immunotherapy approach is proposed, namely tumor-targeted glycosyltransferases to change the glycophenotype of tumors and / or their blood vessels by adding non-self tissue blood group antigen (HBGA) or α-galactose (α-gal) sugar epitopes. This can effectively transform tumors into allografts or xenografts that are incompatible with HBGA. Exemplary embodiments of this multifunctional agent can target PSMA / FOLH1 and transform tumor neovascularization into mismatched HBGA or xenografts, thereby initiating hyperacute rejection reactions. Half a century of transplantation experience has shown that HBGA-incompatible allografts or xenografts expressing α-gal will stimulate a strong immune rejection reaction process.

[0011] As described herein, in order to generate xenogeneic or allogeneic antigen expression by tumors, xenogeneic or allogeneic glycosyltransferases, such as alpha gal transferase (αgalT) or allogeneic glycosyltransferase A and / or B enzymes, which are usually located in the Golgi apparatus, are delivered to the surface of tumor cells - in effect, ectopic allogeneic / xenogeneic transplants at the molecular level. In addition to the targeted glycosyltransferases (αgalT, glycosyltransferase A and / or B enzymes), the corresponding sugar-nucleotide donors (UDP-gal or UDP-NAcGal) are also required. In the presence of glycosyltransferases at the tumor, sugars (gal or NAcGal) are added to existing glycoproteins and glycolipids, including products secreted by targeted cells, to produce allogeneic or xenogeneic antigens, thereby triggering a strong immune response. The converted allo / xenoproteins secreted into the microenvironment bind to abundant natural antibodies, triggering complement activation, immune response, and antibody-dependent cytotoxicity (ADCC), and serving to transform the "cold" microenvironment into a "hot" microenvironment.

[0012] The glycosyltransferase A and B enzymes differ in only four of their 353 amino acid residues (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 AminoAcids From Blood Group B to Blood Group A Glycosyltransferase Boosts Catalytic Activity and Progressively Modifies Substrate Recognition in Human Recombinant Enzymes." Chem. 272:14133-14138 (1997), which is hereby incorporated by reference in its entirety), making them unlikely to be immunogenic. Studies of patient sera have confirmed that, as predicted, these enzymes are not immunogenic. In fact, although their HBGA carbohydrate products are highly immunogenic, the transferase A and B enzymes have never been reported to be immunogenic. Therefore, targeted delivery of non-immunogenic transferase A or B enzymes to tumors provides a method for altering the immune phenotype of tumors or neovascularization to express highly immunogenic non-self HBGAs - thereby presenting the phenotype of an incompatible allograft and eliciting a strong rejection response from the host.

[0013] As described herein, for proof of concept, the method has been validated with humanized GTA or GTB. Alternatively, a xenogeneic α-galactosyltransferase (α1,3 galactosyltransferase; α-galT) can be utilized, which is mutated / inactivated in humans and is the cause of rejection of xenotransplanted organs in other mammals. The use of the α-galT enzyme may require humanization or deimmunization of α-galT, and there are known methods in the art to achieve this, including but not limited to using sequences of homologous regions of other glycosyltransferases that are non-immunogenic to humans. Such humanization or deimmunization methods have been widely and successfully used for humanization or deimmunization of exogenous antibodies before they are used as therapeutic agents for humans. However, studies on patient sera have shown that these enzymes themselves are not immunogenic.

[0014] The present disclosure provides a novel immunotherapy approach in which tumor-targeted glycosyltransferases alter the tissue-blood group antigen expression of the tumor and / or its blood supply. This effectively converts the tumor into an HBGA-incompatible allograft.

[0015] As described herein, a complementary, orthogonal immunotherapy approach is based on the strong immune response to xenogeneic or allogeneic transplants and the model established by the understanding of the rejection process over the past half century. To achieve this goal, the most extreme form of host response to transplantation: hyper-acute rejection (HAR) was selected as a model.

[0016] HAR occurs as a result of ancestral mutations in either of two highly related genes: in the case of xenografts, mutations in α1,3 galactosyltransferase (α1,3GalT) (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 in the case of allografts, mutations in the well-known tissue and blood group antigen (HBGA) locus (Milland et al., "ABO Blood Group and Related Antigens, Natural Antibodies and Transplantation." Transplantation," Tissue Antigens, 68:459-466 (2006), which is hereby incorporated by reference in its entirety).These two highly related genes are located 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", Journal of Biological Chemistry, 265:19257-19262 (1990); Yamamoto et al., "Genomic Organization of Human Histo-Blood Group ABO Genes", These alleles encode glycosyltransferases that post-translationally add terminal sugar moieties to carbohydrate (CHO) chains present on nascent proteins and lipids intended for cell membrane expression or secretion. Due to mutations, the αGalT enzyme was inactivated approximately 28 million years ago in humans and Old World monkeys, but not in other mammals (Macher et al., "The Gal Alpha1,3Gal Beta1,4GlcNAc-R (α-Gal) Epitope: a Carbohydrate of Unique Evolution and Clinical Relevance", Acta Biochimica et Biophysica Sinica 1780:75-88 (2008), which is hereby incorporated by reference in its entirety). Therefore, xenotransplanted organs and tissues derived from non-primate mammals express αgal epitopes that are foreign to humans. For the HBGA locus, a small number of mutations have produced alleles classically referred to as A, B, and O.The B allele encodes glycosyltransferase B (GTB), which, like its α1,3GalT homologue, adds terminal galactose (Gal) to CHO chains, except that it only adds Gal when 1,2-fucose is present on the adjacent Gal. GTB differs from GTB in function only in that it adds N-acetylated terminal Gal (NAcGal). The O gene product is inactivated by a frameshift mutation (. Figure 1 ).

[0017] The α-Gal, HBGA A, and HBGA B epitopes produced by these three active enzymes are widely expressed in nature, including by bacteria that inhabit the human gut (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). Therefore, humans who lack αGalT and the A and / or B alleles are continuously immune to these bacterially derived epitopes. This results in very high levels of natural antibodies (Abs) produced against these non-self epitopes, which account for more than 1% of plasma immunoglobulin (Ig) (Galili et al., "One Percent of Human Circulating BLymphocytes 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 entirety). Given that the diversity of the Ab repertoire is estimated to be as high as billions of different specificities, this represents a huge proportion of endogenous Ig activity.These Abs consist of IgM and IgG, which can activate the complement cascade and thus 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". Supplement), "J. Thrombosis & Haemostasis (J. Thromb. Haemost.)" 13(Supplement 1): S121-S32 (2015), which is hereby incorporated by reference in its entirety). Other immunoglobulin classes such as IgA and IgE can also target these glycosyl epitopes. In fact, the evolutionary mutations of these two genes have created a critical immune state in which the immune system is ready to respond rapidly, vigorously and destructively to the appearance of any of these non-self epitopes. The immunological effects of these mutations make xenotransplantation in humans unsuccessful and explain why HBGA matching has become the single most important match in solid organ transplantation since the early days of renal allotransplantation in the 1960s, since Starzl first recognized its importance in "Experience In Renal Transplantation." (WB Saunders & Co., Philadelphia, Pennsylvania), Chapter 6 (1964), which is hereby incorporated by reference in its entirety. Since then, HBGA mismatches in solid organ transplantation have occurred only in rare cases of iatrogenic errors (Altman, Doctors Discuss Transplant Mistake. New York Times, February 22, 2003, which is hereby incorporated by reference in its entirety).This background has prompted the goal of inducing expression of these non-self epitopes by host cancer cells and / or vascular endothelial cells supplying the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The strict acceptor substrate specificity of glycosyltransferases is shown. B (or A)-transferase will only add its corresponding sugar to the glycosylation site expressing H antigen. Fortunately, this essential H antigen is lacking in many normal tissues, thus preventing off-target conversion to HBGA A or B. Alternatively, in cases where it is desired to intentionally target normal or cancer cell types that naturally lack H antigen, this can be achieved in a manner similar to the addition of A or B, that is, simultaneously targeting α1-2 fucosyltransferase and providing GDP-fucose as a fucose donor. The addition of fucose / H antigen can be performed simultaneously with the targeted A or B transferase, or the addition can be performed stepwise (e.g., fucose is added first, then A or B).

[0019] Figure 2A-2B The chimeric Ab-GTB protein was shown to maintain immunoreactivity and enzymatic activity. Figure 2A is a graph showing that the J591-GTB chimeric protein maintained comparable binding immunoreactivity to PSMA relative to the parental J591 antibody as measured by ELISA. Figure 2B is a bar graph showing that the chimeric protein also retains enzymatic activity, which is catalyzed by 14 C-galactose from the nucleotide donor UDP- 14 This was demonstrated by the ability of anti-4D5(her2)-GTB to transfer C-galactose to 2'-fucosyl-lactose (2-FL). This incorporation occurred at high levels only in the presence of the J591-GTB fusion protein and its acceptor substrate 2-FL. Similar results were obtained with anti-4D5(her2)-GTB.

[0020] Figure 3 Graph showing that GTB activity can be modulated by C-terminal extension. J591-GTB activity (% of control) as a function of increasing C-terminal amino acid extension length and by adding UDP- 14 C-gal 14 C-gal incorporation into 2'-fucosyl-lactose (2-FL) was measured.

[0021] Figure 4is an image showing J591-GTB specific transformation antigen positive tumor cells. Tissue sections of CWR22Rv1 xenografts (heterogeneous PSMA+ / HBGA O) were incubated with J591-GTB+UDP-gal and immunohistochemically stained for HBGA B expression (left). Negative control sections (lacking mouse anti-HBGA B (middle) or J591-GTB (right), respectively, but containing secondary antibody anti-mouse Ig-peroxidase, but) were not stained.

[0022] Figure 5 are images showing the effects of J591-GTB on LNCaP and PC3 cells. LNCaP cells (PSMA + / HBGAO; left) converted from J591-GTB to HBGA B; PC3 cells (PSMA - / HBGA O; right panel) transformation without J591-GTB.

[0023] Figures 6A-6D 6A shows phase contrast images. FIG. 6B shows cells expressing PSMA. FIG. 6C shows HBGA B antigen expression. FIG. 6D is a merge of FIG. 6B and 6C. Only those cells expressing PSMA will be converted to express HBGA B. PSMA negative cells, mainly located in the center left and top center, are still HBGA B negative.

[0024] Figure 7 Is an image showing LNCaP cells incorporated into O-type RBC suspension and incubated with J591 (top row), J591-GTB (middle row) or J591-GTB-54aa extension (bottom row). The left column shows phase contrast images. The middle column shows DAPI nuclear staining. The right column shows mouse anti-HBGA B + goat anti-mouse IgM-alexa488 nuclear staining. When PSMA positive LNCaP cells are converted to HBGA B positive by J591-GTB (with or without C-terminal extension) and bound to its plasma membrane, PSMA negative RBCs are not converted.

[0025] Figures 8A-8D8A is an image showing the lysis of complement-mediated in vitro. LNCaP cells were incubated with natural mAb J591 or mAbJ591-GTB fusion protein. All holes also had UDP-gal. Subsequently, serum from type A patients was added as the source of natural anti-B Ab and complement. The combination of J591-GTB plus type A serum (Fig. 8A) causes LNCaP to be completely lysed. In the absence of type A serum, J591-GTB fusion protein does not induce lysis (Fig. 8B). In the absence of fusion protein, no matter there is (Fig. 8C) or there is no type A serum (Fig. 8D), no lysis is detected.

[0026] Fig. 9 are images showing complement-mediated cytotoxicity of several cell lines. Complement-mediated cytotoxicity of several cell lines observed by trypan blue exclusion is shown. The upper figure was treated with J591-GTB+UDP-gal+human O-type serum. The cells in the lower figure were treated with the same O serum, but without J591-GTB+UDP-gal. The proportion of dead cells determined by FACS is reported below each photo (Figure 10). For FACS, O-type serum without J591-GTB+UDP-gal was used as a negative control, and 0.1% triton treatment provided a complete lysis control.

[0027] Figures 10A-10H are images showing the conversion of prostate and breast cancer to HBGA B in vivo. Figures 10A-10D show serial sections of LNCaP xenografts 24 hours after administration of PBS + UDP-gal (Figure 10A), b) J591 + UDP-gal (Figure 10B), and J591-GTB + UDP-gal (Figure 10C) to SCID mice. Figures 10A-10C are immunohistochemical staining for HBGA B (all 10x). Figure 10D shows serial sections of the same sample stained for PSMA. See also for higher magnifications and additional xenograft lines Figures 12A-12E Figures 10E-10H show MD-MB361 breast cancer (HER2) after treatment with PBS+UDP-gal (Figure 10E), 4D5+UDP-gal (Figure 10F), 4D5-GTB+UDP-gal (Figures 10G and 10H). + ) xenografts. Sections were stained for HBGA B expression by immunohistochemistry. Discrete plasma membrane staining was evident. In Figures 10C and 10H, adjacent connective tissue was not transformed, indicating that the specificity of the immunophenotypic transformation was limited to the targeted tumor.

[0028] Figures 11A-11B is shown as determined by FACS and trypan blue exclusion (see Fig. 9 ) Bar graph showing lysis of B-transformed cell lines by type O sera as determined by propidium iodide uptake measured by Fig.11A ) and histogram ( Fig. 11B ). In the absence of B conversion, O-type serum did not cause lysis. After treating PSMA-positive cells with J591-GTB+UDP-gal, O-type serum completely lysed all PSMA-positive / B-converted cell lines; PC3, which is HBGA O-positive / PSMA-negative, did not convert to HBGA B and was not lysed.

[0029] Figures 12A-12E are images showing in vivo conversion of LNCaP, C4-2, and CWR22Rv1 xenografts by J591-GTB. Figures 12A-12B show LNCaP xenografts treated in vivo: with J591 [without GTB] (Figure 12A) or J591-GTB (Figure 12B), both with the addition of UDP-gal, and immunohistochemically stained with mouse anti-HBGA B; high magnification. Figure 12C shows C4-2 prostate cancer treated in vivo with J591-GTB plus UDP-gal, and immunohistochemically stained with mouse anti-HBGA B; high magnification. Figures 12D-12E show CWR22Rv1 prostate cancer treated in vivo with J591-GTB plus UDP-gal, which is heterogeneous and weakly PSMA positive. Adjacent connective tissue does not convert to HBGA B.

[0030] Figures 13A-13B is a diagram showing the in vivo transformation and processing of HBGA ( Fig.13A ) and in vivo images of mice ( Fig. 13B ). 6 C4-2-luc cells suspended in Matrigel were implanted into mice by intraperitoneal injection (IP). After several days, bioluminescence was measured and 10 mice with confirmed viable tumors were randomly assigned to one of 2 treatment groups. All tumor-bearing mice received a single dose of J591-GTB + UDP-gal + human type O serum; in half of the mice, the serum was heat-inactivated before injection. In mice treated with active type O serum, mean photon flux gradually declined over the following 13 days, while those treated with inactivated serum experienced mean tumor progression. At the end of the experiment on day 13, bioluminescence was significantly different between the groups (p<0.0032). Repeated experiments yielded consistent results.

[0031] Figures 14A-14B are in vivo images and graphs showing the results of experiment #2, in which C4-2-luc cells were IP implanted into mice followed by a single treatment with J591-GTB+UDP-gal+human type O serum (upper row) ( Fig.14A ). Fig.14A Images of mice receiving active O-type serum or previously heat-inactivated O-type serum are shown. Mice receiving heat-inactivated serum showed tumor progression (see photon flux graph; Fig. 14B ), while those mice receiving active serum experienced tumor regression; the results of Experiment 1 were Figures 13A-13B Shown in.

[0032] Fig.15 is a FACS histogram showing the expression of CD19, CD20 and CD38 in MM1-S cells. Flow cytometric analysis showed that the MM1-S multiple myeloma cell line was CD38 positive, CD19 positive and CD20 negative.

[0033] Figures 16A-16B is a FACS histogram showing ABO expression of MM1-S cells. Fig.16A The MM1-S multiple myeloma cell line is shown to be A / B negative. Fig. 16B The MM1-S multiple myeloma cell line is shown to be O positive.

[0034] Fig.17 is a FACS histogram showing CD19 + / O + MM1-S myeloma cells can be transformed into B cells by GTB+UDP-gal + GTB can be targeted to myeloma cells using anti-CD19, anti-CD38, or anti-BCMA.

[0035] Fig.18 is an image showing the use of ACUPA (a small molecule ligand that binds to PSMA) conjugated to GTB (ACUPA-GTB) to direct the conversion of LNCaP from HBGA O to HBGA B. This suggests that in addition to antibodies (or antibody derivatives), small molecule ligands or peptides that bind to target antigens on tumor cells or new vascular endothelium can also be used for the purpose of targeting the enzyme. The left image shows cells treated with ACUPA-PEG-1500-GTB. The right image shows cells treated with GTB alone.

[0036] Fig.19 is an image showing the specificity of conversion from HBGA O to HBGA B. SK-BR5 breast cancer cells (PSMA - / O + ) and LNCaP prostate cancer cells (PSMA + / O +) co-cultured. The two cell types can be distinguished by morphology: SK-BR5 are round, while LNCaP cells are oval / fusiform. In addition, LNCaP cells were labeled with green fluorescent protein (GFP). Only PSMA was transformed after incubation with J591-GTB and UDP-gal + LNCaP cells without transforming neighboring cells that lack the PSMA target. The figures show DAPI (left), GFP (middle), and anti-B (Cy5) (right) imaging, respectively.

[0037] Fig. 20 is an image showing the specificity of conversion from HBGA O to HBGA B. For example, only PSMA + Cells were transformed into B by J591-GTB / UDP-gal + .

[0038] Figures 21A-21B is a FACS histogram showing the specificity of conversion from HBGA O to HBGA B. The expression of HBGA O in SK-BR5 (PSMA negative) cells was compared by flow cytometry. Fig.21B ) LNCaP (PSMA + ) into HBGA B( Fig.21A ) to quantify the specificity of transformation. + FACS histograms are shown. SK-BR5 did not undergo B transformation even at concentrations as high as 100 μg / mL of J591-GTB. In contrast, concentrations as low as 0.012 μg / mL induced transformation of PSMA-positive LNCaP cells.

[0039] Fig. 22 is a table and graph showing the specificity of conversion from HBGA O to HBGA B. Figures 21A-21B Table (left) and MFI histogram (right). The specificity index exceeds 8,000:1.

[0040] Fig.23 are tables and graphs showing that both cell surface and secreted glycoproteins are glycosylated by the methods of the present disclosure. A cell count graph (upper graph) and a table (lower graph) are shown.

[0041] Figures 24A-24B is a graph showing the testing of anti-α1,3GalT antibodies in serum samples. Fig. 24B yes Fig.24A A magnified view showing the lower optical density.

[0042] Fig.25is a picture of SDS-PAGE gel showing the expression and purification of the recombinant protein.

[0043] Figures 26A-26B The figure shows the interaction between scfv-CD19-αGal and CD19 - MM1.S cells (Figure 26A) and CD19 + Figure 26B shows binding of Raji cells.

[0044] Fig. 27 It is shown that in CD19 + and CD19 - Diagram of a galactose transfer assay performed on a mixture of cells.

[0045] Fig.28 It shows that CD19 + Histogram of galactose transfer assay performed on cells.

[0046] Fig.29 is a scatter plot showing the binding of scfv-αGT to human B cells and the αGal transfer assay.

[0047] Fig.30 It shows that CD19 + Dot plots of cells subjected to serum-mediated lysis assay.

[0048] Figures 31A-31B is a graph showing a lysis assay performed on αGal transferred B cells. Fig.31A is a graph showing the percentage (%) of lysis. Fig.31B is a graph showing IgG levels (MFI) and IgM levels.

[0049] Figures 32A-32C In vitro checkerboard assay of scfv-CD19-αGT and UDP-Gal is shown. Fig.32A Measuring combination, Fig.32B αgal expression was measured, and Fig.32C Measurement of lysis of human PBMCs.

[0050] Fig.33 is a bar graph showing the % of B cells remaining at baseline and at 1 hour, 4 hours, 1 day, 7 days, 14 days, 30 days and 60 days after administration of anti-CD19 scFv-αGal transferase fusion protein and UDP-gal. + / CD3 - B cell counts were determined by fluorescence. CD20 was used to avoid interference with B cell counts due to the presence of anti-CD19 scFv. DETAILED DESCRIPTION

[0051] The present disclosure teaches a bifunctional therapeutic agent for treating cancer, the bifunctional therapeutic agent comprising: a targeting component, the targeting component targeting a tumor-associated antigen; and an enzyme, the enzyme enzymatically converting the tumor phenotype into the phenotype of an incompatible allograft or xenograft when delivered to the tumor by the targeting component. The enzyme is coupled to the targeting component.

[0052] The present disclosure also relates to a method for treating cancer. The method involves selecting a subject suffering from cancer and providing a dual-function therapeutic agent according to the present disclosure. The dual-function therapeutic agent is administered to the selected subject under conditions effective to treat the cancer.

[0053] As used herein, the term "treatment" refers to applying or administering the bifunctional therapeutic agents of the present disclosure to a subject, such as a patient. Treatment can be curing, healing, alleviating, slowing, altering, remedying, alleviating, palliating, improving or affecting cancer, cancer symptoms or cancer predisposition.

[0054] As used herein, the term "subject" is intended to include humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0055] As used herein, the term "cancer" includes all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness.

[0056] As used herein, "incompatible allograft" refers to a tissue or tumor that induces hyperacute, acute, and / or chronic immune rejection. Hyperacute rejection occurs within minutes to hours after organ transplantation, or as described herein, after the delivery of a bifunctional therapeutic agent, resulting in transformation of a tumor or tissue. This rapid rejection is characterized by vascular thrombosis, leading to graft / tumor necrosis. Hyperacute rejection is caused by anti-donor antibodies present in the recipient prior to transplantation / transformation.

[0057] As used herein, a "targeting component" is a component that is capable of binding to or otherwise associating with a tumor-associated antigen. Such tumor-associated antigens include, but are not limited to, the following and peptide fragments thereof: 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, melanoma A / MART-1, MAGE, NY-ESO-1, PSA, tyrosinase, HER-2 / neu, HER-3, EGFR, hTERT, mesothelin, adhesion protein-4, TROP-2, tissue factor, MUC-1, CA-125 and its peptide fragments, 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 carcinoembryonic 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, non-mutated p53, truncated epidermal growth factor receptor (EGFR), chimeric protein p210BCR-ABL, telomerase, survivin, WT1 protein, LMP2 protein, HPV E6 E7 protein, 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 enzyme-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 alpha (TGF-α), TGF-α receptor; transforming growth factor beta (TGF-β), TGF-β receptor; interleukin beta receptor alpha 2 chain (IL13Rα2), interleukin-6 (IL-6), IL-6 receptor, interleukin-4, IL-4 receptor, cytokine receptors, class I (hematopoietin family) and class II (interferon / IL-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 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 mutated (HSP70-2M), KIAA0205, MART2, melanoma commonly mutated 1, 2, 3 (MUM-1, 2, 3), new PAP, class 1 myosin, 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-acetylglucosamine transferase V, MGATS), HERV-K MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, antigen recognized by CTL 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 lipophilin, interferon-inducible protein 2 absent 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, 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, 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 signet ring tumor-2 (HST-2), PR1, claudin family (such as claudin1, claudin3, claudin4, claudin6, claudin7, claudin18.2, 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. In some embodiments, the tumor-associated antigen is a neoantigen. In some such embodiments, the targeting domain is a TCR that binds to an MHC / neoantigen complex on the surface of a tumor cell. In some embodiments, the neoantigen is derived from KRAS, G12D Mutation, MART-1, gp100, NY-ESO-1, CEA, MAGE-A3, MAGE-A4 or WT1. See, e.g., Leko and Rosenberg, Cancer Cell 38:454 (2020). The above list exemplifies examples of tumor-associated antigens; additional tumor-associated antigens are known to those skilled in the art.

[0058] The antigen can be, for example, an antigen or epitope present on a tumor cell located 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 can be an antigen or epitope present on a cell membrane, a secreted protein or a non-membrane-bound protein. Examples of secreted proteins include, but are not limited to, hormones, enzymes, toxins and antimicrobial peptides.

[0059] Targeting components can be localized or concentrated at a specific target site, such as a tumor, disease site, tissue, organ, a certain type of cell, infectious bacteria or virus, etc.

[0060] For example, the targeting component considered includes peptides, polypeptides, proteins, glycoproteins, aptamers, carbohydrates or lipids. The targeting component can be a natural or synthetic ligand of a cell surface receptor, such as a growth factor, a hormone, LDL, transferrins, etc. The targeting component can be an antibody, and the "antibody" is intended to include antibody fragments and derivatives, characteristic parts of antibodies, single-chain targeting parts can (for example, using programs such as phage display to identify). The targeting component can also be a targeting peptide, a targeting peptide mimic or a small molecule, whether naturally occurring or artificially produced (for example, by chemical synthesis).

[0061] In one embodiment, the targeting component is selected from the group consisting of antibodies or antigen-binding fragments thereof, proteins, peptides and nucleic acid aptamers, and small molecules.

[0062] Antibodies against tumor-associated antigens are known. For example, antibodies and antibody fragments specifically bound to markers produced by or associated with tumors have been disclosed, especially in U.S. Patent No. 3,927,193 to Hansen and U.S. Patent No. 4,331,647, No. 4,348,376, No. 4,361,544, No. 4,468,457, No. 4,444,744, No. 4,818,709 and No. 4,624,846 to Goldenberg, which are hereby incorporated by reference in their entirety. It is particularly advantageous to use antibodies against tumor-associated antigens (e.g., gastrointestinal, lung, breast, prostate, ovary, testis, brain or lymphatic or hematogenous tumors, sarcomas or melanomas). Antibodies against tumor-associated antigens are well known to those skilled in the art.

[0063] Antibodies of the present disclosure may exist in a variety of 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, and 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, 1999). 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 which is hereby incorporated by reference in its entirety).

[0064] The antibodies of the present disclosure may also be produced using recombinant DNA technology, such as antibodies or fragments thereof expressed by phage. Alternatively, synthetic antibodies are produced by synthesizing DNA molecules encoding and expressing the antibodies of the present disclosure or synthesizing amino acid sequences specifying the antibodies, wherein the DNA or amino acid sequences have been obtained using synthetic DNA or amino acid sequence technology available and well known in the art.

[0065] Methods for producing monoclonal antibodies can be performed using the techniques described herein or techniques well known in the art (MONOCLONAL ANTIBODIES-PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman, eds., 1995), which is hereby incorporated by reference in its entirety). Typically, the methods involve obtaining immune cells (lymphocytes) from the spleen of a mammal that has been previously immunized in vivo or in vitro with an antigen of interest.

[0066] Alternatively, monoclonal antibodies can be prepared using recombinant DNA methods, such as described in U.S. Pat. No. 4,816,567 to Cabilly et al., which is hereby incorporated by reference in its entirety. For example, oligonucleotide primers that can specifically amplify the heavy and light chain genes encoding the antibody are used by RT-PCR to separate polynucleotides encoding the monoclonal antibody from mature B cells or hybridoma cells. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, and when these expression vectors are transfected into host cells (such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not originally produce immunoglobulins), the host cells will produce monoclonal antibodies. Similarly, recombinant monoclonal antibodies or fragments thereof of a desired species can also 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 entireties).

[0067] The polynucleotide encoding the monoclonal antibody can be further modified with recombinant DNA technology to produce alternative antibodies or derivatives. For example, the constant domains of the light chain and heavy chain of the mouse monoclonal antibody can be replaced by those regions derived from human antibodies to produce chimeric antibodies. Alternatively, the constant domains of the light chain and / or heavy chain of the monoclonal antibody can be replaced by non-immunoglobulin polypeptides to produce fusion antibodies. In other embodiments, the constant region is truncated or removed to produce the desired antibody fragment of the monoclonal antibody. In addition, site-directed mutagenesis or high-density mutagenesis of the variable region can be used to optimize the specificity and affinity of the monoclonal antibody.

[0068] The monoclonal antibodies disclosed herein can be humanized antibodies. Humanized antibodies are antibodies containing minimal sequences from non-human (e.g., mouse) antibodies in the variable region. When administered to a human subject, such antibodies are used therapeutically to reduce antigenicity and human anti-mouse antibody responses. In fact, humanized antibodies are typically human antibodies with very few or even no non-human sequences. Human antibodies are antibodies produced by humans, or antibodies with amino acid sequences corresponding to antibodies produced by humans.

[0069] In addition to intact antibodies, the present disclosure also encompasses antigen-binding portions of such antibodies. Such binding portions include monovalent Fab fragments, Fv fragments (e.g., single-chain antibodies, scFv), and single variable V H and V L Domains and F(ab') 2 Fragments, bi-scFv, diabodies, tri-antibodies, mini-antibodies, etc. These antibody fragments can be prepared by conventional methods, such as proteolytic fragmentation methods, such as those described in Monoclonal Antibodies: Principles and Practice by James Goding. NTIBODIES :P RINCIPLES AND P RACTICE ) 98-118 (Academic Press, 1983) and Ed Harlow and David Lane, Antibodies: A Laboratory Manual. NTIBODIES : A LABORATORY MANUAL) (Cold Spring Harbor Laboratory Press, 1988), which is hereby incorporated by reference in its entirety), or other methods known in the art.

[0070] Particularly for antibody fragments, it may be desirable to further modify the antibody to increase its serum half-life. This can be achieved by mutating appropriate regions in the antibody fragment, for example, by incorporating a salvage receptor binding epitope into the antibody fragment, or incorporating the epitope into a peptide tag, which is then fused to the end or middle of the antibody fragment (e.g., by DNA or peptide synthesis).

[0071] Antibody mimics are also applicable to the situations described in the present disclosure. Many antibody mimics are known in the art, including but not limited to those antibody mimics known as single domain antibodies, which are derived from the tenth human fibronectin type III domain ( 10 Fn3) (Koide et al., "The Fibronectin Type III Domain as a Scaffold for Novel Binding Proteins", Journal of 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 which is hereby incorporated by reference in its entirety); and those antibody mimics known as affibodies, which are derived from the stable α-helical bacterial receptor domain Z of Staphylococcus protein A (Nord et al., "Binding Proteins Selected from Combinatorial Libraries of an α-helical bacterial receptor domain alpha-helical Bacterial Receptor Domain", Nature Biotechnol. 15(8):772-777 (1997), which is hereby incorporated by reference in its entirety).

[0072] In certain embodiments, the targeting component is a peptide that can bind to a tumor-associated antigen. Exemplary peptides include, but are not limited to, glutamic acid-urea-lysine derivatives, such as 2-(3-99S)-5-amino-1-carboxypentyl)ureido)glutaric 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 integrins.

[0073] The peptides used in conjunction with the present disclosure can be obtained by known isolation and purification schemes from natural sources, can be synthesized by standard solid or liquid phase peptide synthesis methods based on the known peptide sequence of the peptide, or can be obtained from commercially available preparations or peptide libraries. The peptides encompassed herein exhibit the biological binding properties of natural peptides and retain the specific binding characteristics of natural peptides. As used herein, derivatives and analogs of peptides include modifications in the composition, properties and derivatization of individual amino acids in the peptide, provided that the peptide retains the specific binding properties of the natural peptide. Examples of such modifications include: D-stereoisomer modification of any amino acid, substitution in the aromatic side chain of an aromatic amino acid, derivatization of the amino or carboxyl group on the side chain of an amino acid containing an amino or carboxyl group in the side chain, substitution at the amino or carboxyl terminus of the peptide, linkage of the peptide to a second peptide or a biologically active moiety, and cyclization of the peptide (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).

[0074] As used herein, "small molecules" are generally organic peptide or non-peptide molecules with 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. Such modulators include chemically synthesized molecules, such as compounds from combinatorial chemical libraries.

[0075] In certain embodiments, the targeting component is an aptamer. Aptamers are small single-stranded DNA or RNA oligonucleotides that can specifically bind to their target molecules (eg, tumor-associated antigens) with high affinity and specificity. Aptamers are generated using an in vitro screening method known as 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 nucleic acid aptamers capable of targeting tumor-associated antigens have been developed, including but not limited to MUC1, HER2, HER3, EpCAM, NF-kB, PSMA, CD44, PD-1, CD137, CD134, PDGF, VEGF, and NCL (Jayasena, "Nucleic Acid Aptamers: An Emerging Class of Molecules Comparable to Antibodies in Diagnosis," Clinical Chemistry 45:1628-1650 (1999), which is hereby incorporated by reference in its entirety).

[0076] In some embodiments, the targeting component is a TCR specific for a tumor antigen. Human TCR comprises two variable domains (Vα and Vβ) associated by a constant region (Cα and Cβ). In some embodiments, the TCR targeting component comprises Vα and Vβ domains bound to the MHC / neoantigen complex on the surface of the tumor cell. In some such embodiments, the TCR targeting component can be a single-chain TCR (scTCR), in which the Vα and Vβ domains are connected by a flexible peptide. In some embodiments, the targeting component is a single variable domain TCR, such as a TCR containing only Vβ. 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).

[0077] Certain common tumor-associated neoantigens are known in the art, including those derived from KRAS G12D , MART-1, gp100, NY-ESO-1, CEA, MAGE-A3, MAGE-A4 and WT1 neoantigens. See, for example, He et al., J. Hematol. & Oncol. 12:139 (2019); Oh et al., Scientific Reports 9:17291 (2019), which are hereby incorporated by reference in their entirety.

[0078] In some embodiments, the targeting component is Anticalin is based on human lipocalin, i.e., an abundant plasma protein characterized by a central β-barrel and four variable loops forming a binding site. See, for example, Rothe and Skerra, 2018, BioDrugs 32(3): 233-243, which is hereby incorporated by reference in its entirety. Using random library design, Anticalin binding to a range of tumor antigens has been developed, including Anticalin binding to CTLA-4, PSMA, VEGFR-3 and Hsp70. Anticalin is a polypeptide with mutations in the amino acid positions of the lipocalin family at four peptide loop regions, which are arranged at the ends of a cylindrical β-barrel structure covering a binding pocket, and correspond to those segments containing amino acid positions 28 to 45, 58 to 69, 86 to 99 and 114 to 129 in the linear polypeptide sequence of the bile triene binding protein of Pieris brassicae. See, e.g., WO2005019255A1; WO2012065978A1; and WO1999016873A1, which are hereby incorporated by reference in their entireties.

[0079] In some embodiments, the targeting component is a single-domain antibody ("single-domain antibody, sdAb", also known as a heavy chain-only antibody), which can be derived from Camelidae, such as described in Eyer, L. and K. Hruska. "Single-domain antibody fragments derived from heavy-chain antibodies: a review." Veterinary Medicine (VeterinarniMedicina) 57.9 (2012): 439. Due to the lack of light chains, the antigen binding site of heavy chain antibodies is composed of only three complementary determining regions (CDRs) compared to the six CDRs in conventional antibodies. Single-domain antibody fragments exhibit high affinity for binding to crevices and cavities on the surface of proteins, which provides the possibility of developing selective therapeutic agents for regulating the activity of cell surface proteins (such as receptors, ion channels and leukocyte extracellular enzymes involved in cancer and inflammatory diseases). 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.

[0080] In certain embodiments, the targeting component targets the prostate-specific membrane antigen (PSMA) receptor.

[0081] As used herein, "PSMA" or "prostate specific membrane antigen" protein refers to mammalian PSMA, preferably human PSMA protein. PSMA is sometimes referred to as folate hydrolase 1 (FOLH1) because PSMA is encoded by the FOLH1 gene. The long transcript of PSMA encodes a protein product with a molecular weight of approximately 100-120 kDa that is characterized as a type II transmembrane receptor with sequence homology to the transferrin receptor and with NAALAD enzymatic 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 Res. 53:227-230 (1993), which are hereby incorporated by reference in their entireties).

[0082] As used herein, the term "enzyme" encompasses any enzyme, protein, or peptide that, when delivered to a tumor or tissue via a targeting component, is capable of catalyzing the transformation of a tumor or tissue into an incompatible allograft.

[0083] In one embodiment, the enzyme is an enzyme involved in post-translational modification and is selected from the group consisting of transferases and glycosyltransferases.

[0084] A transferase is any of a class of enzymes that transfer a specific functional group (eg, a methyl group or a sugar group) from one molecule (called the donor) to another molecule (called the acceptor).

[0085] One group of exemplary transferases includes, but is not limited to, glycosyltransferases. Glycosyltransferases catalyze the stepwise addition of activated sugars (donor NDP sugars) to the non-reducing ends of proteins, glycoproteins, lipids or glycolipids or growing oligosaccharides (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.

[0086] 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.

[0087] For enzymatic sugar synthesis involving glycosyltransferase reactions, glycosyltransferases can be cloned or isolated from any source. Many cloned glycosyltransferases and their polynucleotide sequences are known (see, e.g., "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). Glycosyltransferase amino acid sequences and nucleotide sequences encoding glycosyltransferases from which the amino acid sequences can be derived are also well known in the art.

[0088] Glycosyltransferases that can be used in the methods of the present disclosure include, but are not limited to, galactosyltransferases, fucosyltransferases, glucosyltransferases, N-acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases, glucuronyltransferases, sialyltransferases, mannosyltransferases, glucuronyltransferases, galacturonosyltransferases, and oligosaccharyltransferases. Suitable glycosyltransferases include those derived from eukaryotic organisms as well as prokaryotes.

[0089] Glycosyltransferases are essential to the formation of the ABO blood group antigen system. As described above, the ABO blood system is a major antigen system that is very important in blood transfusion and solid organ transplantation. This tissue-blood group antigen (HBGA) system is controlled by the activity of GTA and / or GTB glycosyltransferases, which attach sugar residues (N-acetylgalactosamine or galactose) to a common substrate (H antigen). This enzyme has several phenotypic variants that change the attached carbohydrate (N-acetylgalactosamine (A) vs. galactose (B)) or result in loss of enzyme function so that the H antigen is not modified (O). One variant of A, A2, has reduced N-acetylgalactosamine activity and NAc-gal addition levels. These variants can currently be distinguished by serology and lectin binding (distinguishing Al from A2). Serology can detect both modification of the H antigen and the presence of natural antibodies to A and / or B (for example, a person with type B glycosylation will have antibodies to A).

[0090] In humans, the glycosyltransferase locus, referred to herein as the ABO locus or ABO glycosyltransferase locus, is located on chromosome 9 and contains seven exons spanning over 18 kb of genomic DNA. Exon 7 is the largest and contains the majority of the coding sequence. The ABO locus has three major allelic forms: A, B, and O. The A "allele" (also referred to as Al or A2) encodes a glycosyltransferase that enzymatically adds N-acetylgalactosamine to the D-galactose end of the H antigen, thereby producing the so-called A antigen. The B allele encodes a glycosyltransferase that enzymatically adds D-galactose to the D-galactose end of the H antigen, thereby producing the so-called B antigen. The O allele encodes a non-functional form of the glycosyltransferase, resulting in an unmodified H antigen, producing the so-called O antigen phenotype.

[0091] At the genomic level, the ABO glycosyltransferase gene has many alleles (about 300). These naturally occurring allelic variants are described in the following references: Yip, "Sequence Variation at the Human ABO Locus," Ann. Hum. Genet. 66: 1-27 (2002); Hakomori, "Antigenic Structure and Genetic Basis of Histogroups A, B, and O: Changes in Their Association with Human Cancer," Acta Biochimica et Biophysica Sinica 1473: 247-266 (1999); Seto et al., "Sequential Interchange of Four Amino Acids from Group B to Group A Glycosyltransferase Enhances Catalytic Activity of Human Recombinant Enzyme and Gradual Alters Substrate Recognition," Journal of Biological Chemistry 272: 14133-14138 (1997), which are hereby incorporated by reference in their entirety, and the naturally occurring alleles are contemplated for use in the bifunctional therapeutic agents of the present disclosure. The sequence encoding the catalytic site of the enzyme is located in exon 7 of the gene; key amino acid residues 176, 235, 266, and 268 control the specificity of this active site. In addition, common nucleotide deletions in exon 6 generate a stop codon that terminates the synthesis of full-length glycosyltransferase, resulting in an O or null phenotype.

[0092] 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 as described above are also not considered.

[0093] In some embodiments, the glycosyltransferase used in the methods of the present disclosure is a fucosyltransferase. Fucosyltransferases are known to those skilled in the art. Exemplary fucosyltransferases include enzymes that transfer L-fucose from GDP-fucose to the hydroxyl position of an acceptor sugar. Fucosyltransferases that transfer non-nucleotide sugars to acceptors can also be used in the present disclosure.

[0094] In some embodiments, the glycosyltransferase is a humanized or deimmunized glycosyltransferase.Methods for humanizing and / or deimmunizing proteins are known in the art.

[0095] Thus, one embodiment of the present disclosure is directed to altering blood group antigen expression on a tumor and / or the blood supply of the tumor by tumor-targeted glycosyltransferases. As described above, this effectively converts the tumor phenotype to that of an incompatible allograft or xenograft, thereby inducing a hyperacute rejection response.

[0096] The bifunctional therapeutics described herein can be constructed such that the targeting component is a protein or peptide linked to an enzyme via a peptide bond.

[0097] In some embodiments, the protein or peptide targeting component connected to the enzyme by a peptide bond can be referred to as a chimeric or fusion protein. As used herein, the term "chimeric protein" or "fusion protein" covers a polypeptide with a single continuous polypeptide chain, i.e., a series of continuous amino acids connected by a peptide bond, or a series of polypeptide chains (i.e., polypeptide complexes) covalently or non-covalently connected to each other, the polypeptide comprising at least a portion of the full-length sequence of the first polypeptide sequence and at least a portion of the full-length sequence of the second polypeptide sequence, wherein the first polypeptide and the second polypeptide are different polypeptides. Chimeric polypeptides also cover polypeptides comprising two or more non-continuous parts derived from the same polypeptide. Chimeric polypeptides or proteins also cover polypeptides with at least one substitution, wherein the chimeric polypeptide includes a first polypeptide sequence, wherein a portion of the first polypeptide sequence is replaced by a portion of the second polypeptide sequence. The series of polypeptide chains can be covalently linked using suitable biochemical linkers or disulfide bonds.

[0098] The coupling of the targeting component and the enzyme can also be prepared using chemical linkage (Brennan et al., "Preparation of Bispecific Antibodies by Chemical Recombination of Monoclonal Immunoglobulin G1 Fragments", Science, 229:81-3 (1985), which is hereby incorporated by reference in its entirety) or chemical coupling (Shalaby et al., "Development of Humanized Bispecific Antibodies Reactive With Cytotoxic Lymphocytes and Tumor Cells Overexpressing the HER2 Protooncogene", Journal of Exp Med 175:217-225 (1992), which is hereby incorporated by reference in its entirety).

[0099] In other embodiments, the targeting component and the enzyme may be linked by non-covalent bonds, including but not limited to hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0100] Therefore, the fusion or connection between the targeting component (e.g., antibody) and the enzyme can be achieved by conventional covalent or ionic bonds, protein fusions performed by genetic engineering, or heterobifunctional cross-linking agents (e.g., carbodiimide, glutaraldehyde, etc.). Conventional inert linker sequences (e.g., peptide linkers) can also be used, which only provide the required spacing between the targeting component and the enzyme. 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. A variety of coupling agents or cross-linking agents can be used for covalent conjugation of proteins. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phthalenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfoSMCC) (see, e.g., Karpovsky et al., "Production of Target-Specific Effector Cells Using Hetero-Cross-Linked Aggregates Containing Anti-Target Cell and Anti-Fc Gamma Receptor Antibodies"). =Antibodies", Journal of 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 78, 1 18-132 (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," Journal of Immunology 139:2367-2375 (1987), which are hereby incorporated by reference in their entirety.

[0101] Many other linkers can be used to couple 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 lower pH in endosomes or tumor environments, including hydrazones, ketals, and / or aconitic acid, can also be used. Hybrid linkers, such as linkers with two or more potential cleavage sites, such as disulfides and hydrazones, can also be used. Linkers sensitive to peptidases, such as tumor-specific peptidases, such as linkers sensitive to PSA cleavage 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, e.g., Hamann et al., Bioconjugate Chem. 13:47-58 (2002); Hamann et al., Bioconjugate Chem. 13(1):40-6 (2002), which are hereby incorporated by reference in their entireties); SPP (Immunogen); and various linkers available from Pierce Biotechnology, Inc. In some embodiments, the linker is SSP (a disulfide linker available from Immunogen), and the ratio of linker to antibody can vary, e.g., from 7:1 to 4:1. Various 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.

[0102] The term "peptide linker" or spacer refers to a short peptide fragment of a targeting component and an enzyme portion in a polypeptide that is connected or coupled to a bifunctional therapeutic agent. The linker is preferably composed of amino acids linked together by peptide bonds. For example, the peptide linker can include 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.

[0103] In one example, the linker is composed of a large number of amino acids that are less sterically hindered, such as glycine and alanine. Thus, in additional examples, the linker is polyglycine, polyalanine or polyserine.

[0104] Those skilled in the art will appreciate that many commonly used peptide linkers can be used in embodiments of the present disclosure. In certain embodiments, short peptide linkers can include repeating units to increase linker length. For example, double, triple, or quadruple repeat linkers. In one example, the linker comprises the formula (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 1) or comprises the formula (Ser-Gly-Gly-Gly-Gly)n Ser (SEQ ID NO: 2), wherein n is an integer from 3 to 6. In some embodiments, the linker is (G 4 S) 3 Linker (SEQ ID NO: 67).

[0105] Non-peptide linkers or spacers are also possible. For example, alkyl linkers such as -NH-(CH 2 )sC(O)-, wherein s=2-20. These alkyl linkers can be further substituted by any sterically unhindered groups such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH 2 An exemplary non-peptide linker is a PEG linker or spacer having a molecular weight of 100 to 5000 kD, preferably 1000 to 2000 kD, more preferably 1500 kD.

[0106] According to the bifunctional therapeutic agent of the present disclosure, the N-terminus coupled to the C-terminus may be included. The N-terminus and C-terminus used herein refer to the N-terminal region or part and the C-terminal region or part of the bifunctional therapeutic protein of the present disclosure, respectively. In some embodiments of the present disclosure, the C-terminal part and the N-terminal part of the bifunctional therapeutic agent of the present disclosure are continuously connected. In alternative embodiments, the C-terminal part and the N-terminal part of the bifunctional therapeutic agent of the present disclosure are coupled by an intermediate spacer. In one embodiment, the spacer can be a polypeptide sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues. In some embodiments, the C-terminal part and / or the N-terminal part of the bifunctional therapeutic agent of the present disclosure can include an additional part coupled to the C-terminal residue and / or the N-terminal residue of the chimeric protein of the present disclosure, respectively. In some embodiments, the additional part can be a polypeptide sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues. In some embodiments, the N-terminal portion and / or C-terminal portion having such additional portions will maintain the activity of the corresponding naturally occurring N-terminal portion and / or C-terminal portion of the targeting component of the enzyme, respectively. In some embodiments, the N-terminal portion and / or C-terminal portion having such additional portions will have enhanced and / or prolonged activity compared to the corresponding naturally occurring N-terminal portion and / or C-terminal portion of the targeting component of the enzyme, respectively. In other embodiments, the C-terminal portion and / or N-terminal portion of the bifunctional therapeutic agent of the present disclosure may not include any additional portion coupled to the C-terminal residue and / or N-terminal residue of the chimeric protein of the present disclosure, respectively.

[0107] In one embodiment, the N-terminal region comprises a targeting component. In certain embodiments, 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 mini antibodies, diabodies and triabodies. The antibody or antigen-binding fragment may maintain or lack an FcRn binding domain. In certain embodiments, the targeting component is a TCR, a single domain antibody or anticalin.

[0108] In some embodiments, the C-terminal region comprises an enzyme.

[0109] In one embodiment, the bifunctional therapeutic comprises the catalytic domain of glycosyltransferase B (GTB) and has the amino acid sequence of SEQ ID NO: 7 (GenBank Accession No. AM423112.1, which is hereby incorporated by reference in its entirety herein), or a portion thereof, as follows:

[0110] MAEVLRTLAG KPKCHALRPM ILFLIMLVLV LFGYGVLSPR SLMPGSLERG

[0111] FCMAVREPDH LQRVSLPRMV YPQPKVLTPC RKDVLVVTPW LAPIVWEGTF

[0112] NIDILNEQFR LQNTTIGLTV FAIKKYVAFL KLFLETAEKH FMVGHRVHYY

[0113] VFTDQPAAVP RVTLGTGRQL SVLEVGAYKR WQDVSMRRME MISDFCERRF

[0114] LSEVDYLVCV DVDMEFRDHV GVEILTPLFG TLHPSFYGSS REAFTYERRP

[0115] QSQAYIPKDE GDFYYMGAFF GGSVQEVQRL TRACHQAMMV DQANGIEAVW

[0116] HDESHLNKYL LRHKPTKVLS PEYLWDQQLL GWPAVLRKLR FTAVPKNHQA

[0117] VRNP

[0118] In another embodiment, the bifunctional therapeutic agent comprises a "cis A, B" sequence that produces a hybridization sequence of GTB and GTA and has an amino acid sequence of SEQ ID NO: 8 (GenBank Accession No. ABL75287.1, which is hereby incorporated by reference in its entirety herein), or a portion thereof, as follows:

[0119] YVAFLKLFLE TAEKHFMVGH RVHYYVFTDQ PAAVPRVTLG TGRQLSVLEV

[0120] GAYKRWQDVS MRRMEMISDF CERRFLSEVD YLVCVDVDME FRDHVGVEIL

[0121] TPLFGTLHPS FYGSSREAFT YERRPQSQAY IPKDEGDFYY MGGFFGGSVQ

[0122] EVQRLTRACH QAMMVDQANG IEAVWHDESH LNKYLLRHKP TKVLSPEYLW

[0123] DQQLLGWPAVLRKLRFTAVP KNHQAVRNP

[0124] In another embodiment, the bifunctional therapeutic comprises the catalytic domain of glycosyltransferase A (GTA) and has the amino acid sequence of SEQ ID NO: 9 (GenBank Accession No. AFB74122.1, which is hereby incorporated by reference in its entirety), or a portion thereof, as follows:

[0125] MAEVLRTLAG KPKCHALRPM ILFLIMLVLV LFGYGVLSPR SLMPGSLERG

[0126] FCMAVREPDH LQRVSLPRMV YPQPKVLTPC RKDVLVVTPW LAPIVWEGTF

[0127] NIDILNEQFR LQNTTIGLTV FAIKKYVAFL KLFLETAEKH LMVGHRVHYY

[0128] VFTDQPAAVP RVTLGTGRQL SVLEVRAYKR WQDVSMRRME MISDFCERRF

[0129] LSEVDYLVCV DVDMEFRDHV GVEILTPLFG TLHPGFYGSS REAFTYERRP

[0130] QSQAYIPKDE GDFYYLGGFF GGSVQEVQRL TRACHQAMMV DQANGIEAVW

[0131] HDESHLNKYL LRHKPTKVLS PEYLWDQQLL GWPAVLRKLR FTAVPKNHQA

[0132] VRNP

[0133] In certain embodiments, a tumor with a tumor-associated antigen expresses an H antigen. As used herein, "H antigen" refers to an oligosaccharide chain with a terminal disaccharide (fucose-galactose) in which the fucose has an α-(1-2)-linkage. The H antigen is produced by fucosyltransferase and is the basic structural unit for the production of A or B antigens within the ABO blood group system.

[0134] Therefore, the present disclosure also relates to a method for treating cancer. The method involves selecting a subject suffering from cancer and providing a dual-function therapeutic agent according to the present disclosure. The dual-function therapeutic agent is administered to the selected subject under conditions effective to treat the cancer.

[0135] Virtually any tumor expressing the H antigen can be treated with the bifunctional therapeutic agents described herein, including but not limited to prostate tumors, adrenocortical carcinoma tumors, anal tumors, appendix tumors, astrocytomas (cerebellum or brain in children), basal cell carcinomas, bile duct tumors, bladder tumors, bone tumors, osteosarcoma / malignant fibrous histiocytoma, brain stem gliomas, ependymomas, medulloblastomas, breast tumors, bronchial adenomas / carcinoids, Burkitt's lymphomas, carcinoid tumors, cervical tumors, childhood tumors, chondrosarcomas, colon tumors, cutaneous T-cell lymphomas, desmoplastic small round cell tumors, endometrial tumors, esophageal tumors, Ewing's sarcoma (Ewing's sarcomas), retinoblastoma, 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 carcinoma (endocrine pancreas), Kaposi sarcoma sarcomas), kidney tumors, laryngeal tumors, lip and oral cavity tumors, non-small cell lung tumors, small cell lung tumors, lymphomas, melanomas, Merkel cell tumors, mesothelioma, multiple endocrine tumors, multiple myeloma, nasopharyngeal tumors, neuroblastomas, oligodendrogliomas, oral tumors, oropharyngeal tumors, ovarian tumors, pancreatic tumors, pleural lung tumors, primary central nervous system lymphomas, retinoblastoma, rhabdomyosarcoma, salivary gland tumors, soft tissue sarcomas, uterine sarcomas, skin tumors (non-melanoma), small intestine tumors, squamous cell carcinomas, stomach tumors, testicular tumors, pharyngeal tumors, thymoma and thymic carcinoma, thyroid tumors, trophoblastic tumors, and urethral tumors.

[0136] Some cancers, including but not limited to hematopoietic or lymphoid cancers, mesodermal cancers, sarcomas, neuroectodermal cancers, etc., may not express the H antigen. This can be easily determined by flow cytometry or immunohistochemistry of tumor samples, using Ulex lectin binding to reveal the presence or absence of H. When H is not present, treatment using the present application can be accomplished in two ways: Targeted fucosyltransferases can be employed so that the H antigen is added prior to or concurrently with the action of the targeted glycosyltransferases described above. Alternatively, the αgalT enzyme can be targeted, which can add terminal galactose and does not require the presence of 1,2-fucose (i.e., the H antigen).

[0137] In one embodiment, the targeting component of the bifunctional therapeutic agent targets the PSMA receptor on the tumor vascular endothelium. It is reported that PSMA is expressed in tumor neovascularization of a variety of tumors, but is not expressed in normal tissue blood vessels. Exemplary tissue types with PSMA-positive vascular endothelium include, but are not limited to, kidney, lung, colon, stomach, breast, brain, pancreas, liver, bladder, esophagus, adrenal glands, head and neck, melanoma, and brain tumors. Other embodiments include targeting PSMA expressed on the surface of prostate cancer cells, targeting HER2 on breast and other HER2-positive cancers, targeting CD19 on B-cell lineage cancers, and targeting CEA on colorectal cancer. Other applicable targets are as described above.

[0138] In some embodiments, the heavy and / or light chain variable regions of the antibody-based molecules described herein further comprise human or humanized immunoglobulin heavy and / or light chain framework regions, respectively.

[0139] Suitable amino acid modifications of the heavy chain CDR sequences and / or light chain CDR sequences of the targeting domain disclosed herein include, for example, conservative substitutions or functionally equivalent amino acid residue substitutions that produce CDR variant sequences having similar or enhanced binding properties to the CDR sequences disclosed herein as described above. Conservative substitutions refer to those that occur within a family of amino acids related to the side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) non-polar (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 collectively classified as aromatic amino acids. Alternatively, the amino acid library can be grouped into (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), wherein serine and threonine are optionally grouped as aliphatic hydroxyl groups, respectively; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amides (asparagine, glutamine); and (6) sulfur-containing (cysteine ​​and methionine) (Stryer (ed.), Biochemistry, 2nd ed., WH Freeman & Co., 1981, which is hereby incorporated by reference in its entirety). Non-conservative substitutions can also be made. Non-conservative substitutions involve replacing one or more amino acid residues of a 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 light chain variable region CDR may further include one or more internal neutral amino acid insertions or deletions that maintain or enhance target binding.

[0140] The targeting domains of the present disclosure can be described or specified in terms of their binding affinity. Thus, in some embodiments, the targeting domains of the present disclosure include a dissociation constant or K D Those targeting domains that are 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.

[0141] In some embodiments, the targeting component includes a signal peptide, optionally wherein the signal peptide has a sequence of amino acids 1-19 of SEQ ID NO:34.

[0142] In some embodiments, the glycosyltransferase is selected from glycosyltransferase A (α1-3-N-acetylgalactosaminyltransferase) and glycosyltransferase B (α1-3-galactosyltransferase).

[0143] 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:

[0144] EPDHLQRVSLPRMVYPQPKVLTPCRKDVLVVTPWLAPIVWEGTFNIDILNEQFRLQNTTIGLTVFAIKKYVAFLKLFLETAEKHFMVGHRVHYYVFTDQPAAVPRVTLGTGRQLSVLEVRAYKRWQDVSMRRMEMISDFCERRFLSEVD YLVCVDVDMEFRDHVGVEILTPLFGTLHPGFYGSSREAFTYERRPQSQAYIPKDEGDFYYLGGFFGGSVQEVQRLTRACHQAMMVDQANGIEAVWHDESHLNKYLLRHKPTKVLSPEYLWDQQLLGWPAVLRKLRFTAVPKNHQAVRNP.

[0145] 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:

[0146] EPDHLQRVSLPRMVYPQPKVLTPCRKDVLVVTPWLAPIVWEGTFNIDILNEQFRLQNTTIGLTVFAIKKYVAFLKLFLETAEKHFMVGHRVHYYVFTDQPAAVPRVTLGTGRQLSVLEVGAYKRWQDVSMRRMEMISDFCERRFLSEVD YLVCVDVDMEFRDHVGVEILTPLFGTLHPSFYGSSREAFTYERRPQSQAYIPKDEGDFYYMGAFFGGSVQEVQRLTRACHQAMMVDQANGIEAVWHDESHLNKYLLRHKPTKVLSPEYLWDQQLLGWPAVLRKLRFTAVPKNHQAVRN.

[0147] Suitable additional glycosylases are described below. In some embodiments, the glycosyltransferase is a marmoset α-1,3 galactosyltransferase (amino acids 90-376) and has the amino acid sequence of SEQ ID NO: 66 or a portion thereof, as follows:

[0148] ELRLWDWFNPKKRPEVMTVTQWKAPVVWEGTYNKAILENYYAKQKITVGLTVFAIGRYIEHYLEEFVTSANRYFMVGHKVIFYVMVDDVSKAPFIELGPLRSFKVFEVKPEKRWQDISMMRMKTIGEHILAHIQHEVDFLFCMDVD QVFQDHFGVETLGQSVAQLQAWWYKADPDDFTYERRKESAAYIPFGQGDFYYHAAIFGGTPIQVLNITQECFKGILLDKKNDIEEWHDESHLNKYFLLNKPSKILSPEYCWDYHIGLPSDIKTVKLSWQTKEYNLVRKNVGGGS.

[0149] Another aspect of the present disclosure relates to a bifunctional therapeutic agent for treating cancer, the bifunctional therapeutic agent comprising: a targeting component that targets a member of the human epidermal growth factor receptor (HER) family; and a glycosyltransferase that enzymatically converts the tumor phenotype to an incompatible allograft or xenograft phenotype when delivered to the tumor by the targeting component, the glycosyltransferase being coupled to the targeting component. This aspect of the present disclosure can be used to treat subjects with breast cancer or any cancer expressing HER2.

[0150] It should be understood that the exact dosage of the dual-functional therapeutic agent of the present disclosure is selected by the individual physician according to the patient to be treated. Generally, the dosage and mode of administration are adjusted to provide an effective amount of the agent to the patient being treated. As used herein, the "effective amount" of a dual-functional therapeutic agent refers to the amount necessary to induce a desired biological response. As will be understood by those of ordinary skill in the art, the effective amount of the dual-functional therapeutic agent of the present disclosure can vary according to factors such as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. For example, the effective amount of a dual-functional therapeutic agent can be an amount that reduces the size of a tumor to a desired degree within a desired time period. Additional factors that may be considered include the severity of the disease state; the age, weight, and sex of the subject being treated; diet; the time and frequency of administration; drug combinations; reaction sensitivity; and tolerance / response to therapy.

[0151] An "effective amount" may also be a "prophylactically effective amount," which refers to an amount of a dual-function therapeutic agent as described herein that is effective in preventing or delaying the onset or recurrence of a disorder (e.g., cancer) or treating its symptoms when administered to a subject in a single dose or multiple doses.

[0152] Typically, when administered as a single agent, the dose may be in the range of about 25% to about 100% of the maximum tolerated dose (MTD) of the dual-function therapeutic agent. Based on the composition, the dose may be delivered once, continuously (such as by a continuous pump), or at periodic intervals. The dose may be appropriately adjusted to achieve the desired drug level locally or systemically. In the event that the subject's response is insufficient at such a dose, even a higher dose (or an effective higher dose achieved by a different, more localized delivery route) may be used within the range permitted by the patient's tolerance. For example, continuous IV administration over 24 hours or multiple administrations per day are also contemplated to achieve appropriate systemic levels of the compound. For example, the dosage regimen can be changed so that the dual-function therapeutic agent is applied once, twice, three times or more times per week for any number of weeks, or the dual-function therapeutic agent can be applied once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks or every ten weeks, with cumulative application exceeding once (e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-two times or twenty-four times). For example, the dual-function therapeutic agent can be applied at least twice, three times or four times at the above dosage levels, wherein it is applied once every four to eight weeks. If the subject does not show adverse reactions to the dual-function therapeutic agent, and / or one or more symptoms of cancer improve or remain unchanged, additional dosages can be given. In some embodiments, as the dosing interval is prolonged, the dosage of the dual-function therapeutic agent can be increased.

[0153] For different targeting components, the biodistribution and pharmacokinetics of bifunctional therapeutic agents may be different. For example, large bifunctional therapeutic agents comprising full-length and intact antibodies will have longer plasma and systemic half-lives and tend to remain in the circulatory system. Such bifunctional therapeutic agents are also more likely to be excreted by the liver and less likely to penetrate into normal tissues. On the contrary, small bifunctional therapeutic agents such as those comprising targeting peptides or small molecule ligands will tend to have shorter half-lives, be excreted by the kidneys / urinary tract, and more easily penetrate normal tissues and tumors.

[0154] When practicing the method of the present disclosure, the application step is implemented to treat the cancer of the subject. In one embodiment, the subject suffering from cancer is selected before the application step. Such application can be systemically applied, or by direct or local application to the tumor site. For example, suitable systemic application mode includes but is not limited to oral, topical, transdermal, parenteral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or by intranasal instillation, by intracavity or intravesical instillation, intraocular application, intraarterial application, intralesional application or mucosal application. Suitable local application mode includes but is not limited to catheterization, implantation, direct injection, skin / transdermal application or portal vein application to related tissues, or by any other local application technology, method or procedure known in the art. The mode affecting the delivery of the dual-function therapeutic agent will change according to the type (for example, with antibody targeting component or peptide targeting component) of the dual-function therapeutic agent and the disease to be treated.

[0155] The dual-function therapeutic agent of the present invention can be administered orally, for example, with an inert diluent or with an absorbable edible carrier, or can be encapsulated in a hard or soft shell capsule, can also be compressed into a tablet, or can be directly mixed with daily food. The dual-function therapeutic agent of the present invention can also be administered in a sustained release manner, such as being loaded into a device such as a sustained release capsule or nanotube. Such devices are flexible with respect to time and dosage. For oral therapeutic administration, the medicament of the present invention can be mixed with an excipient and used in the form of tablets, capsules, elixirs, suspensions, syrups, etc. Such compositions and preparations should contain at least 0.1% of the medicament, although lower concentrations may also be effective and may actually be the optimal concentration. Of course, the percentage of the medicament in these compositions can vary and can be conveniently between about 2% and about 60% of the unit weight. The content of the dual-function therapeutic agent of the present invention in such a composition for therapeutic use should be such that a suitable administration dose can be obtained.

[0156] When the dual-function therapeutic agent of the present disclosure is administered parenterally, it can be appropriately mixed with a surfactant such as hydroxypropylcellulose in water to prepare a solution or suspension of the agent. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and its mixture with oils. Exemplary oils are oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil or mineral oil. Generally, water, saline, dextran aqueous solution and related sugar solutions and glycols (such as propylene glycol or polyethylene glycol) are preferred liquid carriers, particularly for injectable solutions. Under common storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0157] Pharmaceutical formulations suitable for injection purposes include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the formulation must be sterile and must be sufficiently fluid so that it can be easily injected with a syringe. It must remain stable under manufacturing and storage conditions and must be free from contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium, which includes, for example, water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol), a suitable mixture thereof, and vegetable oils, etc.

[0158] When it is desired to deliver the dual-function therapeutic agent of the present disclosure systemically, it can be formulated into a formulation suitable for administration by parenteral injection (e.g., by bolus injection or continuous infusion). Injectable formulations can be present in unit dosage form, such as in ampoules or in multi-dose containers, and preservatives are added. These compositions can be present in oily or aqueous vehicles in the form of suspensions, solutions, or emulsions, and can include formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0159] Intraperitoneal or intrathecal administration of the dual-function therapeutic agents of the present disclosure can also be achieved using an infusion pump device. Such a device can achieve continuous infusion of the desired compound, thereby avoiding multiple injections and multiple manipulations.

[0160] In addition to the formulations described above, the bifunctional therapeutic agent can also be formulated as a depot preparation. Such long-acting formulations can be formulated with suitable polymers or hydrophobic materials (e.g., emulsions made in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives (e.g., sparingly soluble salts).

[0161] Another aspect of the present disclosure relates to a pharmaceutical composition comprising the bifunctional therapeutic agent of the present disclosure and a pharmaceutically acceptable carrier.

[0162] The bifunctional therapeutic agents are as described above.

[0163] The pharmaceutical composition containing the bifunctional therapeutic agent used 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 microspheres and helical liposomes.

[0164] In one embodiment of the present disclosure, the pharmaceutical composition or formulation is encapsulated in a lipid formulation to form a nucleic acid-lipid particle, as described in Semple et al., "Rational Design of Cationic Lipids for siRNA Delivery," Nature Biotechnology, 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.

[0165] In another embodiment of the present disclosure, the delivery medium is a nanoparticle. A variety of nanoparticle delivery media 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 polymer nanoparticles for tumor-targeted drug delivery (Multi-functional PolymericNanoparticles for Tumour-Targeted Drug Delivery)", "Expert Opin. Drug Deliv. " 3 (2): 205-216 (2006), the document is hereby incorporated by reference in its entirety).Suitable nanoparticles include, but are not limited to, poly(β-amino esters) (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) copolymers (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-Loaded Nanoparticles shRNA-Loaded PEI-alt-PEG Nanoparticles Suppressed Colon Carcinoma Established Subcutaneously in Mice", Journal of Nanosci. Nanotechnology, 10(5):3280-3 (2010), which is hereby incorporated by reference in its entirety) and liposome-encapsulated siRNA nanoparticles (Kenny et al., "Novel Multifunctional Nanoparticle Mediates siRNA Tumor Delivery, Visualization and Therapeutic Tumor Reduction In Vivo", Journal of Controlled 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 microencapsulated nanotube devices disclosed in US Patent Publication No. 2010 / 0215724 to Prakash et al., which is hereby incorporated by reference in its entirety.

[0166] In another embodiment of the present disclosure, the pharmaceutical composition is contained in a liposome delivery vehicle. The term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical monolayer or multilayer bilayer. Liposomes are monolayer or multilayer vesicles with a membrane formed by a lipophilic material and an internal water-containing membrane. Its aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with cell walls. Although non-cationic liposomes cannot fuse with cell walls as effectively as cationic liposomes, they can be taken up by macrophages in the body.

[0167] Several advantages of liposomes include their biocompatibility and biodegradability, their ability to encapsulate a wide range of water-soluble and lipid-soluble drugs, and their ability to protect encapsulated drugs from metabolism and degradation. When preparing liposome formulations, lipid surface charge, vesicle size, and the aqueous volume of the liposomes are important factors to consider.

[0168] Liposomes can be used to transfer and deliver active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissues, the liposomes begin to fuse with the cell membrane, and as the fusion of liposomes and cells progresses, the liposome contents are released into the cells, where the active agents act.

[0169] Methods for preparing liposomes for use in the present disclosure include those disclosed in Bangham et al., "Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids," Journal of Molecular Biology 13:238-52 (1965); U.S. Pat. No. 5,653,996 to Hsu; U.S. Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613 to Holland et al.; U.S. Pat. No. 5,631,237 to Dzau and Kaneda; and U.S. Pat. No. 5,059,421 to Loughrey et al., which are hereby incorporated by reference in their entirety.

[0170] In another embodiment of the present 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 agents. Suitable gene therapy vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and herpes virus vectors.

[0171] Adenovirus viral vector delivery vehicles can be readily prepared and utilized 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 Epitheliium 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., which are hereby incorporated by reference in their entireties.Adeno-associated virus delivery vehicles can be constructed and used to deliver the bifunctional therapeutic agents of the present disclosure to cells, 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 Research 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 Adeno-Associated Virus Antisense Vectors." 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", Journal of 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.The in vivo use of these agents is described in Flotte et al., "Stable in Vivo Expression of the Cystic Fibrosis Transmembrane Conductance Regulator With an Adeno-Associated Virus Vector," Proc. Natl. Acad. Sci. USA 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 entireties. Other types of adenoviral vectors are described in U.S. Pat. No. 6,057,155 to Wickham et al.; U.S. Pat. No. 6,033,908 to Bout et al.; U.S. Pat. No. 6,001,557 to Wilson et al.; U.S. Pat. No. 5,994,132 to Chamberlain et al.; U.S. Pat. No. 5,981,225 to Kochanek et al.; U.S. Pat. No. 5,885,808 to Spooner et al.; and U.S. Pat. No. 5,871,727 to Curiel, which are hereby incorporated by reference in their entirety.

[0172] Retroviral vectors modified to form infectious transformation systems can also be used to deliver nucleic acid molecules to target cells. 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 herein. Other nucleic acid delivery vehicles suitable for the present disclosure include those disclosed in U.S. Patent Publication No. 20070219118 to Lu et al., which is hereby incorporated by reference herein in its entirety.

[0173] Regardless of the type of infectious transformation system used, the nucleic acid should be delivered to the desired cell type. For example, in order to deliver to a cell cluster (e.g., cancer cells), a high titer of the infectious transformation system can be injected directly into the location of these cells to increase the possibility of cell infection. The infected cells will express nucleic acid molecules targeting tumor-associated antigens. The expression system may further contain a promoter to control or regulate the intensity and specificity of expression of the nucleic acid molecule in the target tissue or cell.

[0174] As described above, the effective dose of the compositions of the present disclosure for treating metastatic disease varies according to many different factors, including the type and stage of cancer, the mode of administration, the target site, the patient's physiological state, other drugs or therapies administered, and the patient's physical state relative to other medical complications. The therapeutic dose needs to be adjusted to optimize safety and efficacy.

[0175] The pharmaceutical compositions of the present disclosure may include a "therapeutically effective amount" or a "prophylactically effective amount" of the bifunctional therapeutic agent of the present disclosure. A "therapeutically effective amount" refers to an effective amount that can achieve the desired therapeutic result at the necessary dosage and time period. The therapeutically effective amount of the bifunctional therapeutic agent can vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the bifunctional therapeutic agent to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutic beneficial effect exceeds any toxic or deleterious effect of the bifunctional therapeutic agent. Relative to untreated subjects, a "therapeutically effective dose" preferably results in at least about 20% inhibition of a measurable parameter (e.g., tumor growth rate), more preferably at least about 40%, even more preferably at least about 60% inhibition, and still more preferably at least about 80% inhibition. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be assessed in an animal model system that is predictive of its efficacy in human tumors. Alternatively, this property of the composition can be assessed by testing the ability of the compound to inhibit (as determined by an in vitro assay known to those skilled in the art).

[0176] "Prophylactically effective amount" refers to an effective amount that can achieve the desired preventive result at the necessary dosage and time period. Usually, since the preventive dose is used for subjects before or in the early stages of the disease, the preventive effective amount will be lower than the therapeutic effective amount.

[0177] In certain embodiments, the administering step further comprises administering the nucleotide sugars uridine diphosphate galactose (UDP-gal), uridine diphosphate-N-acetylgalactosamine (UDP-NAcGal), and / or guanosine diphosphate-fucose (GDP-fucose).

[0178] 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. In addition, UDP-gal, UDP-NAcGal and / or GDP-fucose can be administered simultaneously with the bifunctional targeted enzyme or after the bifunctional targeted enzyme. In the latter case, i.e., subsequent administration, the interval between the targeted enzyme and the nucleotide sugar can be 1 minute to 1 week. In a preferred embodiment, the interval is in the range of 1 minute to 48 hours.

[0179] The bifunctional therapeutic agents described herein can be used in combination with other therapies. As used herein, "combination" administration means delivering two (or more) different treatments to a subject during the process of the subject suffering from the disease, for example, after the subject has been diagnosed as suffering from the disease and before the disease has been cured, eliminated or the treatment has stopped treatment for other reasons, the two or more treatments are delivered. In some embodiments, the delivery of a treatment is still carried out when the delivery of the second treatment is started, so that there is overlap in the application. This is referred to as "simultaneous delivery" or "parallel delivery" in this article. In other embodiments, the delivery of a treatment ends before the delivery of another treatment begins. In some embodiments, treatment is more effective due to combined administration. For example, the second treatment is more effective, for example, compared to the application of the second treatment in the absence of the first treatment, a small amount of the second treatment can achieve the same effect, or the second treatment can alleviate symptoms to a greater extent, or similar situations are also seen in the case of the first treatment. In some embodiments, the combined administration makes the degree of alleviation of symptoms or the degree of improvement of other parameters related to the disease greater than the observed situation when only one treatment is used without using another treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. Delivery may be such that the effect of the first treatment is still detectable while the second treatment is being delivered.

[0180] Exemplary therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, The invention relates to hydroxyproline, anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansine compounds (e.g., maytansinol, see U.S. Pat. No. 5,208,020, which is hereby incorporated by reference in its entirety), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 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., mechlorethamine, thiotepa 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, mithramycin, and anthramycin (AMC), and antimitotic agents (e.g., vincristine, vinblastine, paclitaxel, and maytansine compounds).

[0181] In other embodiments, the dual-function therapeutic agent is administered in combination with other treatments, including surgery, radiation, cryosurgery, and / or thermal therapy. Such combination therapies can advantageously utilize lower doses of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with various monotherapies.

[0182] In other embodiments, the bifunctional therapeutic is administered in combination with an immunomodulator (eg, IL-1, IL-24, IL-6, or IL-12, or interferon alpha or gamma).

[0183] Another aspect of the present disclosure provides nucleic acid (e.g., polynucleotide) molecules encoding the bifunctional therapeutic agent of the present disclosure. The polynucleotide can be, for example, DNA, cDNA, PNA, RNA or a combination thereof, single-stranded and / or double-stranded form, or a natural or stable form of the polynucleotide, for example, a polynucleotide with a thiophosphate backbone, and as long as it encodes a bifunctional therapeutic agent, it may or may not contain introns. Of course, only peptides containing naturally occurring amino acid residues connected by naturally occurring peptide bonds can be encoded by polynucleotides. Another aspect of the present disclosure provides a recombinant expression vector capable of expressing a bifunctional therapeutic agent according to the present disclosure. A variety of methods have been developed to connect polynucleotides (particularly DNA) to a carrier, such as by connecting to complementary sticky ends. For example, complementary homopolymer pieces can be added to a DNA fragment to be inserted into a carrier DNA. The carrier and the DNA fragment are then combined together by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

[0184] Synthetic linkers containing one or more restriction endonuclease sites provide an alternative method for connecting DNA fragments to vectors. Synthetic linkers containing various restriction endonuclease sites are commercially available from many sources, including International Biotechnologies Inc. New Haven, CN, USA. An ideal method for modifying the DNA encoding the bifunctional therapeutic agent of the present disclosure employs 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 Research 16(15):7351-67 (1988), which is hereby incorporated by reference in its entirety. This method can be used to introduce DNA into a suitable vector, for example, by constructing a suitable restriction endonuclease site, or can be used to modify DNA in other useful ways known in the art.

[0185] The nucleic acids disclosed herein can be selected based on the preference of a particular expression system for codons, which can be preferred or non-preferred. For example, the nucleic acid can be a nucleic acid in which at least one codon (preferably at least 10% or 20% of the codons) is changed so that the sequence is optimized for expression in E. coli, yeast, humans, insects, NS0 or CHO cells.

[0186] Typically, the polynucleotide encoding the bifunctional therapeutic agent is placed under the regulation of a functional promoter in the desired host cell. A variety of promoters are well known and can be used in the expression vectors of the present disclosure according to the specific disclosure. Typically, the selected promoter depends on the cell type in which the promoter is to be active. Other expression control sequences, such as ribosome binding sites, transcription termination sites, etc., may also be included as needed. Constructs containing one or more such control sequences are referred to as "expression vectors." Therefore, the present disclosure provides some expression vectors into which nucleic acid molecules encoding bifunctional therapeutic agents are integrated for achieving high-level expression in desired host cells.

[0187] Expression control sequences suitable for specific host cells are usually obtained by cloning genes expressed in the cells. Commonly used prokaryotic control sequences, which are defined herein as including a promoter for transcription initiation, optionally with an operator, and a ribosome binding site sequence, include commonly used promoters such as β-lactamase (penicillinase) and lactose (lac) promoter systems (Change et al., Nature 198: 1056 (1977), which are hereby incorporated by reference in their entirety), tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Research 8: 4057 (1980), which are hereby incorporated by reference in their entirety), tac promoters (DeBoer et al., Proceedings of the National Academy of Sciences of the United States of America 80: 21-25 (1983), which are hereby incorporated by reference in their entirety); and the PL promoter and N gene ribosome binding site derived from lambda phage (Shimatake et al., Nature 292: 128 (1981), which are hereby incorporated by reference in their entirety). However, any available promoter that functions in prokaryotes may be used.

[0188] In order to express bifunctional therapeutic agents in prokaryotes other than E. coli, a promoter that works in a specific prokaryotic species is required. Such promoters can be obtained from genes cloned from that species, or heterologous promoters can be used. For example, the hybrid trp-lac promoter can work in Bacillus in addition to E. coli.

[0189] A ribosome binding site (RBS) is conveniently included in the expression cassette of the present disclosure. For example, the RBS in Escherichia coli is composed of a nucleotide sequence of 3-9 nucleotides in length, which is located 3-11 nucleotides upstream of the start codon (Shine and Dalgarno, "Determinant of Cistron Specificity in Bacterial Ribosomes", Nature 254:34-38 (1975); Steitz, Biological regulation and development: Gene expression (ed. R. F. Goldberger), Vol. 1, p. 349, 1979, Plenum Publishing, New York, which is hereby incorporated by reference in its entirety).

[0190] For mammalian cells, the regulatory sequences will include a promoter and, preferably, an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may also include splice donor and acceptor sequences.

[0191] In the present disclosure, either constitutive promoters or regulatable promoters can be used. Regulatable promoters may be advantageous because host cells can grow to high density before inducing expression of the bifunctional therapeutic agent. In some cases, high-level expression of heterologous proteins slows cell growth. Inducible promoters are promoters that direct gene expression, and their expression levels can be affected by environmental or developmental factors (e.g., temperature, pH, anaerobic conditions or aerobic conditions, light, transcription factors, and chemicals, etc.) and change. Such promoters are referred to herein as "inducible" promoters, which allow artificial control of the expression time of the bifunctional therapeutic agent. For E. coli and other bacterial host cells, inducible promoters are known to those skilled in the art. These inducible promoters include, for example, the lac promoter, the P promoter of bacteriophage λ, and the P promoter of phage λ. L promoter, the hybrid trp-lac promoter (Amann et al., Gene 25:167 (1983); de Boer et al., Proc. Natl. Acad. Sci. USA 80:21 (1983), which are hereby incorporated by reference in their entireties), and the bacteriophage T7 promoter (Studier et al., J. Mol. Biol. (1986); Tabor et al., Proc. Natl. Acad. Sci. USA 82:1074-8 (1985), which are hereby incorporated by reference in their entireties).

[0192] Selective markers are usually incorporated into expression vectors for expressing the bifunctional therapeutic agents of the present disclosure. These genes can encode a gene product, such as a protein, which is necessary for the survival or growth of transformed host cells grown in selective culture media. Host cells that are not transformed by vectors containing selection genes will not be able to survive in culture media. The protein encoded by typical selection genes can confer resistance to antibiotics or other toxins (such as ampicillin, neomycin, kanamycin, chloramphenicol or tetracycline) on host cells. Alternatively, selective markers can encode proteins that can make up for nutritional deficiencies or provide key nutrients that cannot be obtained from complex culture media, such as genes encoding D-alanine racemase in Bacillus. Typically, the vector will have a selective marker that can function in, for example, Escherichia coli or in other cells that replicate before the vector is introduced into the host cell. Many selective markers are known to those skilled in the art.

[0193] The suitable nucleic acid constructs comprising one or more components listed above can be constructed by standard connection techniques as described in the references cited above. Isolated plasmids or DNA fragments can be cut, tailored and reconnected in a desired form to generate required nucleic acid constructs (e.g., plasmids). In order to confirm whether the sequence in the constructed plasmid is correct, these plasmids can be analyzed by standard techniques such as restriction endonuclease digestion and / or sequencing according to known methods. Molecular cloning techniques for realizing these purposes are known in the art. A variety of clones and in vitro amplification methods that are applicable to construct recombinant nucleic acids are well known to those skilled in the art. Examples of such instructions, which are sufficient to guide the skilled artisan in performing many cloning procedures, are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); and Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley Sons, Inc., (1998 Supplement) (Ausubel), which are hereby incorporated by reference in their entirety.

[0194] A variety of common vectors suitable for use as starting materials for constructing 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 TM , 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 used plasmids, including pSV2, pBC12BI and p91023, as well as lytic viral vectors (e.g., vaccinia virus, adenovirus and baculovirus), free viral vectors (e.g., bovine papilloma virus) and retroviral vectors (e.g., mouse retrovirus).

[0195] Then, the nucleic acid can be expressed in a suitable host to produce a polypeptide comprising the bifunctional therapeutic agent of the present disclosure. Therefore, the nucleic acid encoding the bifunctional therapeutic agent of the present disclosure can be used to construct an expression vector according to known techniques and after being appropriately modified according to the teachings contained herein, which is then used to transform a suitable host cell to express and produce the bifunctional therapeutic agent of the present disclosure. Such technology is described below, and also includes, for example, 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, and the document is hereby incorporated by reference as a whole.

[0196] The method for introducing the expression vector into the selected host cell is not particularly critical, and such methods are known to those skilled in the art. For example, the expression vector can be introduced into prokaryotic cells (including E. coli) by calcium chloride transformation, and introduced into eukaryotic cells by calcium phosphate treatment or electroporation. Other transformation methods are also applicable.

[0197] The bifunctional therapeutic agents disclosed herein can also be further linked to other bacterial proteins. This approach often results in high yields because normal prokaryotic control sequences can guide transcription and translation. In E. coli, lacZ fusions are commonly used to express heterologous proteins. Suitable vectors are readily available, such as pUR, pEX, and pMR100 series vectors. For some 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 by cyanogen bromide, proteases, or factor X. aCleavage can be performed by (see, e.g., 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 entireties). A cleavage site can be constructed at the desired cleavage point in the gene for the fusion protein.

[0198] More than one bifunctional therapeutic agent can be expressed in a single host cell by placing multiple transcription cassettes in a single expression vector or utilizing a different selectable marker in the cloning strategy for each expression vector used.

[0199] The bifunctional therapeutic agent can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity chromatography columns, column chromatography, gel electrophoresis, etc. (see, R. Scopes, "Protein Purification", Springer-Verlag, New York (1982), Deutscher, "Enzymology Methods" Vol. 182: "Guide to Protein Purification.", Academic Press, Inc., New York City (1990), which are hereby incorporated by reference in their entirety). Substantially pure compositions of at least about 70 to 90% homogeneity are preferred, and compositions of 98 to 99% or greater homogeneity are most preferred. For example, when the targeting component of the bifunctional therapeutic agent 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 MacroPrep High Q DEAE Sepharose Fast and Macro-Prep Types of cation exchange chromatography include, but are not limited to, SP Sepharose Fast Source CM Sepharose Fast Macro-Prep CM and Macro-PrepHigh S

[0200] To facilitate purification of the bifunctional therapeutic agents of the present disclosure, the nucleic acid encoding the bifunctional therapeutic agent may also include a coding sequence for an epitope or "tag" that can be used to obtain an affinity binding agent, i.e., a purification tag. Examples of suitable epitopes include myc and V-5 reporter genes; expression vectors that can be used to recombinantly produce fusion proteins with 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). Other expression vectors and corresponding detection systems suitable for linking tags to the bifunctional therapeutic agents of the present disclosure are known to those skilled in the art, and some are commercially available (e.g., the "FLAG" tag (Kodak, Rochester NY). Another example of a suitable tag is a polyhistidine sequence that is capable of binding to a metal chelating affinity ligand. Typically, six consecutive histidines are used, although more or less than six histidines may be used. Suitable metal chelating affinity ligands that can be used as a polyhistidine tag binding portion include nitrilo-tri-acetic acid (NTA) (Hochuli, E. (1990) "Purification of recombinant proteins with metal chelating adsorbents" in Genetic Engineering: Principles and Methods, JK Setlow, ed., Plenum Press, New York New York, which is hereby incorporated by reference in its entirety; commercially available from Qiagen (Santa Clara, CA).

[0201] Purification tags also include maltose binding domains and starch binding domains. Methods for purifying maltose binding domain proteins are known to those skilled in the art. Starch binding domains are described in WO 99 / 15636, which is hereby incorporated by reference in its entirety.

[0202] Example

[0203] The following examples are intended to illustrate the practice of embodiments of the present disclosure, but are in no way intended to limit the scope thereof.

[0204] Materials and methods

[0205] Cell lines: 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. Breast cancer cell line MDA-MB-361 was a gift from Christel Larbouret (Montpellier Cancer Institute (France)). LNCaP, PC3 and CWR22Rv1 were maintained in RPMI1640 medium supplemented with 2 mM / L-glutamine, 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum (FBS) (all supplements were purchased 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.

[0206] Antibodies: Murine and deimmunized monoclonal antibodies (mAbs) J591 anti-FOLH1 / PSMA were generated as described in Liu et al., "Monoclonal Antibodies to the Extracellular Domain of Prostate Specific Membrane Antigen Also React With Tumor Vascular Endothelium," Cancer Research 57:3629-3634 (1997), U.S. Pat. No. 7,045,605 to Bander et al., and U.S. Pat. No. 7,514,078 to Bander et al., which are hereby incorporated by reference in their entireties. 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 and anti-B antibodies were purchased from Ortho Diagnostic Systems (Raritan, NJ). Ulex oleraceus agglutinin, which recognizes α-linked fucosyl residues, for detection of O / H antigens, 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 from Sigma-Aldrich. IRDye 800CW-goat anti-mouse secondary antibody was purchased from LI-COR Biosciences (Lincoln, NE).

[0207] DNA Plasmids used in this study: A series of plasmids can be constructed for any desired targeting Ab or Ab construct, or peptide plus any glycosyltransferase (including but not limited to GTB, GTA, and fucosyltransferase (FUT1 or FUT2), as outlined in the Examples below.

[0208] The DNA plasmids and their protein products that are overexpressed in host cells after transfection or co-transfection are listed below and briefly described. Each plasmid is described as: Name of DNA plasmid (its protein product): Brief description. pMG 145 (H chain): Transfection of this plasmid into host cells will produce huJ591 heavy chain. pMG 135 (L chain): Transfection of this plasmid will produce huJ591 light chain (L). pMG 145 and pMG 135 (huJ591 antibody): Co-transfection of these two plasmids will result in co-expression of heavy and light chains, thereby forming functional huJ591 antibody. pMG 181 (H chain-GTB): Transfection of this plasmid will produce a fusion protein with huJ591 heavy chain (H) at the N-terminus and GTB at the C-terminus (see below). pMG 181 and pMG 135 (huJ591-GTB fusion antibody): Co-transfection of these two plasmids will produce heavy and light chains containing GTB.

[0209] Construction of GTB and huJ591 or 4D5 heavy chain-GTB (H-GTB) fusion expression plasmids: The α1,3 galactosyltransferase (GTB) region containing the catalytic domain (amino acids 57-354) was subcloned by PCR using the GTB-encoding plasmid pBBBB as a template. If desired, a Flag tag and a His tag can be added to the 3' end to track or aid in the purification of the fusion protein. 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 generate the plasmid pMG181. The same steps were also followed to generate 4D5-GTB or any Ab (or Ab derivative or peptide)-GTB (or the catalytic domain of GTA for A antigen). Alternatively, in the case of desired targeted synthesis of the H antigen, the catalytic domain of FUT1 or FUT2 can be incorporated. Glycosyltransferases are preferentially attached to the C-terminus of the heavy or light chain of the Ab construct.

[0210] Between the Ab sequence and the enzyme sequence, (G 4 S) 3 Spacer sequence. Alternatively, various fusion protein linkers or spacers may be used, as described by Chen et al., "Fusion Protein Linkers: Property, Design and Functionality", Advanced Drug Delivery Reviews 65(10): 1357-69 (2013), which is hereby incorporated by reference in its entirety.

[0211] DNA transfection and fusion protein expression: For huJ591-GTB (and similarly for 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. Supernatants containing fusion antibodies were collected 5 days after transfection and concentrated through Amicon Ultra 10K centrifugal filters (Merck Millipore).

[0212] Purification of 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, supernatants containing fusion proteins were incubated with M2 affinity gel for 2 hours, followed by washing, elution with 3xFLAG peptide (Sigma-Aldrich), and dialyzed against PBS.

[0213] Western blot analysis: Supernatants containing fusion proteins or purified fractions were separated by 4-20% SDS-PAGE gels (Life Technology) under reducing and non-reducing conditions and transferred to polyvinylidene difluoride membranes (PVDF) (Millipore, Billerica, MA). The membranes were blocked with 5% nonfat dry milk / PBST for 60 minutes. Anti-flag M2 antibody was incubated with the membrane for 60 minutes. After washing, IRDye 800CW-goat anti-mouse secondary antibody was incubated with the membrane for 60 minutes. After washing again, the membranes were analyzed using the Odyssey infrared imaging system (LI-COR Biosciences).

[0214] Immunostaining: Before the experiment, cells (2×10 512-well plates (cells / well) were grown on coverslips in 12-well plates for 24 hours. The cells were fixed with PBS containing 4% paraformaldehyde (PFA) followed by 3 PBS washes. For the detection of human tissue blood group antigens, mouse monoclonal anti-A or anti-B antibodies were added at room temperature (RT) for 60 minutes. After washing with PBS, the cells were incubated with FITC-conjugated donkey anti-mouse immunoglobulin for 60 minutes and washed with PBS. The expression of HBGA O was detected by incubating the cells with FITC-conjugated oleander agglutinin for 60 minutes at RT and then observing under a UV microscope. For the detection of PSMA, huJ591 was added at RT for 60 minutes. After PBS washing, the cells were stained with FITC-conjugated anti-human Ig for 60 minutes and washed with PBS. Coverslips were mounted and examined under a UV microscope.

[0215] For immunostaining of tissue sections of xenograft tumors, monoclonal antibody 3E6 was used to detect PSMA expression in paraffin sections, and huJ591 was used to detect the expression of the antigen in frozen sections. The antibodies used to detect blood group antigens were the same as above. Paraffin sections were dewaxed by placing the slides in Histo-Clear, then rehydrated in different concentration gradients of ethanol, and washed in Tris buffered saline containing Tween 20 (TBST). The dewaxed and rehydrated sections were placed in an antigen retrieval solution (purchased from Dako) at pH 9.0 and heated in a water bath (95-99°C) for 30 minutes. The sections were washed with TBST. Peroxidase blocker was added for 5 minutes. After TBST washing, mAb was added at RT for 60 minutes. Antibody binding was detected using peroxidase-labeled polymer-conjugated goat anti-mouse Ig and 3,3'-diaminobenzidine (DAB) substrate. The sections were observed after counterstaining with 10% hematoxylin. Cryosections were used to detect J591-GTB fusion antibodies that bind to cell surface PSMA in vivo. Cryosections were fixed with pre-cooled acetone for 10 minutes and then washed with PBS. Peroxidase blocker was added for 5 minutes. After washing with PBS, J591-GTB was detected with horseradish peroxidase-conjugated rabbit anti-human IgG, followed by DAB color development and counterstaining as described above. Sections incubated directly with huJ591 were used as positive controls.

[0216] Competition ELISA: Plates were coated overnight at 4°C with 7E11 antibody (an antibody that binds to the N-terminal / cytoplasmic domain of PSMA) at a concentration of 15 μg / ml in 0.05 M carbonate buffer (pH 9.5). Wells were blocked with PBS containing 2% HSA for 30 minutes at RT and washed. LNCaP cell lysate (containing PSMA) diluted 1:8 was added at RT for 60 minutes. After washing with PBS, a dilution of murine J591 antibody (30 μl) was added for 60 minutes and then co-incubated with supernatant containing J591-GTB or huJ591 (1.6 μg Ig / ml; 30 μl) at 4°C overnight. After washing, donkey anti-human IgG-alkaline phosphatase (1:1,000) was added at RT for 60 minutes. After washing, the plate was incubated with pNPP (purchased from Sigma) and the data were read at 405 nm.

[0217] In vitro blood group antigen conversion: cells (2×10 5 ) were grown on coverslips in 12-well plates for 24 hours. The coverslips were washed with PBS and transferred to a wet chamber. The cells were then incubated with huJ591-GTB (or 4D5-GTB) fusion antibodies and UDP-galactose at 37°C for 30 minutes. After washing with PBS, the cells were fixed with PFA. The conversion of B antigens on the cell surface was detected by immunostaining as described above.

[0218] Lytic activity of normal human type O or type A serum after in vitro conversion of cancer cells to HBGA B: LNCaP cells were seeded in 60-well microtiter plates. Cells were incubated with native J591 or J591-GTB fusion protein, or without either agent; UDP-gal was added to all wells. Subsequently, serum from type A or type O patients was added as a source of native anti-B Ab and complement; control wells were added with J591 without GTB or without serum. After 3 hours, wells were washed, fixed with methanol, and incubated with 2% Giemsa stain for 25 minutes before washing and reading. Similar methods were used to test more prostate and breast cancer cell lines in suspension. Lytic activity was assessed by trypan blue exclusion and propidium iodide uptake detected by FACS.

[0219] In vivo blood group antigen switching: 6-8 week old NOD SCID mice (Charles River, Wilmington, MA) were injected subcutaneously with 5 × 10 IgG suspended in 200 μl of Matrigel (Corning Life Sciences, Bedford, MD) according to a protocol approved by the Institutional Animal Care and Use Committee (IUCUC). 6 cells. Cell lines LNCaP, CWR22Rv1, PC3 and MDA-MB-361 were used for animal experiments. After 14 to 21 days, the diameter of the formed tumors reached 8 to 10 mm. HuJ591, huJ591-GTB, 4D5 or 4D5-GTB were injected intravenously (IV) or intratumorally. UDP-gal was injected by IV, intraperitoneal (IP) or subcutaneous (SQ). Mice were euthanized on the 1st, 2nd or 3rd day, and tumors and other organs were collected. Half of each tumor was treated with OCT compound to prepare frozen sections; the other half was placed in phosphate-buffered formalin for preparation of paraffin sections. Immunostaining was described above.

[0220] An intraperitoneal xenograft model has also been established in NOD / SCID mice using the castration-resistant human PC cell line C4-2-luciferase. In this model, human plasma can be injected IP to provide natural Abs and complement without the fluid overload that would result if the IV route were used. 6 After several days of inoculation of C4-2-Luc cells, and after confirmation of tumor uptake by bioluminescent imaging, two groups of five animals with similar median / range bioluminescent photon flux were treated with a single IP injection of 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 per animal was measured every 3-4 days for approximately 2 weeks.

[0221] Example 1 - Production of Antibody-Glycosyltransferase Fusion Proteins

[0222] First, a chimeric protein consisting of a tumor-targeting Ab and a glycosyltransferase was generated, which is a prototype construct that provides a highly versatile modular system with multiple functions: (1) The Ab specificity is interchangeable to allow targeting of different tumor-associated antigens. 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 proteins known in the art. The targeting portion of the structure can vary from intact (full-length dimer) to monomeric single-chain Ab structures, Fab, Fab'2, scFv or other Ab fragment derivatives (such as minibodies, diabodies, triabodies, etc.). They may retain or lack the FcRn binding domain. Alternatively, the targeting moiety may be a peptide that binds to the targeting antigen; examples include, but are not limited to, glutamate-urea-lysine derivatives such as ACUPA (2-(3-((S)-5-amino-1-carboxypentyl)ureido)glutaric acid) that binds to FOLH1 / PSMA, somatostatin derivatives that bind to SSTR2, Arg-Gly-Asp (RGD) peptides that bind to α-v / β-3 integrins expressed on proliferating endothelial cells, and other targeting peptides known in the art. The variety of these targeting agents and their different physical properties allow for the adjustment of different pharmacokinetics and biodistribution. For example, larger molecular constructs such as full-length intact antibodies containing an FcRn (neonatal receptor) binding site will have longer plasma and systemic half-lives and tend to remain in the circulation; they are more likely to be excreted through the liver rather than the kidneys; and are less likely to penetrate into normal tissues due to being between normal cell layers and tight junctions. In contrast, smaller constructs lacking FcRn binding prepared with targeting peptides rather than antibodies will tend to have shorter half-lives, be more likely to be excreted via the kidneys / urinary tract, and more easily penetrate normal tissues and tumors. In addition to the specificity of target binding, these different physical properties, PK, and biodistribution profiles will affect the adverse event profile of the constructed agent. (2) The glycosyltransferase component can vary based on a large amount of knowledge about naturally occurring allelic variants and their corresponding properties that can be used to adjust its function. It may also include an α-galactosyltransferase that produces a highly immunogenic α-galactosyl epitope that does not naturally occur in humans. It is possible to use any enzyme that participates in post-translational modification. In addition to glycosylation, other embodiments include phosphorylation and lipidation.

[0223] As an additional alternative to producing a genetically engineered fusion protein, the linkage of the targeting agent and the post-translational modifying enzyme can be achieved by using a chemical bond of the two separate moieties. Such chemical linkages are known to those skilled in the art.

[0224] For initial proof of concept, three well-characterized and clinically validated Abs were selected: J591 (anti-FOLH1 / PSMA (folate hydrolase-1 / prostate-specific membrane antigen), 4D5 (trastuzumab; anti-her2), and obexelimab (anti-CD19); four Ab structures: intact dimer, intact monomer, Fab, and scFv, and four glycosyltransferase variants: α1,3 galactosyltransferase (GTB; AF134414), α1-3-N-acetylgalactosaminyltransferase (GTA; AF134415), α-1,3-galactosyltransferase (α-1,3-galactosyl T or α-galactosyl T; EC 2.4.1.87), as well as the novel structures described below. GTB transfers a galactose moiety from the nucleotide donor UDP-gal to the acceptor H antigen via an α1,3 linkage to form Galα(1–3)[Fucα(1–2)]Galβ1,4GlcNAc-R (HBGA B); GTB activity requires an α1–2-linked fucose modification on the H antigen because the B transferase will not add to the unmodified type 2 precursor. α-1,3-GalT transfers a galactose moiety from the nucleotide donor UDP-gal to Galβ1,4GlcNAc-R via an α1,3 linkage; this enzyme activity does not require an α1–2-linked fucose modification on the H antigen. GTB was chosen because HBGA type O and A individuals account for 85–90% of the population (Galili et al., “A Unique Natural Human IgG Antibody With Anti-Alpha-Galactosyl Specificity”). Specificity), "Journal of Experimental Medicine 160: 1519-1531 (1984), which is hereby incorporated by reference in its entirety), and as previously described, these individuals have high levels of anti-HBGA B antibodies. α-1,3-GalT was chosen because it can add terminal Gal to cells that do not form H-antigen, such as those derived from hematopoietic cells or mesenchymal cells.Based on essentially identical structures, the selection of GTB further benefited from the high level of polyclonal anti-gal activity (responsible for hyperacute rejection of xenografts) as a result of cross-reactivity with HBGA B (Macher et al., "The Gal alpha1,3Gal beta1,4GlcNAc-R(alpha-Gal) Epitope: a Carbohydrate of Unique Evolution and Clinical Relevance," Biochimica et Biophysica Sinica 1780:75-88 (2008), which is hereby incorporated by reference in its entirety). From the GTB (or GTA, α-galactosyl T or FUT) sequence, short cytoplasmic, transmembrane and stem regions that are not essential for enzyme activity are partially removed and replaced with corresponding antibody (or derivative) or peptide sequences to generate chimeric proteins whose membrane binding can be reconstructed by binding of the antibody or peptide domain to its cognate antigen located on the plasma membrane. ELISA assays of chimeric proteins confirm that the corresponding Ab binding specificity and immunoreactivity remain intact regardless of whether the complete or antibody fragment is used (. Figure 2A-2B ). For example, 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", Journal of Biological Chemistry 271: 10515-10520 (1996), which is hereby incorporated by reference in its entirety. Figure 2A-2B ), the incorporation of 14C-gal from UDP-14C-gal into a synthetic substrate, fucosyl-lactose (FL), was also measured, confirming the maintenance of high GTB activity.

[0225] In addition to producing such fusion proteins by genetic engineering, one skilled in the art can chemically link targeting proteins, peptides or other biological substances to effector enzymes (eg, glycosyltransferases) that can post-translationally modify cellular proteins.

[0226] Example 2 - Modulation of the function of glycosyltransferase activity

[0227] A deep understanding of the A, B and O alleles provides ample opportunity to further refine the function of this component. For example, among the allele variants available, a so-called "cis A, B" sequence can be selected, in which the two most critical amino acid residues (aa 266 and 268 of GTA (leu and gly) and GTB (meth and ala)) are exchanged, resulting in 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 two specific antigens of HBGA A and B.

[0228] It is known that other sequences can modulate the activity of the enzyme, thereby allowing one to modulate its potency. For example, a completely new type of GTB was developed based on two naturally occurring mutant alleles of GTA, named Ae101 and A201, respectively. Ae101 has a single base insertion and A201 has a single base deletion; both result in frameshifts. The transferases produced by the frameshifts produce extensions of 37 and 21 amino acids, respectively, at their C-termini. The enzymatic activity of these transferases that produce the extensions is reduced by 30-50 times or more (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). Although these two mutant alleles were determined in the context of GTA, there is no description of such mutant alleles in the case of GTB. However, by inserting amino acid sequences of varying lengths before the stop codon, a C-terminal sequence extension was directly incorporated into GTB, resulting in completely novel sequences. When four versions of GTB incorporating extensions of 2, 7, 14, and 54 amino acids were tested, GTB activity was progressively reduced, with a maximum reduction of 93% ( Figure 3), which provides a mechanism by which one can adjust to the desired activity level by incorporating a cleavable sequence (which removes the extension in the presence of tumor or tissue-associated endoproteinases or endopeptidases (such as PSA, metalloproteinases, etc.) as described above to achieve a switch-like function. The choice of the optional extension sequence is determined by the practitioner, and the only requirement is that it is selected to achieve the desired level of enzyme activity, which can be measured as described below, and that it is non-immunogenic. Non-immunogenicity can be achieved by using sequence information of a naturally non-immunogenic protein (e.g., albumin), or immunogenicity can be obtained or determined by methods known in the art, such as by eliminating T cell binding motifs.

[0229] Example 3 - In vitro induction of HBGA B alloantigen expression

[0230] To demonstrate construct functionality, human prostate cancer cell lines LNCaP (PSMA-high), CWR22Rv1 (PSMA-heterogeneous and low), and PC-3 (PSMA-negative), all of which are native HBGA O, were incubated with chimeric J591 (anti-FOLH1 / PSMA)-GTB or J591 (no GTB), both with UDP-gal, in vitro and on xenograft tissue sections derived from SCID mice. Cell lines and tissue sections incubated with chimeric J591-GTB+UDP-gal transformed to HBGA B, while those incubated with J591 (no GTB)+UDP-gal did not transform, demonstrating that GTB is required for transformation ( Figure 4 ).

[0231] In vitro, although J591-GTB was able to convert LNCaP (PSMA-high) cells from HBGA O to HBGA B, PC3 (PSMA-negative) cells were not converted ( Figure 5 ). The high specificity of HBGA transformation was confirmed by testing PC3 cells transfected with PSMA (PC3-PSMA). Among these cells heterologously expressing PSMA, only those PSMA-positive cells were transformed; adjacent PSMA-negative cells were not transformed (Figure 6).

[0232] HBGA O LNCaP cells were also incubated with type O whole blood plus UDP-Gal, J591, J591-GTB, and J591-GTB with a 54 amino acid extension. Figure 7 As shown, while J591 did not transform any cells, J591-GTB, with or without extension, transformed LNCaP cells, but not RBCs, from the O type to the HBGA B type.

[0233] Example 4 - Lytic activity of normal human type O or type A serum after cancer cells are converted into HBGA B

[0234] The in vitro assay was used to test the ability of normal human type O and type A sera to lyse prostate or breast cancer cells after they had been transformed to express HBGA B. Figures 8A-8D It is shown that when LNCaP cells (HBGA O) are incubated with J591-GTB+UDP-gal and human A (or O) serum as a source of anti-B antibodies and complement components, the cells are lysed. If no human A or O serum is added, and / or J591 without GTB is used instead of J591-GTB, no lysis will result.

[0235] Trypan blue exclusion method ( Fig. 9 ) and propidium iodide uptake by FACS analysis (Figure 10), more prostate cancer cell lines (all HBGA O) were tested. Four of these cell lines (LNCaP, VCaP, MDA-PCa-2b, and CWR22Rv1) expressed different levels (high to low) of PSMA, and all were lysed when they were incubated with J591-GTB + human O or A type serum containing natural anti-B Ab plus endogenous complement. The fifth cell line, PC3, which is PSMA negative, did not transform and was not lysed ( Figures 11A-11B Similar results were obtained after the breast cancer cell line MDA-MB-361 was transformed with the chimeric agent mAb4D5-GTB.

[0236] Example 5 - Conversion of HBGA expression in vivo

[0237] Since the rapid rejection and destruction of HBGA-mismatched solid organ transplants is a well-documented and widely recognized phenomenon in humans since the early days of renal allotransplantation (T. Starzl, Experience In Renal Transplantation. WB Saunders Company, Philadelphia, PA, Chapter 6 (1964); L. Altman, Doctors Discuss Transplantation Errors The New York Times (2003), which are hereby incorporated by reference in their entirety), key in vivo experiments were designed to demonstrate that HBGAs of established human cancers can be converted into highly immunogenic HBGAs by "molecular transplantation" of allogeneic glycosyltransferases that normally function in the Golgi / ER onto the plasma membrane of tumor cells using a systemically administered tumor targeting approach. To prove this concept, two clinically defined tumor-associated antigens (FOLH1 / PSMA and HER2) derived from the two most common solid tumors (prostate cancer and breast cancer) were selected, respectively. A variety of tumor cell lines expressing different levels of target antigens were tested. PSMA-positive prostate cancer LNCaP, C4-2 and CWR22Rv1 and her2-positive breast cancer MDA-MB361 were inoculated into the subcutaneous site of NOD SCID mice. J591-GTB or 4D5 / trastuzumab-GTB was administered by IV route; UDP-gal was administered by IV, IP or subcutaneous injection (SQ) route. J591-GTB and 4D5 / trastuzumab-GTB transformed PSMA-positive prostate cancer and her2-positive breast cancer, respectively (Figures 10A-10H, also see Figures 12A-12E ).

[0238] HBGA B conversion was poor after IP administration of UDP-gal relative to IV or SQ administration. HBGA expression was evident at the plasma membrane. As expected, replacement of Ab-GTB with the corresponding Ab alone did not result in expression of HBGA B. No conversion was detected in any other tissues, and the experimental animals did not show any signs of toxicity.

[0239] Example 6 - In vivo antitumor activity

[0240] Since both mice and rats express the cis A, B alleles as well as the α1,3GalT allele, there are several obstacles to testing the antitumor activity resulting from Ab-GTB-directed transformation of HBGA expression in animal models. Therefore, these rodent models are all HBGA A- and B-positive and α1,3gal-positive, and are therefore tolerant to all of these glycostructures. In addition, the complement system of mice is abnormally weak or even inactive (Bergman et al., Cancer Immunol. Immunother. (2000) 49:259-266; 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". In some cases, it may even inhibit complement activity in other species, including humans (Ratelade et al., "Inhibitors(s) of the Classical Complement Pathway in Mouse Serum Limit the Utility of Mice as Experimental Models of Neuromyelitis optica", Mol. Immunol. 62:104-113 (2014), which is hereby incorporated by reference in its entirety). In fact, the complement activity of normal human plasma was measured in the presence of C57BL / 6 plasma, and it was found that the lytic activity of human complement was reduced by approximately 33%. To overcome the lack of natural Ab and the weak or even inhibitory complement system in these animal models, it is necessary to almost completely replace the animal's plasma with human type O or type A plasma to provide the necessary natural Ab and functional complement proteins.However, such plasma replacement is impractical in practice, causes fluid overload, does not provide the appropriate immunoglobulin biodistribution balance that reflects human homeostasis, and is affected by the inhibitory effect of the mouse's own plasma. These issues were overcome by establishing an intraperitoneal xenograft model in NOD / SCID mice using the castration-resistant human PC cell line C4-2-luciferase, in which human plasma can be injected IP to provide natural Ab and complement without causing fluid overload. 10 × 10 were injected IP. 6 After several days of inoculation with C4-2-Luc cells and after confirmation of tumor uptake by bioluminescent imaging, two groups of five animals with similar median / range bioluminescent photon flux were treated with a single IP injection of J591-GTB, UDP-gal, and human type O serum. For the control group, the type O serum was heat-inactivated prior to injection. The total flux was measured for each animal every 3-4 days for approximately 2 weeks. Although animals receiving heat-inactivated serum experienced significant tumor progression on day 13, those treated with serum containing active complement had an 80% regression of flux relative to the control group (p=0.0032; p=0.0034). Figures 13A-13B Repeated experiments yielded consistent results ( Figures 14A-14B ).

[0241] Discussion of Examples 1 to 6

[0242] The immune response of the body to cancer is completely different from the response to incompatible allografts. As described herein, the present application proposes a strategy to selectively modify tumor cells to express non-autologous, highly immunogenic phenotypes, i.e., to express incompatible HBGA strategies. Starzl found that human allografts incompatible with HBGA were rapidly destroyed in early renal allograft work (T. Starzl, WB Saunders Company, Philadelphia, Pennsylvania, "Renal Transplantation Experience", Chapter 6 (1964), which is hereby incorporated by reference in its entirety), which makes HBGA compatibility testing an indispensable and important part of donor-recipient matching. Only in the case of very few errors and violations of HBGA compatibility requirements, people will once again experience and strengthen this lesson (L. Altman, Doctors Discuss Transplantation Errors "New York Times" (2003), which is hereby incorporated by reference in its entirety).

[0243] To implement the strategy, clinically validated tumor-restricted antibodies (e.g., anti-FOLH1 / PSMA and anti-HER2) were fused to GTB to generate a single bifunctional protein. Targeted glycosyltransferases (GTs) were similarly constructed using various antibody fragments, peptides / ligands, and constructs. While the consequences of incompatible ABO allografts in humans are well established, the challenge of this work is to "transplant" post-translational glycosyltransferase functional molecules that normally reside in the Golgi apparatus to the surface of tumor (or neovascular endothelial) cells, and to do so in a systemic, tumor-targeted manner.

[0244] These chimeric proteins have successfully changed the HBGA of various cancer cell lines in vitro and in vivo. In animal experiments, no off-target HBGA conversion or toxicity was found. As shown herein, HBGA incompatible cells trigger complement-mediated lysis, as has been confirmed many times in clinical transplant settings, which is a response that is expected to occur in cancer patients (L.Altman, doctors discuss transplant errors "The New York Times" (2003); T.Starzl, "Kidney Transplantation Experience" WB Sanders Company of Philadelphia, Pennsylvania, Chapter 6 (1964), the document is hereby incorporated by reference as a whole).

[0245] The biosynthesis of new HBGA requires the presence of both GT and (fucosylated) H antigen "receptor structures" on target cell glycoproteins and glycolipids (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) in order to add HBGA. Because many cancers express H antigens, including lung cancer, gastric cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, bladder cancer, pancreatic cancer, etc., these tumor types are suitable for this strategy. Normal non-target cells do not undergo HBGA conversion for the following reasons: (1) lack of binding of the targeted GTB (or GTA) enzyme, and (2) many normal cell types (e.g., bone marrow, liver, spleen, kidney, myocardium, central and peripheral nervous system, etc.) lack the required H Ag, which precludes GTB (or GTA) transferase activity in these sites. FOLH1 / PSMA is reported to be expressed in tumor neovasculature of a variety of tumors, but not in normal tissue vessels. Examples of tumor types with FOLH1 / PSMA positivity in tumor neovasculature 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, melanomas, and brain tumors. FOLH1 positivity is relatively rare in testicular tumors, lymphoid and sarcomas, but occasionally exists. Targeting FOLH1 / PSMA expression in tumor neovasculature provides a means for altering HBGA expression within the vascular bed of a variety of tumors. This in turn can lead to hyperacute rejection phenomena similar to those seen in mismatched solid tissue allografts of HBGA (L. Altman, Physicians Discuss Transplant Errors, New York Times (2003); T. Starzl, The Kidney Transplant Experience, WB Saunders & Co., Philadelphia, PA, Chapter 6 (1964), which are hereby incorporated by reference in their entireties).

[0246] The enzymatic nature of the reaction creates an amplification effect, as each targeted enzyme molecule is able to convert many receptor molecules. Furthermore, not only is the Ab-targeted tumor-associated antigen itself enzymatically converted, but so are all neighboring molecules within the enzyme’s range of action. And since most cell surface molecules have multiple glycosylation sites—for example, FOLH1 / PSMA has 10 glycosylation sites (20 if one considers that FOLH1 / PSMA is typically expressed as a homodimer)—the number of non-self HBGA sites that can be generated by this approach is considerable. In addition, glycoproteins secreted by targeted neovascularization or tumor cells can also undergo HBGA conversion, leading to complement activation in the tumor microenvironment, which in turn enhances the immune environment surrounding the tumor.

[0247] These factors—enzyme amplification, conversion of the directly targeted molecule as well as neighboring molecules and secreted glycoproteins, and the multiplication effect of abundant glycosylation sites—should result in unprecedented high levels of immunogenic antigen expression in tumor cells and the tumor microenvironment, even in the presence of weak expression of tumor-associated antigen targets. Because HBGA O and A patients comprise approximately 85% of the total population, GTB was utilized in the proof-of-concept work. Furthermore, since the polyclonal anti-gal activity that impedes xenografting 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," Acta Biochimica et Biophysica Sinica 1780:75-88 (2008), which is hereby incorporated by reference in its entirety), tumors and / or their blood supply induce production of HBGA B, making it an unprecedented target for complement-fixing antibodies that can mediate high levels of inflammation and hyperacute rejection. This strategy can be expanded to cover HBGA O, A, and B patients (approximately 95% of the population) by using GTs with both A and B activity. This can be achieved by a single nucleotide / amino acid change in GTA (803G>C (Gly268Ala), a naturally occurring so-called cis-AB GT mutation that produces HBGA A and B. This approach works for all but AB patients (approximately 5% of the population) who have neither natural anti-A nor natural anti-B antibodies.

[0248] 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, the methods of the present invention utilize the lysis mechanism of the complement cascade. In addition to direct lysis, triggering the complement cascade in the tumor microenvironment also serves as a bridge to enhance the efficacy of cellular immune responses because C3 can activate APCs (Baudino et al., “C3 Opsonization Regulates Endocytic Handling of Apoptotic Cells Resulting in Enhanced T-cell Responses to Cargo-Derived Antigens”, Proceedings of the National Academy of Sciences of the United States of America 111:1503–1508 (2014); Surace et al., “Complement is a Central Mediator of Radiotherapy-Induced Tumor-Specific Immunity and Clinical Responses”. Response", Immunity 42:767–777 (2015), which is hereby incorporated by reference in its entirety), thereby promoting T cell priming (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 hereby incorporated by reference in its entirety).In addition, recent studies have shown that the release of free C3d (a fragment of C3) can deplete Tregs through apoptosis, increase the infiltration of CD8+T cells that produce perforin, TNF-α and IFN-γ, and reduce PD-1 expression of T cells (Platt et al., "C3d Regulates Immune Checkpoint Blockade and Enhances Antitumor Immunity", Journal of Clinical Investigation Insight (JCI Insight.) 2: e90201 (2017), which is hereby incorporated by reference in its entirety). In addition, activation of the complement system produces chemokines such as C3a and C5a, which can induce inflammation and recruit inflammatory cells. This will convert the 'cold' tumor microenvironment into a 'hot' tumor microenvironment, thereby further assisting the immune response. In summary, the methods described herein expand the potential for the breadth and intensity of immune attacks against cancer by directly mobilizing the humoral immune system and the complement cascade, as well as through its role in enhancing cellular immune responses.

[0249] Example 7 - B antigen conversion of MM1-S using anti-CD19-GTB

[0250] Figure 15-17 The ability to convert CD19-positive / HBGA O-positive myeloma cells to express HBGA B was shown. In this case, using antibodies targeting CD19, CD20, CD38 ( Fig.15 ), HBGA A, HBGA B (Thermo Fisher Scientific, Inc. (Ortho)), and Ulex-FITC or Ulex-Dylight (Vector Labs) mouse monoclonal antibodies to detect HBGA O ( Figures 16A-16B ), MM1-S myeloma cells that have been passaged in tissue culture were tested by fluorescence activated cell sorting (FACS). MM1-S cells were incubated with each antibody for 1 hour, then the cells were washed and incubated with the appropriate secondary antibody (such as anti-mouse IgM-Alexa488 or 647 (Jackson ImmunoResearch) when the primary antibody is IgM; when the primary antibody is labeled anti-mouse IgG, the corresponding labeled anti-mouse IgG secondary antibody is used), or when the primary antibody is washed again. After washing again, the cells were analyzed by FACS. Fig.17 As shown, MM1-S cells were incubated with anti-CD19-GTB fusion protein and UDP-gal, and the expression of HBGA B was compared with that of untreated cells by FACS.

[0251] Fig.15 It shows that MM1-S myeloma cells are CD20 negative, CD19 + and CD38 + . Figures 16A-16B MM1-S cells are shown to be HBGA A-negative and B-negative ( Fig.16A ), but HBGA O positive ( Fig. 16B ). Fig.17 It is shown that MM1-S cells incubated with anti-CD19-GTB fusion protein and UDP-gal were converted to high-level expression of HBGA B relative to untreated cells.

[0252] These experiments provide another example of targeted transformation of a tumor to express a foreign antigen: HBGA B. In this case, the target is CD19, a B cell marker also present on B cell malignancies. It also represents transformation of a blood-borne tumor type, whereas the other examples provided previously - prostate (PSMA) and breast (HER2) are examples of solid tumors.

[0253] Example 8 - Targeting glycosyltransferases via small molecule ligands as an alternative to antibodies or antibody derivatives

[0254] Fig.18 It was demonstrated that targeting of GTA or GTB or α-gal can be achieved not only by antibody-based constructs, but also by peptide / small molecule ligand-based targeting agents. In this case, the GTB enzyme was conjugated to 2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid (ACUPA), a galactose-urea-lysine-based ligand that binds to PSMA. To achieve high levels of binding, a PEG 1500 spacer was used between the ACUPA and GTB moieties. This provides sufficient steric freedom for ACUPA to bind to the enzymatic active pocket of PSMA without steric interference from the much larger GTA / GTB enzyme.

[0255] Fig.18 It is shown that ACUPA-PEG1500-GTB can convert LNCaP cells from HBGA O to HBGA B (left panel), whereas there is no conversion when pure GTB without the ACUPA moiety for targeting is used (right panel).

[0256] The flexibility to use a variety of targeting moieties, from large antibodies of 150 kd to smaller antibody-derived forms such as monomers (75 Kd), Fab'2 (100 kd), Fab (50 kd), scFv (25 kd), down to short peptides such as ACUPA (1.0 kd), enables the construction of fusion proteins with a variety of pharmacokinetic and biodistribution properties. Larger fusion proteins will circulate longer, tend to stay in the blood compartment longer, and be excreted through the liver, while smaller constructs tend to have shorter serum half-lives, reach / contact tumor targets faster, and are excreted through the kidneys. These different options can be used to tailor therapies to the needs of different tumor types (e.g., hematological tumors vs. solid tumors).

[0257] Example 9 - Specificity and precision of transformation; absence of bystander effect

[0258] like Fig.19 and 20 As shown, 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 by their unique morphology: SK-BR5 is round and Fig.19 Two clusters can be seen near the center and top (red circle) of the mid-field. LNCaP are more elongated, and these cells also express GFP as an identification marker.

[0259] When cultures were treated with J591-GTB+UDP-gal, HBGA B (stained with Cy5 (purple)) appeared only on PSMA-positive cells. Despite being in close proximity to transformed cells, clusters of PSMA-negative SKBR5 cells (highlighted in red circles) were not converted to HBGA B. Similarly, Fig. 20 Shown are cell types that are equally distinguishable. The left panel shows all nuclei stained with DAPI. The middle panel shows spindle-shaped LNCaP cells that have green fluorescence due to the expression of GFP. The right panel shows that after treatment with J591-GTB+UDP-gal, HBGA B is expressed only by PSMA-positive LNCaP cells, while PSMA-negative SK-BR5 cells remain HBGA B-negative.

[0260] This demonstrated both the high specificity of transformation and the absence of a bystander effect—unless cells were directly targeted and bound to the fusion protein, even neighboring cells would not be transformed.

[0261] Example 10 - Quantitative Specificity Index

[0262] Figures 21A-21Band 22 quantified the specificity index of the same 2 PSMA positive and negative cell lines. Different concentrations of anti-PSMA-GTB antibody (from 100 μg / mL to 0.003 μg / mL) were incubated with each cell line in the presence of the nucleotide donor UDP-gal. The specificity of conversion was quantified by FACS by comparing the concentration of J591 (anti-PSMA)-GTB required to convert LNCaP (PSMA+) to HBGA B relative to SK-BR5 (PSMA negative) cells. Both cell lines were O+. The FACS histograms are shown in Figures 21A-21B It should be noted that the data with concentrations greater than 12.5 ug / mL overlap with the curve of 12.5 ug / mL and are removed from the FACS histogram to simplify the view.

[0263] Even at concentrations as high as 100ug / mL of J591-GTB, SK-BR5 did not undergo B transformation. In contrast, anti-PSMA-GTB at concentrations as low as 0.012ug / mL began to induce transformation of PSMA-positive cells. This data is presented in the form of a histogram in Fig. 22 This indicates that the fusion protein is at least 8,196 times more specific for PSMA-positive cells than for PSMA-negative cells. This is due to the ability of the fusion protein to bind directly to PSMA-positive cells, where it aggregates on the cell surface and exerts its enzymatic function. When the enzyme is not bound to the targeted cell surface, the enzymatic reaction is much weaker or absent. For practical reasons, concentrations above 100ug / mL were not tested; in practice, the calculated specificity index is likely to be much greater than 8,000 times.

[0264] Figure 19-22 The transformation reaction was shown to be extremely specific, limited to target-positive cells, and devoid of a bystander effect, whereby even if cells were adjacent to transforming / target-positive cells, they would not be transformed if those cells were target-negative and did not bind to the fusion protein.

[0265] Example 11 - Both cell surface and secreted glycoproteins are glycosylated by this method

[0266] Since both cell surface and secreted glycoproteins are glycosylated by the same cellular processes in the Golgi / endoplasmic reticulum, in addition to HBGAs that transform cell surface molecules, glycoproteins secreted by target cells will also undergo HBGA transformation. In this exemplary embodiment, secreted glycoproteins are converted to HBGA B positive. LNCaP cells were treated with J591-GTB plus UDP-gal for 5 hours (10ug / ml anti-PSMA-GTB+100μM UDP-gal). As a negative control, another group of LNCaP cells was incubated with 10μg / ml anti-PSMA-GTB antibody, but UDP-gal was not added. As a positive control, untransformed cells were used as background controls for measurement of cell surface transformation. After 5 hours of incubation, the spent culture medium containing secreted glycoproteins was collected from each group of cells and concentrated 10 times using an Amicon with a molecular weight cutoff of 3,000 Daltons. The spent culture medium was adsorbed to the wells of an ELISA plate and the presence of HBGA B was determined by ELISA using IgM anti-HBGA B followed by anti-mouse IgM alkaline phosphatase.

[0267] Fig.23 It is shown that secreted proteins in the transformed medium were HBGA B positive relative to the negative control (spent medium without transformation).

[0268] This demonstrates that HBGA B conversion is not limited to the cell surface, but also includes glycoproteins secreted by target cells. In vivo, this suggests that these converted secreted glycoproteins will penetrate into the tumor extracellular space, bind to natural anti-B antibodies, trigger complement responses and generate a pro-inflammatory microenvironment, recruit inflammatory and immune cells through chemotaxis, and further transform the tumor microenvironment into a "hotspot" environment.

[0269] Example 12 - Analysis of the utilization of α1,3 galactosyltransferase (aGalT)

[0270] In addition to using human glycosyltransferase A or B, another embodiment utilizes α1,3 galactosyltransferase (aGalT, EC 2.4.1.87), which is active in all mammals but inactive in humans and Old World monkeys due to mutations during evolution. GTA, GTB, and α1,3GalT are highly homologous and are thought to be derived from a common ancestral gene. Like GTB, αGalT adds terminal α1,3 galactose to carbohydrate chains of cell surface and secreted glycoproteins and glycolipids, but unlike GTB, αGalT can add Gal in the absence of an H-antigen fucose acceptor structure. Because all humans lack functional αGalT and therefore lack expression of this terminal α1,3Gal epitope, humans carry high levels of anti-αGal antibodies, including IgM, IgG, IgA, and IgE types, estimated to account for approximately 1% of all circulating immunoglobulins. It is the immunogenicity of this α1,3Gal epitope that prevents xenotransplantation from other mammals that possess functional aGalT and express terminal α1,3Gal on tissues including their blood vessels.

[0271] The use of αGalT eliminates the need to select GTA or GTB based on the subject's blood type. It also allows the use of this treatment approach in patients with AB blood type who do not carry natural antibodies to HBGAA or B but carry antibodies to α1,3Gal. In the case of αGalT, since no H-antigen fucose is required as a receptor, this also expands the types of tissues that can be used. For example, hematopoietic and mesenchymal-derived cells (and tumors derived from these cell types) as well as other tissues lack expression of the H antigen receptor. These tissues / tumors cannot be treated with GTA or GTB, but can be treated with α1,3GalT.

[0272] One concern with the use of αGal transferase is whether the enzyme itself is immunogenic in humans if humans do not express a functional version of the enzyme. If this is the case, repeated administration would require humanization or deimmunization of the enzyme. This concern was assessed by assaying sera from 50 randomly selected patients of different blood types to see if any anti-αGalT antibodies were present.

[0273] The ELISA assay was performed by coating α1,3GalT (500 ng / ml) in a 96-well half-area high-binding microplate overnight at 37°C. 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 microplate for 2 hours at room temperature (RT). These serum donors included patients from 21 O type, 20 A type, 3 B type and 6 AB type. After washing, anti-α1,3GT antibodies were detected by adding anti-human IgG+IgM-Alk Phos antibody solution, followed by PNPP and reading the plate at 405nm. To ensure that α1,3GT was properly coated, anti-His-Tag antibody was used for detection because α1,3GT is labeled with a His tag (positive control). As another control, to ensure that the anti-human IgG+IgM (AP) was functional, BSA was coated at 500 ng / ml and anti-BSA antibodies from human serum (HBGA A) were measured using the same method.

[0274] The results showed that none of the 50 sera contained antibodies against α1,3GalT ( Figures 24A-24B ). It is speculated that this is due to the homology between various glycosyltransferases (including but not limited to GTA and GTB). This result suggests that the αGalT enzyme can be used in fusion proteins to generate the αGal epitope without worrying that the fusion protein will be immunogenic. Therefore, deimmunization or humanization treatment is unlikely to be required.

[0275] Example 13 - Expression and purification of recombinant α1,3 galactosyltransferase (aGalT)

[0276] An anti-CD19 scFv fused to a portion of the α1,3GalT sequence (amino acids 90-376) was constructed using a method similar to that used for GTA and GTB. The scFv sequences used were derived from Denintuzumab (Den) from Seattle Genetics and Obexelimab (Obx) from Xencor, which recognize and bind to cynomolgus monkey and human CD19. The same (G 4 S) 3 Spacer / Linker. For αGalT, the marmoset sequence was chosen to have 376 amino acid residues and to be consistent with the general topology of glycosyltransferases: a 6aa cytoplasmic domain, a 16aa transmembrane domain, and a 354aa luminal domain containing the enzymatic activity.

[0277] The stem region of marmoset α1,3GT contains 67 amino acids and covers amino acids 23-89 of the lumenal portion of the enzyme; it can be removed without affecting enzyme activity. A truncated α1,3GT at positions 90-376 is functional and was therefore chosen for the construction of the fusion protein. A His tag was added to the enzyme. The construct was expressed in Expi293F cells and purified using a metal affinity chromatography column.

[0278] SDS-PAGE electrophoresis with anti-his detection revealed that the obtained preparation of the target construct was of high purity and had a predicted molecular weight ( Fig.25 ).

[0279] Example 14 - Testing the functionality and specificity of the anti-CD19 scFv-αGalT construct

[0280] To demonstrate the functionality and specificity of the anti-CD19 scFv-αGalT construct, a hematopoietic cell target was chosen that does not express the H antigen receptor structure nor HBGA A or B: CD19 on Raji B lymphoma cells. CD19 is also a validated tumor target. In addition to functionality, specificity was assessed by comparing the amount of αGal added to CD19+ Raji cells co-incubated with CD19-negative MM1.S cells.

[0281] CD19 positive cancer cell line Raji-GFP was mixed with CD19 negative cancer cells (MM1.S) at different ratios and incubated with scfv-αGalT construct (10 μg / ml) and UDP-Gal (5 mM) for 1 hour at 37° C. The presence of α1,3Gal epitope was then assessed by flow cytometry using anti-α1,3Gal antibody; Raji-GFP cells were used to distinguish them from MM1.S cells.

[0282] The fusion protein binds to CD19-positive Raji cells and reaches saturation 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-positive Raji cells but not to CD19-negative MM1.S cells ( Fig. 27 ), even when the latter was in 30-fold excess relative to the former. Even at a high ratio of MM1.S to Raji cells, MM1.S cells did not become αGal positive in the presence of scfv-αGalT fusion protein + UDP-Gal.

[0283] α1,3GalT itself does not add the Gal moiety in the absence of the scFv binding domain, indicating that binding via the antibody (or fragment) portion of the fusion protein is necessary for the addition of α1,3GalT ( Fig.28 ). In addition, when UDP-gal was not added, no gal was added to the target cells. In the presence of UDP-gal, the α1,3Gal moiety was added, but it did not generate the HBGA B epitope, as evidenced by the lack of antibody binding to HBGA B ( Fig.28 ).

[0284] Example 15 - Ability of anti-CD19 scFv-aGalT to transform fresh human lymphocytes

[0285] Similarly, the ability of anti-CD19 scFv-aGalT to transform fresh human lymphocytes was also tested. In order to avoid biasing the results due to the presence of anti-CD19 constructs, anti-CD20 was used to identify B cells in this experiment. Cells were incubated with UDP-gal alone, Obx CD19-αGalT alone, or Obx CD19-αGalT plus UDP-gal at the indicated concentrations. Dual-channel FACS was used to measure the binding of CD19-αGalT (X axis) and the expression of the αGal epitope (Y axis) ( Fig.29 A negative control without treatment was also set up.

[0286] CD20-negative cells neither bind to the fusion protein nor transform to express α1,3Gal( Fig.29 , upper panel). CD20 positive cells ( Fig.29 , lower panel) shows binding of the fusion protein and is switched to express α1,3Gal only when UDP-gal is simultaneously added.

[0287] Example 16 - Lysis function of human sera from different blood type donors on CD19 positive Raji-GFP cells

[0288] The lytic function of human sera from different blood type donors on CD19 positive Raji-GFP cells was tested. CD19+Raji-GFP cells were incubated with Obx-αGT (10 μg / ml) with or without UDP-Gal (5 mM) at 37°C for 1 hour. Sera from different donors were then added to the cells and incubated at 37°C for 4 hours. Cell viability was assessed by measuring the GFP expression of the cells by flow cytometry.

[0289] Unless UDP-gal is provided as a nucleoside donor to complete the transformation of anti-CD19 scFv-aGalT to express the terminal α1,3Gal, only background cleavage is seen ( Fig.30 ). However, when UDP-gal was included and αgal conversion occurred, human serum lysed the transformed cells. In this experiment, type O and type A sera were found to cause greater lysis than type B or AB sera.

[0290] Example 17 - Conversion and lysis of fresh human B cells using autologous serum

[0291] The above was extended to study the transformation and lysis of fresh human B cells using autologous serum (from the same donor), and 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 (10μg / ml) and UDP-Gal (5mM) for 1 hour at 37°C. One cell sample was taken and analyzed for binding of human IgG and human IgM by FACS using anti-γ or anti-μ chain specific antibodies. Serum from the same donor was then added to another cell sample and incubated for 4 hours at 37°C. B cell depletion was measured by flow cytometry using anti-CD20 antibodies.

[0292] The highest degree of lysis of transformed cells was found in patients with genotype A and O, and this was consistent with the anti-α1,3Gal levels of individual patients ( Figures 31A-31B ). This suggests that measuring anti-αgal levels prior to treatment will help predict the extent to which a patient is likely to respond to this treatment approach. It also suggests that some patients, particularly those with type B or AB, may benefit from pre-treatment by exposure to the α1,3gal antigen to stimulate higher levels of anti-αgal antibodies. This can be achieved by subcutaneously administering an α-Gal containing polysaccharide or glycoprotein at least one week prior to this treatment approach, or alternatively, induction of the αgal epitope in an initial treatment cycle can be used to stimulate the production of anti-α-Gal antibodies in vivo for subsequent cycles. One skilled in the art can assess pre-treatment anti-α-Gal levels in a series of patients and their relationship to response, and determine a threshold below which a response is unlikely in the absence of pre-treatment.

[0293] Example 18 - Determination of the optimal concentrations of anti-CD19 scFv-aGalT and UDP-gal when lysing human donor CD19 cells using autologous serum

[0294] The optimal concentrations of anti-CD19 scFv-aGalT and UDP-gal were determined for lysis of human donor CD19 cells using autologous serum. Human PBMCs from an HBGA type A donor known to induce serum-mediated lysis of αGal-transformed cells were incubated with different concentrations of Obx-αGT and UDP-Gal for 1 hour at 37°C. Serum from the same donor was then added to the cells and incubated for 4 hours at 37°C. Binding, α1,3Gal transfer, and B cell depletion were assessed by flow cytometry.

[0295] Anti-CD19 scFv-aGalT saturated CD19 cells at a concentration of approximately 10 ug / mL ( Figures 32A-32C ). Expression of α1,3gal was optimal at a UDP-gal concentration of approximately 10 mM. B cell lysis was greatest with 10 ug / mL of anti-CD19 scFv-aGalT and a 5-20 mM range of UDP-gal. B cell lysis gradually diminished when the anti-CD19 scFv-aGalT concentration was >10 ug / mL, and especially > / =25 ug / mL (above the saturation binding point of CD19). This is likely due to unbound anti-CD19 scFv-aGalT competing for UDP-gal, thereby reducing the UDP-gal available for anti-CD19 scFv-aGalT bound to cells.

[0296] The concentrations of scFv-aGalT and UDP-gal can vary depending on the cancer target antigen, and their density and lytic effect on the tumor cell membrane can vary depending on the level of anti-α1,3Gal antibodies (IgM and / or IgG and / or IgA and / or IgE). All of these parameters can be tested before treatment, and those skilled in the art can determine the optimal concentrations of the various components for treating each individual patient.

[0297] Example 19 - Engineering of anti-CD19 scFv-αGal transferase

[0298] Obelimonab-scFv-α-1,3Gal (SEQ ID NO: 63, Table 1) was prepared by (G 4 S) 3 The linker was constructed by fusing the obelizumab single chain variable fragment (scFv) to the N-terminus of marmoset-derived α-1,3 galactosyltransferase (aa90-376) in the vH-vL orientation. A 6His tag was added to the C-terminus of the fusion protein to enable purification by affinity chromatography.

[0299] Protein preparation was performed at WuXi Biologics. Briefly, the target DNA sequence encoding obelimonab-scFv-α-1,3Gal (SEQ ID NO: 63) was codon optimized, synthesized, and subcloned into WuXi Biologics' proprietary expression vector. The fusion protein was expressed in CHO cells scaled up to 2 L by transient transfection. Obelimonab-scFv-α-1,3Gal was purified from the cell culture supernatant by a three-step column purification process. Nickel affinity chromatography was first used in the initial capture step, followed by anion exchange chromatography, and then size exclusion chromatography, and finally a protein with a purity of 95% and an endotoxin level of <1EU / mg was obtained. The purified protein was formulated into a 20mg / ml solution in histidine buffer at pH 6.0. Protein purity was evaluated by SDS-PAGE and SEC-HPLC, and endotoxin levels were tested.

[0300] Table 1. Sequences of the bifunctional therapeutic protein of obelizumab

[0301]

[0302]

[0303] Example 20 - In vivo treatment of non-human primates using anti-CD19 scFv-αGal transferase fusion protein plus UDP-Gal

[0304] Next, we investigated whether in vivo treatment of nonhuman primates with anti-CD19 scFv-αGal transferase fusion protein plus UDP-Gal would result in CD19 + Decrease in B-cell counts or toxicity.

[0305] Two cynomolgus monkeys (5 kg each) underwent baseline blood tests to confirm that each laboratory index was within acceptable range and to measure B cell and T cell counts at baseline. Next, at time 0, the cynomolgus monkeys received an intravenous injection of anti-CD19 scFv-αGal transferase (SEQ ID NO: 63), followed by an injection of UDP-gal 1 hour later.

[0306] Complete blood count, serum chemistry, liver function tests, and total lymphocyte, B-cell, and T-cell counts were performed at 1, 4, and 24 hours after treatment and on days 7, 14, 30, and 60 ( Fig.33 ). By examining CD20 + / CD3 - B cell counts were determined by fluorescence. CD20 was used to avoid interference with B cell counts due to the presence of anti-CD19 scFv.

[0307] The first monkey was treated with anti-CD19 scFv-α-Gal transferase plus UDP-Gal. On day 7 after treatment, the CD19 / CD20 + B cells decreased by 70%, and this decrease lasted for 8 weeks before returning to their baseline levels. The second monkey recently received a higher dose of anti-CD19 scFv-α-Gal transferase (3mg / kg) and had an 80% decrease in B cells when first tested 4 hours after treatment. The veterinarian observed no obvious signs of toxicity in the test monkey. The weight of the test monkey remained unchanged. No measurable signs of toxicity were found in the blood tests. For the first monkey, laboratory parameters of other blood tests remained stable during the 2 months of follow-up, except for the CD19 / CD20 counts. The second monkey also remained stable and there was no observable or laboratory-measured toxicity.

[0308] These results demonstrate that non-human primates can be safely treated by the methods disclosed herein and that the treatment results in a significant reduction in target cells.

[0309] Example 21 - Engineering of Tumor Targeting Bifunctional Therapeutic Proteins

[0310] Tumor targeting fusion proteins are constructed by fusing the H chain of a tumor targeting antibody with a glycosyltransferase gene. In the embodiments 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). The post-translational modification enzymes used in the embodiments presented herein are all naturally expressed in the Golgi apparatus and / or the endoplasmic reticulum capsule membrane. In order to construct the bifunctional fusion proteins described herein, those parts that are not necessary for the enzyme function, such as the extracapsular region, the transmembrane region, and the stem region, are omitted.

[0311] As described herein, the tumor targeting portion of the fusion protein can be a full-length ab, Fab'2, Fab, scFv, monomeric Ab, or any Ab / immunoglobulin derivative thereof. The enzyme portion of the fusion protein can be any post-translational modification enzyme; its sequence is typically human, humanized, primatized (from non-human primates), or deimmunized. The connection of the targeting portion to the enzyme may or may not have a linker / spacer. In the examples provided herein, (G 4 S) 3 (SEQ ID NO: 67) linker / spacer, but any linker / spacer known to those skilled in the art may be used.

[0312] The preparation of these fusion proteins is modular, so any tumor / tissue targeting moiety can be fused to any post-translational enzyme according to the several embodiments provided herein. Sequences for the engineering and generation of huJ591 and 4D5 bifunctional therapeutics are provided in Table 2. Sequences of bifunctional therapeutics are provided in Tables 3 and 4.

[0313] Table 2. Sequences

[0314]

[0315]

[0316]

[0317] Table 3. J591 bifunctional therapeutic protein sequences

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326] Table 4.4D5 bifunctional therapeutic protein sequences

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333] huJ591-GTB

[0334] huJ591-GTB (H chain) (SEQ ID NO: 34) was constructed by ligating the huJ591 heavy chain (SEQ ID NO: 68) to the N-terminus of human GTB (aa 57-354) (SEQ ID NO: 69). huJ591-LC (L 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.

[0335] The DNA sequences encoding the H and L chains were subcloned into the pcDNA3.1 expression vector. Protein production was performed by co-transfection of the H and L chains into CHO cells 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.

[0336] huJ591Fab-GTB

[0337] huJ591Fab-GTB (H chain) (SEQ ID NO: 37) was constructed by linking a truncated fragment of the huJ591 heavy chain (VH-CH1 - partial hinge sequence) (SEQ ID NO: 72) to the N-terminus of human GTB (aa 57-354) (SEQ ID NO: 69). A Myc / his tag (SEQ ID NO: 73) was added to the C-terminus to facilitate monitoring of expression and affinity chromatography purification. huJ591-LC (L chain) (SEQ ID NO: 39) encodes the huJ591 light chain sequence (SEQ ID NO: 71).

[0338] The DNA sequences encoding H and L chains were subcloned into pcDNA3.1 expression vectors. Protein production was performed by cotransfection of H and L chains into CHO cells using a transient expression method. Fab-GTB fusion proteins were purified from cell culture supernatants by nickel affinity chromatography and evaluated by SDS-PAGE.

[0339] huJ591-HC67-GTB

[0340] By (G 4 S) 3huJ591-HC67-GTB (H chain) (SEQ ID NO: 40) was constructed by connecting the huJ591 heavy chain (SEQ ID NO: 74) to the N-terminus of human GTB (aa57-354) (SEQ ID No: 69) with a (SEQ ID NO: 67) linker. The changed aa is marked with bold double underline in Table 3 and with bold text in Table 2. J591-LC (L chain) (SEQ ID NO: 42) was constructed by adding a 6His-tag (SEQ ID NO: 70) at the C-terminus of J591-LC (SEQ ID NO: 71) to facilitate affinity chromatography purification.

[0341] The production of monomeric Fc fusion protein was carried out as follows. DNA sequences encoding J591HC67-GTB and L chain were synthesized, subcloned into expression vectors, and then co-transfected into CHO cells. Cell culture supernatant was collected. J591HC67-GTB fusion protein was purified by nickel affinity chromatography and evaluated by SDS-PAGE.

[0342] huJ591-HC67-GTB54aa

[0343] huJ591-HC67-GTB54aa (H chain) (SEQ ID NO: 43) was modified from huJ591-HC67-GTB (H chain) (SEQ ID NO: 74) by adding a tail chain consisting of 54aa at the C-terminus of GTB (SEQ ID NO: 75). huJ591-LC (L chain) (SEQ ID NO: 45) was constructed by adding a 6His-tag (SEQ ID NO: 70) at the C-terminus to facilitate affinity chromatography purification.

[0344] Protein production was performed using a transient expression method by co-transfecting the H and L 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.

[0345] huJ591scFv-Fc67-GTB

[0346] huJ591scFv-Fc67-GTB (SEQ ID NO: 46) encodes huJ591 single-chain variable fragment (scFv) / J591 Fc fragment (SEQ ID NO: 76) and human GTB (aa 57-354) (SEQ ID NO: 69) from N-terminus to C-terminus. (G 4 S) 3(SEQ ID NO:67) linker.

[0347] The DNA sequence encoding huJ591scFv-Fc67-GTB (SEQ ID NO: 46) was synthesized, subcloned into an expression vector, and transfected into CHO cells. The cell culture supernatant was collected. The huJ591scFv-Fc67-GTB (SEQ ID NO: 46) fusion protein was purified by nickel affinity chromatography and evaluated by SDS-PAGE.

[0348] huJ591scFv-GTB

[0349] huJ591scFv-GTB (SEQ ID NO: 48) encodes the deimmunized version of huJ591 single-chain variable fragment (scFv) (SEQ ID NO: 77) and human GTB (aa 57-354) (SEQ ID NO: 69) from N-terminus to C-terminus. Between scFv (SEQ ID NO: 77) and GTB (SEQ ID NO: 69), (G 4 S) 3 (SEQ ID NO: 67) linker. A Myc / His tag (SEQ ID NO: 73) was added to the C-terminus to facilitate monitoring of expression and affinity chromatography purification.

[0350] The DNA sequence encoding huJ591scFv-GTB (SEQ ID NO: 48) was subcloned into the pcDNA3.1 expression vector. Protein production was performed by transfecting the plasmid into CHO cells 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.

[0351] GTA construct

[0352] The human GTA (aa 57-354) sequence (SEQ ID NO:78) was also used to replace the GTB sequence (SEQ ID NO:69) in the DNA constructs described above to produce the following recombinant proteins: huJ591-GTA (SEQ ID NO:35), huJ591Fab-GTA (SEQ ID NO:38), huJ591-HC67-GTA (SEQ ID NO:41), huJ591scFv-Fc67-GTA (SEQ ID NO:47) and huJ591scFv-GTA (SEQ ID NO:49).

[0353] Trastuzumab (4D5) construct

[0354] To target HER2 to breast cancer and other cancers, the trastuzumab (4D5) sequence was used in place of the huJ591 sequence in the DNA constructs described herein to produce the following recombinant proteins: 4D5-GTA (SEQ ID NO: 51), 4D5Fab-GTA (SEQ ID NO: 54), 4D5HC67-GTA (SEQ ID NO: 57), 4D5scFv-Fc67-GTA (SEQ ID NO: 60), 4D5scFv-GTA (SEQ ID NO: 62), 4D5-GTB (SEQ ID NO: 50), 4D5Fab-GTB (SEQ ID NO: 53), 4D5HC67-GTB (SEQ ID NO: 56), 4D5scFv-Fc67-GTB (SEQ ID NO: 59), and 4D5scFv-GTB (SEQ ID NO: 61).

[0355] While preferred embodiments have been depicted and described herein in detail, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like are possible without departing from the spirit of the present disclosure, and are therefore considered to be within the scope of the present disclosure as defined in the following claims.

Claims

1. A dual-function therapeutic agent for treating cancer, the dual-function therapeutic agent comprising: a targeting component that targets a tumor-associated antigen; and an enzyme that, when delivered to a tumor via the targeting component, enzymatically converts the tumor phenotype to a phenotype of an incompatible allograft or xenograft, the enzyme being coupled to the targeting component; The targeting component is a TCR, a single domain antibody or 2. The bifunctional therapeutic agent according to claim 1, wherein the tumor-associated antigen is FOLH1 / PSMA, VEGFR, CD19, CD20, CD25, CD30, CD33, CD38, CD52, B cell maturation antigen (BCMA), CD79, somatostatin receptor, 5T4, gp100, CEA, melanoma A / MART-1, MAGE, NY-ESO-1, PSA, tyrosinase, HER-2, HER-3, EGFR, hTERT, MUC1, mesothelin, adhesion protein-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 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 alpha (TGF-α), TGF-α receptor; transforming growth factor beta (TGF-β), TGF-β receptor; interleukin beta receptor alpha 2 chain (IL13Rα2), interleukin-6 (IL-6), IL-6 receptor, interleukin-4, IL-4 receptor, cytokine receptors, class I (hematopoietin family) and class II (interferon / IL-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 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 mutated (HSP70-2M), KIAA0205, MART2, melanoma common mutations 1, 2, 3 (MUM-1, 2, 3), new PAP, myocardial infarction class 1 Globulin, 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-acetylglucosamine transferase V, MGATS), HERV-KMEL, KK-LC, KM-HN-1, LAGE, LAGE-1, antigen recognized by CTL 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, TRP2-1 NT2, kallikrein 4, mammaglobin-A, OA1, TRP-1 / ,75, TRP-2 lipophilin, interferon-inducible protein 2 absent in melanoma (AIM-2), 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, NXF2TDRD1, TEX 15, FATE, TPTE, immunoglobulin idiotype, Bence Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD4, CD3, cancer antigen 72-4 (CA72-4), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA125), 1-2 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 signet ring tumor-2 (HST-2), PR1, claudin (such as claudin1, claudin3, claudin4, claudin din6, claudin7, claudin18.2), 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 MHC / neoantigen complex.

3. A dual-function therapeutic agent for treating cancer, the dual-function therapeutic agent comprising: a targeting component that targets a tumor-associated antigen; and an enzyme that, when delivered to a tumor via the targeting component, enzymatically converts the tumor phenotype to a phenotype of an incompatible allograft or xenograft, the enzyme being coupled to the targeting component; The tumor-associated antigens are 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 alpha (TGF-α), TGF-α receptor; transforming growth factor beta (TGF-β), TGF-β receptor; interleukin beta receptor alpha 2 chain (IL13Rα2), interleukin-6 (IL-6), IL-6 receptor, interleukin-4, IL-4 receptor, cytokine receptors, class I (hematopoietin family) and class II (interferon / IL-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 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 mutated (HSP70-2M), KIAA0205, MART2, melanoma common mutations 1, 2, 3 (MUM-1, 2, 3), new PAP, myocardial infarction class 1 Globulin, 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-acetylglucosamine transferase V, MGATS), HERV-KMEL, KK-LC, KM-HN-1, LAGE, LAGE-1, antigens recognized by CTL 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 lipophilin, not present in melanoma Interferon-inducible protein 2 (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 (CA125), 1-2 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 signet ring tumor-2 (HST-2), PR1, claudin (such as claudin1, claudin3, claudin4, cl audin6, claudin7, claudin18.2), 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 complex.

4. The bifunctional therapeutic agent of claim 3, wherein the targeting component is selected from an antibody or an antigen-binding fragment thereof, a TCR, a single domain antibody, anticalin, a protein, a peptide, a nucleic acid aptamer, and a small molecule ligand.

5. The bifunctional therapeutic agent of claim 4, wherein the targeting component is a peptide linked to the enzyme via a peptide bond.

6. The bifunctional therapeutic agent of claim 4, wherein the targeting component is an antibody or an antigen-binding derivative or fragment thereof or a single domain antibody.

7. The bifunctional therapeutic agent according to claim 4, wherein the targeting component is a small molecule ligand chemically linked to the enzyme through an intermediate polyethylene glycol (PEG) spacer.

8. The bifunctional therapeutic agent according to any one of claims 1 to 7, wherein the targeting component and the enzyme are genetically engineered to produce a fusion protein.

9. The bifunctional therapeutic agent according to any one of claims 1 to 7, wherein the targeting component and the enzyme are chemically linked.

10. The bifunctional therapeutic agent according to any one of claims 1 to 9, wherein the enzyme is an enzyme involved in post-translational modification and is a transferase or a glycosyltransferase.

11. The bifunctional therapeutic agent of claim 10, wherein the enzyme involved in post-translational modification is a transferase.

12. The bifunctional therapeutic agent of claim 10, wherein the transferase is a glycosyltransferase.

13. The bifunctional therapeutic agent of claim 12, wherein the glycosyltransferase is glycosyltransferase A (α1-3-N-acetylgalactosaminyltransferase), glycosyltransferase B (α1-3-galactosyltransferase), α-galactosyl-transferase, glycosyltransferase A (Gly268Ala) or fucosyltransferase.

14. The bifunctional therapeutic agent according to any one of claims 1 to 13, wherein the enzyme comprises an additional second amino acid sequence at its C-terminus.

15. The bifunctional therapeutic agent of claim 14, wherein the second amino acid sequence comprises a cleavable amino acid sequence located between the enzyme and the additional second amino acid sequence.

16. The bifunctional therapeutic agent of claim 15, wherein the cleavable amino acid sequence is cleavable by PSA, matrix metalloproteinase or cathepsin B.

17. The bifunctional therapeutic agent of any one of claims 1 to 16, wherein the tumor having the tumor-associated antigen expresses an H antigen.

18. The bifunctional therapeutic agent of any one of claims 1 to 17, wherein the cancer is lung cancer, gastric 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.

19. The bifunctional therapeutic agent of any one of the preceding claims, wherein the targeting component is a TCR, and wherein the TCR is a single-chain TCR (scTCR) or a single-domain TCR (sdTCR).

20. The bifunctional therapeutic agent of any one of the preceding claims, wherein the targeting component is a TCR, and the TCR binds to a neoantigen / MHC complex on the surface of a tumor cell.

21. The bifunctional therapeutic agent of claim 20, wherein the neoantigen is derived from KRAS G12D , MART-1, gp100, NY-ESO-1, CEA, MAGE-A3, MAGE-A4 or WT1.

22. The bifunctional therapeutic agent of any one of claims 1 to 21, wherein the cancer is prostate cancer.

23. The bifunctional therapeutic agent of claim 22, wherein the targeting component is a TCR, a single domain antibody, or an anticalin targeting the prostate specific membrane antigen (PSMA) / folate hydrolase 1 (FOLH1) receptor.

24. The bifunctional therapeutic agent of any one of claims 1 to 21, wherein the cancer is breast cancer.

25. The bifunctional therapeutic agent of claim 24, wherein the targeting component is a TCR, a single domain antibody, or an anticalin targeting a member of the HER receptor family.

26. The bifunctional therapeutic agent of any one of claims 1 to 21, wherein the cancer is a blood cancer of the B-cell lineage.

27. The bifunctional therapeutic agent of claim 26, wherein the targeting component is a TCR, a single domain antibody, or anticalin targeting CD19.

28. A method for treating cancer, the method comprising: The bifunctional therapeutic agent of any one of claims 1 to 27 is administered to a subject suffering from cancer.

29. The method of claim 28, wherein the subject is a human.

30. The method of claim 28 or claim 29, wherein the cancer is lung cancer, gastric 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.

31. The method of any one of claims 28 to 30, wherein the administering further comprises: Uridine diphosphate-galactose (UDP-gal), uridine diphosphate-N-acetylgalactosamine (UDP-NAcGal) and / or guanosine diphosphate-fucose (GDP-fucose) were administered.

32. A pharmaceutical composition comprising: The dual-functional therapeutic agent according to any one of claims 1 to 27; and A pharmaceutically acceptable carrier.

33. A nucleic acid molecule encoding the bifunctional therapeutic agent of any one of claims 1 to 27.

34. An expression vector comprising the nucleic acid molecule according to claim 33.

35. A recombinant host cell transformed with the nucleic acid molecule according to claim 33 or the expression vector according to claim 34.

36. A host cell expressing the bifunctional therapeutic agent of any one of claims 1 to 27.

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