Bispecific dendritic cell engager and uses thereof

Abstract

This disclosure relates to the use of a combination of a bispecific conjugate against dendritic cells (anti-DC) and against immunogenic cell death (anti-ICD) with a tumor ICD inducer to enhance dendritic cell activity for the treatment of cancer patients. An exemplary multivalent protein comprises at least one DC binding site and at least one ICD binding site. In some embodiments, these binding sites may be linked via an immunoglobulin constant region. This disclosure also provides monoclonal antibodies against DC and against ICD.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 238,229, filed on August 30, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to a bispecific dendritic cell engager that targets immunogenic cell death (ICD) markers on dendritic cells (DCs) and tumor cells. Administration of the anti-DC / anti-ICD bispecific engager enhances the activation and maturation of dendritic cells, accompanied by phagocytosis of ICD-expressing tumor cells, thereby further inducing tumor-specific cellular and humoral immunity. This disclosure provides a method for treating cancer using the anti-DC / anti-ICD bispecific engager. [Previous Technology]

[0003] The idea of ​​using bispecific antibodies (BsAbs) to effectively retarget tumor cells by immune cells emerged in the 1980s. Bispecific antibody backbones are generally classified into two main categories with different pharmacokinetic properties based on whether they possess a crystallizable region (Fc) fragment, an IgG-like molecular structure, and a small recombinant bispecific architecture. Most bispecific backbones are derived from single-chain variable fragments (scFvs).

[0004] Dendritic cells (DCs) are highly specialized antigen-presenting cells (APCs) with the unique ability to initiate the development of an adaptive immune response upon antigen stimulation (Steinman, 1991). They are effective stimulators of B lymphocytes and T lymphocytes. When a dendritic cell captures an antigen, the antigen is processed into a peptide for presentation to CD8+ T cells by MHC class I molecules or to CD4+ T cells by MHC class II molecules. Furthermore, cytokines secreted by CD4+ T cells contribute to B cell maturation and cytotoxic T cell activation. Moreover, activated dendritic cells can also secrete interleukins IL-12 and IL-15, as well as type I interferons (IFNs), to activate natural killer (NK) cells (Münz et al., 2005). Furthermore, high density of tumor-infiltrating dendritic cells, along with increased T cell activation (Ladányi et al., 2007), has been found to be a better indicator of clinical prognosis (Dieu-Nosjean et al., 2008). Dendritic cells targeting antigens with C-type lectin 9A (CLEC9A) can significantly enhance anti-tumor immunity. Other evidence shows that antigen-targeting CLEC9A can enhance the immune responses of CD4+ T cells, CD8+ T cells, and B cells (Park HY et al., 2013). Further research shows that CLEC9A can specifically recognize F-actin (a core component of the cytoskeleton exposed by necrotic cells) and initiate cross-sensitization of dendritic cells to CD8+ T cells, thereby activating the CD8+ T cell response to dead cell-associated antigens (Zhang JG et al., 2013). These results indicate that CLEC9A is a unique dendritic cell marker capable of sensing damaged cells and their antigens. Therefore, targeting CLEC9A-positive dendritic cells can promote humoral and cellular immunity. Since dendritic cells play a crucial role in initiating immune responses, they can serve as ideal targets for enhancing endogenous anti-tumor responses to eradicate tumors.

[0005] Immunogenic cell death (ICD) can be defined by exposure to tumor-associated molecular patterns (DAMPs) in the tumor microenvironment that stimulate the host immune system. ICD can be induced by chemotherapy, nanopulse stimulation, nanoparticle-coated immunotherapy, near-infrared phototherapy, and immune attack (Zhou et al., 2019). Induction of ICD in tumors upregulates the expression of endogenous danger signals, such as adenosine triphosphate (ATP), heat shock proteins (HSP), calreticulin (CRT), and high-mobility group box 1 protein (HMGB1) (Krysko et al., 2012). Dendritic cells engulf immunogenic dead tumor cells and present tumor antigens, which then activate the response of tumor-specific cytotoxic T cells (Obeid et al., 2007). Notably, the induction of tumor ICD is associated with the maintenance of long-lasting protective anti-tumor immunity (Zhou et al., 2019). Furthermore, elevated CRT performance is a strong predictor of overall survival (OS) in cancer patients (Fucikova et al., 2016).

[0006] Successful induction of anti-tumor T cell activity via dendritic cells requires three signals: capture and presentation of tumor-associated antigens (TAAs) onto MHC molecules (signal 1), provision of co-stimulatory factors (signal 2), and soluble factors (signal 3) (Palucka and Banchereau, 2012; Palucka et al., 2011). The absence of signals 1, 2, and / or 3 (possibly due to tumor regulation) will result in cross-presentation of tumor antigens that are unfavorable to dendritic cell regulation and will usually induce T cell tolerance, i.e., T cell non-response (Gabrilovich et al., 1997). Several agonists can stimulate dendritic cells (DCs) via toll-like receptors (Schreibelt et al., 2010), interferon gene stimulating protein (STING) (Ishikawa and Barber, 2008), and differentiation cluster 40 (CD40) (O'Sullivan and Thomas, 2002). Some of these agonists have been approved by US regulatory agencies, while others are undergoing clinical evaluation (Hübbe et al., 2020). Therefore, inducing tumor ICDs and then enhancing the phagocytosis and activation of dendritic cells is a promising approach to enhance host anti-tumor immunity and generate immune memory to eliminate tumor cells. This disclosure describes how to synergize dendritic cells and ICD tumors to enhance host anti-tumor immunity. [Summary of the Invention]

[0007] This disclosure relates to an antibody or antigen-binding fragment comprising a region that binds to an immunogenic cell death (ICD) marker on a tumor cell.

[0008] In some embodiments, the ICD marker comprises calreticulin, heat shock protein (HSP), or other proteins that are exposed on the surface of the tumor cells during immunogenic cell death.

[0009] In some embodiments, the HSP is Hsp70 or Hsp90.

[0010] In some embodiments, the ICD tag contains calcium reticulum companion protein.

[0011] In some embodiments, the antibody or antigen-binding fragment provided herein further comprises a heavy chain variable region having an amino acid sequence having at least about 90% sequence homology with SEQ ID NO: 1, and a light chain variable region having an amino acid sequence having at least about 90% sequence homology with SEQ ID NO: 2.

[0012] In some embodiments, this disclosure provides an antibody or antigen-binding fragment comprising a region that binds to a protein marker on a dendritic cell.

[0013] In some embodiments, the protein tag includes CD1a, CD1c, CD11b, CD11c, CD16, CD32, CD103, CD115, CD123, CD207, CD301b, CD317, B220, BDCA1, BDCA2, BDCA3, BDCA4, CADM1, CCR2, CLEC9A, CXCR1, DCIR2, DEC205, EPCAM, Ly6C, SIRP, SiglecH, or XCR1.

[0014] In some embodiments, the protein is labeled as CLEC9A.

[0015] In some embodiments, the antibody or antigen-binding fragment provided herein further comprises a heavy chain variable region having an amino acid sequence having at least about 90% sequence homology with SEQ ID NO: 3, and a light chain variable region having an amino acid sequence having at least about 90% sequence homology with SEQ ID NO: 4.

[0016] In some embodiments, this disclosure provides a bispecific antibody or antigen-binding fragment comprising a region that binds to an immunogenic cell death (ICD) marker on a tumor cell and a region that binds to a protein marker on a dendritic cell.

[0017] In some embodiments, the antibody or antigen binding fragment provided in this disclosure further includes a first binding region specifically binding to the calreticulum companion protein and a second binding region specifically binding to CLEC9A.

[0018] In some embodiments, the antibody or antigen-binding fragment provided herein further comprises a heavy chain variable region having an amino acid sequence having at least about 90% sequence homology with SEQ ID NO: 5, and a light chain variable region having an amino acid sequence having at least about 90% sequence homology with SEQ ID NO: 6.

[0019] In some embodiments, the isotype of the antibody is IgG, IgE, IgM, IgD, or IgA.

[0020] In some embodiments, the antibody is an IgG antibody.

[0021] In some embodiments, the IgG antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0022] In some embodiments, the antibody is a human antibody.

[0023] This disclosure also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment as described above and a pharmaceutically acceptable carrier.

[0024] This disclosure also relates to a method for treating cancer patients. The method includes the step of administering an effective amount of the pharmaceutical composition to a patient in need.

[0025] In some embodiments, the cancer is a cancer that exhibits ICD markers.

[0026] In some embodiments, the cancers exhibiting ICD labeling are selected from the group consisting of malignant sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma multiforme, lung cancer, breast cancer, oral cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, oral cancer, oropharyngeal cancer, laryngeal cancer, and prostate cancer.

Implementation Method

[0040] The articles “a” and “a kind” as used herein refer to one or more (i.e., at least one) grammatically object of the article. For example, “an element” refers to one element or more elements.

[0041] The antibodies described herein may be full-length or may comprise one or more fragments of an antibody having an antigen-binding portion, including but not limited to Fab, F(ab')2, Fab', F(ab)', variable region fragments (Fv), single-chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragments (Ward et al., (1989) Nature, 341:544-546), isolated complementarity-determining regions (CDR), diabodies, tribodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from multiple antibody fragments. This invention also covers single-chain antibodies generated by linking antibody fragments using recombinant methods or synthetic linkers (Bird et al., (1988) Science, 242:423-426; Huston et al., (1988) PNAS, 85:5879-5883).

[0042] This invention covers all antibody isotypes, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, or IgE (all types and subtypes are included in this invention). The antibody or its antigen-binding portion may be a mammalian (e.g., mouse, human) antibody or its antigen-binding portion. The light chain of the antibody may be kappa or lambda type.

[0043] In one embodiment, the antibody or its antigen-binding portion disclosed herein comprises at least one heavy chain variable region and / or at least one light chain variable region.

[0044] This disclosure relates to the combination of a bispecific conjugate of anti-DC / anti-ICD with a tumor ICD inducer for the treatment of cancer patients.

[0045] Therefore, this disclosure is based on the following discovery: the activation and maturation of dendritic cells are enhanced by the anti-DC / anti-ICD bispecific conjugate, thereby increasing the phagocytic activity of dendritic cells against ICD-expressing tumor cells. Targets on dendritic cells include, but are not limited to, CD1a, CD1c, CD11b, CD11c, CD16, CD32, CD103, CD115, CD123, CD207, CD301b, CD317, B220, BDCA1, BDCA2, BDCA3, BDCA4, CADM1, CCR2, CLEC9A, CXCR1, DCIR2, DEC205, EPCAM, Ly6C, SIRP, SiglecH, and XCR1.

[0046] ICD targets (i.e. ICD markers) include, but are not limited to, calreticulin, Hsp70, Hsp90, and proteins expressed on the cell membrane during tumor immunogenic cell death.

[0047] Cancers exhibiting ICD markers include, but are not limited to, malignant sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma multiforme, lung cancer, breast cancer, oral cancer, head and neck cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, oral cancer, oropharyngeal cancer, laryngeal cancer, and prostate cancer.

[0048] The term "subject" can refer to a vertebrate suffering from cancer or a vertebrate considered to require cancer treatment. An individual includes all warm-blooded animals, such as mammals, such as primates, and preferably humans. An individual can also be a non-human primate. The term "subject" includes domesticated animals, such as cats and dogs, livestock (such as cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (such as mice, rabbits, rats, gerbils, guinea pigs, etc.). Therefore, the scope of this disclosure covers veterinary and medical preparations.

[0049] As used herein, “effective amount” means a dose of a pharmaceutical composition sufficient to alleviate symptoms and signs of cancer, such as weight loss, pain, and a palpable mass that is detectable, whether clinically palpable or radiologically detectable by various imaging methods. The terms “effective amount” and “therapeutic effective amount” are used interchangeably.

[0050] The exemplary, non-limiting range of the therapeutic or preventive effective amount of the antibody or antigen binding portion disclosed herein is about 0.05 μg / kg body weight to about 500 mg / kg body weight, about 0.1 μg / kg body weight to about 100 mg / kg body weight, about 1.0 μg / kg body weight to about 10 mg / kg body weight, and about 10 μg / kg body weight to about 1.0 mg / kg body weight.

[0051] In some embodiments, the antibody or its antigen-binding portion comprises a variable region containing an amino acid sequence that has a sequence homology of at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0052] Sequence List serial number Amino acid sequence and description 1 Full-length amino acid sequence of the heavy chain of the anti-CRT (calreticulin) monoclonal antibody (indicated by the underline below the CDR sequence): 5B3-1 vH EVQLVETGGGLVQPKGSLKLSCAASGFSFNNNAMNWVRQAPGKGLEWVARIRSKTNNYEIYYAESVKDRFTISRDDSQSMLYLQMNNLKTDDTAMYYCVRDYNHVGFVYWGQGTQVTVST 2 Full-length amino acid sequence of the light chain of the anti-CRT (calcium reticulum protectant) monoclonal antibody (indicated by the underline below the CDR sequence): 5B3-1 vL DIVMTQTTPSVPVTPGESVSISCRSSKSLLYSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKR 3 Full-length amino acid sequence of the heavy chain of the anti-CLEC9A monoclonal antibody (indicated by the underlined CDR sequence): EC 10 vH EVQLVESDGGFLQPGRSLKLSCAASGFTFSDYYMAWVRQAPTKGLEWVATISSDGSNTYYRDSVKGRFTISRDNAKTTLYLQMDSLRSEDTATYYCAGQAAGFASWGQGTLVTVSS 4 Full-length amino acid sequence of the light chain of the anti-CLEC9A monoclonal antibody (indicated by the underlined CDR sequence): EC 10 vL DIQMTQSPSFLSASVGDRVTINCKASQNINKYLNWYQQKLGEAPKRLIYNTNNLQPGIPSRFSGSGSGTDYTLTISSLQPEDFATYFCLHHNSFPLTFGSGTKLEIKR 5 Full-length amino acid sequence of the heavy chain of the anti-CLEC9A x anti-CRT bispecific antibody (CDR sequences are underlined): EC10 x 5B3-1 BsAb vH EVQLVESDGGFLQPGRSLKLSCAASGFTFSDYYMAWVRQAPTKGLEWVATISSDGSNTYYRDSVKGRFTISRDNAKTTLYLQMDSLRSEDTATYYCAGQAAGFASWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSGGGGSEVQLVETGGGLVQPKGSLKLSCAASGFSFNNNAMNWVRQAPGKGLEWVARIRSKTNNYEIYYAESVKDRFTISRDDSQSMLYLQMNNLKTDDTAMYYCVRDYNHVGFVYWGQGTQVTVSTGGGGSGGGGSGGGGSDIVMTQTTPSVPVTPGESVSISCRSSKSLLYSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKR 6 Full-length amino acid sequence of the light chain of the anti-CLEC9A x anti-CRT bispecific antibody (indicated by the underlined CDR sequence): EC10 x 5B3-1 BsAb vL DIQMTQSPSFLSASVGDRVTINCKASQNINKYLNWYQQKLGEAPKKRLIYNTNNLQPGIPSRFSGSGSGTDYTLTISSLQPEDFATYFCLHHNSFPLTFGSGTKLEIKR Example

[0053] Example 1: Tumor cells expose endogenous danger signals

[0054] Mouse 4T1 breast cancer cells were seeded into 6-well culture dishes. After overnight culture, the cells were treated with 100 μM oxaliplatin or left untreated for two days. Cells were collected and centrifuged, then stained at 4°C for 30 minutes with anti-CRT-Alexa 647 antibody (Abcam, catalog number 0080-012-310) and a purple dye (ThermoFisher, catalog number L34964) to distinguish between live and dead cells. Cells were washed and centrifuged again. The expression of calcium reticulum protectant protein (CRT) and heat shock protein 70 (Hsp70) on the surface of live 4T1 cells was analyzed using a BD FACSCanto clinical flow cytometer system (FACSCANTO II, BD Biosciences). Figure 2A shows the translocation of CRT to the cell surface after oxaliplatin treatment. Figure 2B shows the translocation of Hsp70 to the cell surface after oxaliplatin treatment. These results demonstrate that treatment with 100 μM oxaliplatin increased the expression levels of CRT (from 6.5% to 59%) and Hsp70 (from 3.2% to 11%) on the cell surface.

[0055] Further, the supernatant was collected and centrifuged, and then the adenosine triphosphate (ATP) in the supernatant was measured using CellTiter-Glo fluorescence detection (Promega, catalog number G7570). In short, the collected supernatant was mixed with an equal volume of reagent and reacted at room temperature in the dark for 10 minutes. Fluorescence was measured using a Molecular Device (SpectraMax L). Figure 3 shows the increase in ATP released into the culture medium after two days of treatment with 100 μM oxaliplatin.

[0056] Example 2: Determination of CLEC9A expression on dendritic cells

[0057] Mouse MutuDC 1940 cells were stained with an anti-CLEC9A monoclonal antibody (eBioscience, catalog number 12-5975-82) that binds to the phycoerythrin (PE) fluorophore at 4°C for 30 minutes. Cells were then washed and collected. The binding of CLEC9A to the antibody on MutuDC 1940 cells was analyzed using FACSCANTO II. Figure 4 shows the presence of CLEC9A on MutuDC 1940 cells (approximately 68.4%).

[0058] Example 3: Dendritic cell-regulated phagocytosis of tumor cells

[0059] Mouse 4T1 breast cancer cells were stained with CellTrace far-infrared staining solution (ThermoFisher, catalog number C34564) at 37°C for 30 minutes, followed by washing with phosphate-buffered saline (PBS). Cells were seeded into 6-well culture dishes. After overnight culture, the cells were treated with 100 μM oxaliplatin or untreated for two days. Cells were then collected and counted. Equal numbers of MutuDC 1940 cells and 4T1 cells were mixed and cultured at 37°C for the specified time. After the aforementioned treatment, cells were collected and stained with anti-mouse CD11c-BV421 antibody (Biolegend, catalog number 117330) at 4°C for 30 minutes. Cells were washed, centrifuged, and analyzed using FACSCANTO II. The integration of 4T1 cells into the MutuDC 1940 cell line was assessed by the percentage of CD11c-positive cells stained with far-infrared light. As shown in Figure 5, treatment of 4T1 cells with oxaliplatin increased the dendritic cell-regulated phagocytosis of 4T1 cells by MutuDC 1940 cells (from 31.03% to 43.3%).

[0060] Example 4: Co-culturing with tumor cells to inhibit the expression of activation markers on dendritic cells

[0061] Mouse 4T1 breast cancer cells were seeded into 6-well culture dishes. After overnight culture, the cells were treated with 100 μM oxaliplatin or not for two days. Then, the cells were collected and counted. An equal number of MutuDC 1940 cells and 4T1 cells were mixed and cultured at 37°C for 24 hours. Following the aforementioned treatment, cells were collected and reacted with TruStainFcXTMPLUS antibody (anti-mouse CD16 / 32 antibody) (Biolegend, catalog number 100512) at 4°C for 10 minutes. Then, cells were stained with anti-mouse CD11c-BV421 antibody (Biolegend, catalog number 117330), anti-mouse CD40-PE antibody (Biolegend, catalog number 124610), anti-mouse IA / IE-APC / Cyanine7 antibody (Biolegend, catalog number 107628), and anti-mouse CD86-BV510 antibody (Biolegend, catalog number 105040) at 4°C for 30 minutes. Cells were washed, centrifuged, and then analyzed using FACSCANTO II. As shown in Figure 6, after culturing with 4T1 cells, the expression of CD40, CD86, and MHC-II on MutuDC 1940 cells decreased slightly, but this decrease did not occur after co-culturing with oxaliplatin-treated 4T1 cells.

[0062] Example 5: Enhancing T cell proliferation induced by dendritic cells by adding oxaliplatin to tumor cells.

[0063] Mouse 4T1 breast cancer cells were seeded into 6-well culture dishes. After overnight culture, the cells were treated with 100 μM oxaliplatin or not for two days. Then, the cells were collected and counted. Equal numbers of MutuDC 1940 cells and 4T1 cells were mixed and cultured at 37°C for 24 hours. Mouse pan-T cells were purified from spleen cells of C57BL / 6 mice using pan-T cell isolation reagent kit II (Miltenyi Biotec, catalog number 130-095-130), and then stained with CellTrace far-infrared staining solution (ThermoFisher, catalog number C34564) at 37°C for 30 minutes. The cells were washed and counted. Five-fold far-infrared labeled mouse pan-T cells were added to the co-cultured MutuDC 1940 cells and 4T1 cells, and cultured for another 72 hours. Cells were collected and stained with anti-mouse CD3-FITC antibody (Biolegend, catalog number 100203) and anti-mouse CD4-PE antibody (Biolegend, catalog number 100407) at 4°C for 30 minutes. Cells were washed, centrifuged, and then analyzed using FACSCANTO II. CD4+ T cell proliferation was calculated by selecting CD3+ / CD4+ cells and setting a marker indicating at least 97% cell content in the unstimulated sample. As shown in Figure 7A, co-culturing dendritic cells with 4T1 cells inhibited CD4+ T cell proliferation, but co-culturing with oxaliplatin-treated 4T1 cells did not show this inhibition. Conversely, CD8+ T cell proliferation was calculated by selecting CD3+ / CD4- cells and setting a marker indicating at least 98% cell content in the unstimulated sample. As shown in Figure 7B, co-culturing dendritic cells with oxaliplatin-treated 4T1 cells increased the proliferation of CD8+ T cells, but co-culturing with 4T1 cells in the control group did not increase the proliferation of CD8+ T cells.

[0064] Example 6: ELISA determination of protein binding activity of EC10 antibody (anti-mouse CLEC9A) and 5B3-1 antibody (anti-CRT)

[0065] 200 ng of mouse CLEC9A-ECD protein or human CRT-ECD protein (manufactured in-house by OBI Pharma Inc.) was spread into 96-well plates (Thermo Fisher, catalog number 44-2402-21) and incubated overnight at 4°C. The 96-well plates were washed with PBS containing 0.2% polysorbate 20 (Tween-20) (i.e., PBST) and then blocked with PBST containing 5% bovine serum albumin (BSA) at room temperature for one hour. The 96-well plates were then washed again with PBST and reacted with a three-fold serially diluted anti-mouse CLEC9A antibody (OBI Pharma, Inc., named EC10) or anti-CRT antibody (OBI Pharma, Inc., named 5B3-1) at room temperature for two hours. After washing, goat anti-mouse IgG (H+L) conjugated with wasabi peroxidase (HRP) (Jackson ImmunoResearch, catalog number 115-035-062) (HRP-bound) was added to the 96-well plate and reacted at room temperature for one hour. The 96-well plate was then washed, reacted with 3,3',5,5'-tetramethylbenzidine (TMB) as the reactant, and developed at room temperature for 20 minutes. The absorbance (OD 450 nm) of the 96-well plate was then read using an ELISA reader (Molecular Devices, SpectraMax M2). As shown in Figure 8A, the EC50 of the EC10 antibody against mouse CLEC9A-ECD was 0.1044 nM. As shown in Figure 8B, the EC50 of the 5B3-1 antibody against human CRT-ECD was 0.4068 nM.

[0066] Example 7: Cell binding activity analysis of EC10 antibody (anti-mouse CLEC9A), 5B3-1 antibody (anti-CRT) and EC10 x 5B3-1 bispecific antibody

[0067] 293F cells overexpressing mouse CLEC9A or oxaliplatin-treated cells were stained at 4°C for 30 min with 30 nM mouse IgG2a (Biolegend, catalog number 400202), anti-mouse CLEC9A antibody (OBI Pharma, Inc., EC10), anti-CRT antibody (OBI Pharma, Inc., 5B3-1), or anti-mouse CLEC9A x anti-CRT bispecific antibody (OBI Pharma, Inc., EC10 x 5B3-1 BsAb). The samples were washed and centrifuged, then stained at 4°C for 30 min with FITC-labeled anti-mouse IgG (SouthernBiotech, catalog number 1032-02). Cells were then washed and collected. Antibody binding activity was analyzed using FACSCANTO II. Figure 9A shows that the EC10 antibody (55.1%) and the EC10 x 5B3-1 bispecific antibody (55.8%) have binding activity against 293F cells overexpressing mouse CLEC9A. Figure 9B shows that the 5B3-1 antibody (41.2%) and the EC10 x 5B3-1 bispecific antibody (77.6%) have binding activity against oxaliplatin-treated 4T1 cells, but the EC10 antibody (1.9%) has no binding activity.

[0068] Example 8: Antitumor activity of the combination of EC10 x 5B3-1 bispecific antibody and oxaliplatin in a mouse 4T1 tumor allograft model

[0069] One × 10⁵ 4T1 breast cancer cells were placed in 100 μL of sterile PBS and injected subcutaneously (sc) into the right abdomen of mice. On day 7 post-inoculation, when the tumor size reached 50 mm³, five mice per group were randomly divided into three treatment groups. On days 7, 13, and 20, mice were injected intravenously or intratumorally with PBS, oxaliplatin (MedChemExpress, catalog number HY-17371), or oxaliplatin and EC10 x 5B3-1 bispecific antibody (OBI Pharma, Inc., EC10 x 5B3-1 BsAb). Oxaliplatin was administered intravenously at 5 mg / kg, and the bispecific antibody was administered intratumorally at 20 μg. Tumor length and width were recorded weekly. Tumor volume was calculated using the following formula: Volume (mm³) = ([width]² × length) / 2. Results are expressed as arithmetic mean ± standard error of mean (SEM). Statistical comparisons were performed using a two-tailed Student's t-test. The probability of significance, "p", was set as *p < 0.05, **p < 0.01, and ***p < 0.001. Figure 10A shows a schematic flowchart of the in vivo 4T1 tumor study in BALB / c mice. Mice were sacrificed on day 27. Figure 10B shows the growth curve of the 4T1 tumor. Figure 10C shows photographs and tumor weights for each group. It reveals that there was no difference between the tumors treated with oxaliplatin alone and the control group. However, treatment with the EC10 x 5B3-1 bispecific antibody in combination with oxaliplatin significantly inhibited 4T1 tumor growth and reduced tumor weight.

[0070] Example 9: The combination of EC10 x 5B3-1 bispecific antibody and oxaliplatin enhances the number of immune cells in a mouse 4T1 tumor allograft model.

[0071] Splenic cells were collected from a mouse 4T1 breast cancer tumor allogeneic model on the day of sacrifice. One x 106 cells from each sample were blocked at 4°C for 10 minutes with purified anti-mouse CD16 / 32 antibody (BioLegend, catalog number 101302). Cells were then stained at 4°C for 30 minutes using group A antibodies [anti-mouse CD11c antibody (Biolegend, catalog number 117311), anti-mouse CLEC9A antibody (Biolegend, catalog number 143504), and anti-mouse IA / IE antibody (Biolegend, catalog number 107628)] and group B antibodies [anti-mouse CD3 antibody (Biolegend, catalog number 100210), anti-mouse CD4 antibody (Biolegend, catalog number 100425), anti-mouse CD8a antibody (Biolegend, catalog number 100726), and anti-mouse CD49b antibody (Biolegend, catalog number 103510)]. Cells were washed, centrifuged, and then analyzed using FACSCANTO II. The cDC1 cell population was calculated by selecting CD11c+ / CLEC9A+ cells and setting a marker that included at least 97% of the cells in the isotype control sample. MHC-II on cDC1 cells was calculated using mean fluorescence intensity (MFI). CD4+ T cell populations were calculated as CD3+ / CD4+, CD8+ T cells as CD3+ / CD8+, and NK cells as CD3- / CD49b+. Figure 11A shows that the combination of EC10 x 5B3-1 bispecific antibody (BsAb) and oxaliplatin maximally increased the cDC1 population in the spleen. Figure 11B also shows that the combination of EC10 x 5B3-1 bispecific antibody (BsAb) and oxaliplatin maximally increased MHC-II expression on cDC1 cells. Figure 12 shows that the combination of EC10 x 5B3-1 bispecific antibody (BsAb) and oxaliplatin increased the populations of CD4+ T cells (Figure 12A), CD8+ T cells (Figure 12B), and NK cells (Figure 12C) in the mouse spleen. References

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[0076] 5. Münz, C., Dao, T., Ferlazzo, G., de Cos, M.A., Goodman, K., and Young, J.W. (2005). Mature myeloid dendritic cell subsets have distinct roles for activation and viability of circulating human natural killer cells. Blood 105, 266–273.

[0077] 6. Obeid, M., Tesniere, A., Ghiringhelli, F., Fimia, G.M., Apetoh, L., Perfettini, J.-L., Castedo, M., Mignot, G., Panaretakis, T., Casares , N., et al. (2007). Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13, 54–61.

[0078] 7. Park HY, Light A, Lahoud MH, Caminschi I, Tarlinton DM and Shortman K. (2013). Evolution of B cell responses to Clec9Atargeted antigen. Journal of immunology 191, 4919-4925.

[0079] 8. Schreibelt, G., Tel, J., Sliepen, K.H.E.W.J., Benitez-Ribas, D., Figdor, C.G., Adema, G.J., and de Vries, I.J.M. (2010). Toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy. Cancer Immunol Immunother 59, 1573–1582.

[0080] 9. Steinman, R.M. (1991). The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9, 271–296.

[0081] 10. Zhang JG, Czabotar PE, Policheni AN, Caminschi I, Wan SS, Kitsoulis S, Tullett KM, Robin AY, Brammananth R, van Delft MF, Lu J, O'Reilly LA, Josefsson EC, Kile BT, Chin WJ, Mintern JD, et al. (2012). The dendritic cell receptor Clec9A binds damaged cells via exposed actin filaments. Immunity. 36, 646-657.

[0082] 11. Zhou, J., Wang, G., Chen, Y., Wang, H., Hua, Y., and Cai, Z. (2019). Immunogenic cell death in cancer therapy: Present and emerging inducers. J Cell Mol Med 23, 4854–4865.

[0083] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any combination, method, kit, and information transmission device similar to or equivalent to those described herein may be used in the practice of the invention, preferred combinations, methods, kits, and information transmission devices are described herein only.

[0084] All references cited herein are incorporated herein by reference to the fullest extent permitted by law. Discussion of these references is solely for the purpose of summarizing the arguments of their authors. This document does not acknowledge any reference (or any part thereof) as relating to prior art. The applicant reserves the right to question the accuracy and relevance of any cited references. [Simplified Explanation of the Diagram]

[0027] A more complete understanding of this disclosure can be obtained by referring to the accompanying drawings and the following detailed description. The embodiments illustrated in the drawings are only for illustrating the content of this disclosure and should not be construed as limiting this disclosure to those embodiments.

[0028] Figure 1 is a schematic diagram of the mechanism of action (MOA) of the anti-DC / anti-ICD bispecific conjugate in enhancing host anti-tumor immunity. Cancer patients receive pre-treatment to induce ICDs, including targeted therapy, chemotherapy, radiotherapy, and phototherapy. After inducing ICD labeling in tumors, administering the anti-DC / anti-ICD conjugate to the patient helps dendritic cells to activate and phagocytose ICD-labeled tumor cells, thereby initiating cellular and humoral immunity. The phagocytosed tumor antigens are processed into peptides, which are then presented to CD8+ T cells by type I MHC molecules or to CD4+ T cells by type II MHC molecules. Cytokines secreted by activated CD4+ T cells help mature and activate B cells and cytotoxic T cells, thereby eradicating tumor cells. Enhancing the cancer immune cycle with the anti-DC / anti-ICD bispecific conjugate will provide patients with lasting benefits by enhancing immune surveillance of cancer.

[0029] Figures 2A and 2B show tumor cells exposed to endogenous danger signals. Figure 2A shows tumor cells exposed to calreticulum protectant protein (CRT) after treatment with oxaliplatin. Figure 2B shows tumor cells exposed to heat shock protein 70 (Hsp70) after treatment with oxaliplatin.

[0030] Figure 3 shows the release of ATP from tumor cells after treatment with oxaliplatin.

[0031] Figure 4 shows the expression of CLEC9A on dendritic cells.

[0032] Figure 5 shows that treatment of tumor cells with oxaliplatin enhances the dendritic cell-regulated phagocytosis of tumor cells.

[0033] Figure 6 shows that co-culturing dendritic cells with tumor cells inhibits the expression of activation markers on dendritic cells.

[0034] Figures 7A and 7B show the enhancement of dendritic cell-induced T cell proliferation in tumor cells treated with oxaliplatin (Oxa). Figure 7A shows CD4+ T cells. Figure 7B shows CD8+ T cells.

[0035] Figures 8A and 8B show the enzyme-linked immunosorbent assay (ELISA) binding assays using anti-CELC9A antibody (EC10) and anti-CRT antibody (5B3-1). Figure 8A shows the 50% maximum effective concentration (EC50) of EC10 antibody binding to mouse CLEC9A-ECD. Figure 8B shows the EC50 of 5B3-1 antibody binding to human CRT-ECD.

[0036] Figures 9A and 9B show the characterization of EC10 antibody (anti-mouse CLEC9A), 5B3-1 antibody (anti-CRT), and EC10 x 5B3-1 bispecific antibody by cell binding assays. Figure 9A shows the binding of EC10 antibody, 5B3-1 antibody, or EC10 x 5B3-1 bispecific antibody to 293F cells overexpressing mouse CLEC9A. Figure 9B shows the binding of EC10 antibody, 5B3-1 antibody, or EC10 x 5B3-1 bispecific antibody to oxaliplatin-treated 4T1 cells.

[0037] Figures 10A to 10C show the antitumor activity of the combination of EC10 x 5B3-1 bispecific antibody and oxaliplatin in a mouse 4T1 allogeneic transplantation model. Figure 10A shows the treatment regimen and timeline. Figure 10B shows the tumor growth curve. Figure 10C shows photographs of the dissected tumor and the weight of the removed tumor.

[0038] Figures 11A and 11B show the cDC1 cell population and MHC type II (MHC-II) expression in a mouse 4T1 allograft model after administration of the EC10 x 5B3-1 bispecific antibody in combination with oxaliplatin. Figure 11A shows the percentage of cDC1 cells (CD11c+ / CLEC9A+). Figure 11B shows the mean fluorescence intensity (MFI) of MHC-II on cDC1 cells.

[0039] Figures 12A to 12C show the CD4+ / CD8+ T cell and NK cell populations in a mouse 4T1 allograft model after administration of the EC10 x 5B3-1 bispecific antibody in combination with oxaliplatin. Figure 12A shows the percentage of CD4+ T cells. Figure 12B shows the percentage of CD8+ T cells. Figure 12C shows the percentage of NK cells. [Biomaterial Storage]

[0086] None.

Claims

1. An antibody or antigen-binding fragment that specifically binds to calreticulin (CRT) and C-type lectin 9A (CLEC9A), comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 5; and a light chain comprising the amino acid sequence of SEQ ID NO:

6.

2. The antibody or antigen-binding fragment as described in claim 1, wherein a homotype of the antibody is IgG, IgE, IgM, IgD, or IgA.

3. The antibody or antigen-binding fragment as described in claim 1, wherein the antibody is an IgG antibody.

4. The antibody or antigen-binding fragment as described in claim 3, wherein the IgG antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

5. The antibody or antigen-binding fragment as described in claim 1, wherein the antibody is a human antibody.

6. A pharmaceutical composition comprising: an antibody or antigen-binding fragment as described in any one of claims 1 to 5; and a pharmaceutically acceptable carrier.

7. Use of a pharmaceutical composition as described in claim 6 in the preparation of a medicament for treating an individual suffering from cancer.

8. The use as described in claim 7, wherein the individual is a human being.

9. The use as claimed in claim 7, wherein the effective amount of the pharmaceutical composition administered to the individual is from 0.05 μg / kg body weight to 500 mg / kg body weight.

10. The use as described in claim 7, wherein the cancer is a cancer exhibiting immunogenic cell death (ICD) markers.

11. The use as described in claim 10, wherein the cancers represented by the ICD marker are selected from the group consisting of malignant sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma multiforme, lung cancer, breast cancer, oral cancer, head and neck cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, oral cancer, oropharyngeal cancer, laryngeal cancer, and prostate cancer.

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