Trispecific tumor-targeting polypeptide with enhanced binding affinity comprising tumor-targeting ligand, co-targeting ligand, and effector molecule, and use thereof

Abstract

The present invention relates to a binding protein and use thereof, the binding protein comprising a first binding domain, a second binding domain, and a third binding domain, wherein: the first binding domain is a domain that specifically binds to a tumor-associated antigen (TAA); the second binding domain is both an auxiliary domain that specifically binds to the TAA and a domain that repolarizes the tumor microenvironment (TME) from tolerogenic to pro-inflammatory and, at the same time, specifically binds to an immune checkpoint protein, for in situ T cell priming; and the third binding domain is an effector molecule for inducing T cell response activation and in situ T cell priming in the TME.

Description

Binding-enhancing trispecific tumor-targeting polypeptide comprising a tumor-targeting ligand, a co-targeting ligand, and an effector molecule, and its uses

[0001] The present invention relates to a binding-enhancing triple-specific tumor-targeting polypeptide comprising a tumor-targeting ligand, a co-targeting ligand, and an effector molecule, and to the use thereof.

[0002] In combating all diseases, including cancer and viral infections, the functional immune system is a crucial part of the therapeutic response. Consequently, significant research has been conducted on immunotherapy, including the field of immuno-oncology (IO), which is currently recognized as a strategy for treating cancer. Recently, novel targets and compounds that manipulate the immune response have been studied by researchers and clinicians. For example, IO agents targeting programmed cell death protein 1 (PD1) and programmed death-ligand 1 (PD-L1) have already been approved for the treatment of some advanced malignancies, while compounds interacting with other IO targets are currently under development.

[0003] Nevertheless, these novel immunotherapies are effective only for specific patients. In fact, despite interest in IO agents, response and extended survival in many patients remain very poor. Therefore, in light of the desire to maximize the variability in response and clinical benefits of both long-established therapies and novel immunotherapies, there remains a need for improved treatment options, including more effective combinations with IO therapy.

[0004] Currently, most tumor immunotherapies consist of anti-tumor antibodies, immune checkpoint inhibitors (ICP-B), immunomodulators (cytokines, co-stimulators), and combinations of immune checkpoint inhibitors (ICP-B) and immunomodulators (cytokines, co-stimulators); however, these drugs exhibit off-target tumor toxicity, necessitating improvements in efficacy and safety.

[0005] Despite the high potential of existing IL-2 as an immunotherapeutic agent, it had limitations in that it could not be used in sufficient quantities due to its high toxicity.

[0006] Recently, research results have been reported showing that the combined administration of anticancer antibodies and sustained-release IL-2 can dramatically improve anticancer efficacy.

[0007] The Wittrup group at MIT attempted an approach to localize IL-2 to the tumor by conjugating a tumor-targeting ligand to IL-2, but discovered that tumor targeting did not occur at all because the affinity for the IL-2 receptor was actually higher than that of the tumor cells.

[0008] To overcome these problems, Roche improved the antibody affinity targeting the cancer antigen CEA (carcinoembryonic antigen) to balance the affinity between the tumor targeting ligand and the effector molecule IL-2, used a monomer instead of a dimer for IL-2, and added engineering to the IL-2 substance itself to eliminate binding to receptor α in order to minimize binding affinity.

[0009] Consequently, it was confirmed that the targeting effect on the tumor was enhanced when an IL-2 variant targeted by CEA was engineered, compared to when the CEA-IL-2 wildtype was used. Nevertheless, the tumor targeting effect was not reproduced as expected in clinical practice. In other words, since limited tumor targeting is directly reflected in clinical efficacy, it ultimately failed to lead to dramatic clinical results.

[0010] Ultimately, the conclusion drawn from this case is that the desired results were not achieved because the affinity of the tumor-targeting ligand did not exceed that of IL-2. It can be inferred that if the tumor-targeting affinity could be higher than that of IL-2, the therapeutic index would be maximized by increasing the tumor accumulation of IL-2, thereby improving clinical efficacy. Furthermore, this suggests that achieving such an objective is difficult with existing approaches using monoclonal antibodies.

[0011] [Prior Art]

[0012] Republic of Korea Patent Publication No. 10-2019-0014525

[0013] The present invention was devised to solve the above problems and address the above needs, and the objective of the present invention is to provide a novel tumor immunotherapeutic agent that enhances anti-tumor efficiency and safety through avidity-driven high tumor selectivity, TME repolarization, and in-situ T cell priming.

[0014] To achieve the above objective, the present invention provides a binding protein comprising a first binding domain, a second binding domain and a third binding domain, wherein

[0015] The above-mentioned first binding domain is a domain that specifically binds to a tumor-associated antigen (TAA); the above-mentioned second binding domain is an auxiliary domain that specifically binds to TAA, and simultaneously repolarizes the tumor microenvironment (TME) from tolerogenic to pro-inflammatory while specifically binding to an immune checkpoint protein for in situ T cell priming; and the above-mentioned third binding domain is an effector molecule for inducing T cell response activation and in situ T cell priming within the tumor microenvironment, thereby providing a binding protein.

[0016] In one embodiment of the present invention, it is preferable that the first binding domain, the second binding domain, and the third binding domain include an immunoglobulin binding domain, and it is more preferable that the immunoglobulin binding domain is a human immunoglobulin binding domain, but is not limited thereto.

[0017] In another embodiment of the present invention, the tumor-associated antigen (TAA) is preferably, but is not limited to, one or more tumor-associated antigens selected from the group consisting of Mucin 1, cell surface associated (MUC1), mesothelin (MSLN), ganglioside (GD2), glypican-3 (GPC3), human epidermal growth factor receptor 2 (HER2), carcinoembryonic antigen (CEA), epidermal growth factor receptor and its variants (EGFR / EGFRvIII), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and claudin 18.2.

[0018] In another embodiment of the present invention, the immune checkpoint protein regulates T cell function in the immune system. T cells play a pivotal role in cell-mediated immunity. The immune checkpoint protein interacts with specific ligands that signal T cells and essentially turn off or inhibit T cell function. Cancer cells utilize this system by inducing high expression levels of the immune checkpoint protein, which suppresses the anti-cancer immune response, by causing control of T cells that express immune checkpoint protein receptors on the surface of T cells entering the tumor microenvironment. Thus, inhibition of the immune checkpoint protein by a preparation referred herein as an "immune checkpoint protein inhibitor" or "immune checkpoint inhibitor" will result in the restoration of T cell function and an immune response against cancer cells. Examples of immune checkpoint proteins include, but are not limited to, CD47, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, OX40, B-7 series ligands, or combinations thereof. Preferably, the immune checkpoint inhibitor interacts with a ligand of an immune checkpoint protein that may be CD47, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN15049, CHK1, CHK2, OX40, A2aR, B-7 series ligands, or a combination thereof. Examples of immune checkpoint inhibitors include, but are not limited to, CD47 antagonists, PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, adenosine A2A receptor antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, KIR antagonists, LAG3 antagonists, TIM-3 antagonists, VISTA antagonists, or TIGIT antagonists.

[0019] In another embodiment of the present invention, the effector is preferably, but not limited to, IL-21, IL-2, IL-12, IL-15, CD40L, 4-1BBL, or a combination thereof.

[0020] In another embodiment of the present invention, the binding protein comprises the heavy chain amino acid sequence of SEQ ID NO. 1 or 2 and the light chain amino acid sequence of SEQ ID NO. 3, or

[0021] Comprising the heavy chain amino acid sequence of SEQ ID NO. 4 and the light chain amino acid sequence of SEQ ID NO. 5, or

[0022] It is preferable to include the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO. 7, but if one or more mutations such as substitution, deletion, inversion, or translocation are induced in these amino acid sequences to obtain the binding protein intended for the present invention, said protein is also included within the scope of the present invention.

[0023] In addition, the present invention provides a pharmaceutical composition comprising a binding protein according to the present invention and a pharmaceutically acceptable excipient.

[0024] In one embodiment of the present invention, the composition preferably has an effect on cancer treatment, but is not limited thereto.

[0025] In addition, the present invention provides a method for treating cancer in a subject, comprising the step of administering a binding protein or the composition according to the present invention to a subject who requires it.

[0026] In one embodiment of the present invention, it is preferable to further include the step of administering an antitumor agent to the subject, but is not limited thereto.

[0027] In another embodiment of the present invention, the cancer is preferably selected from the group consisting of pancreatic tumor, liver cancer, lung cancer, esophageal cancer, brain cancer, gastric cancer, and glioblastoma (GBM), but is not limited thereto.

[0028] In another embodiment of the present invention, it is preferable, but not limited to, that the subject has previously been treated with cancer immunotherapy or has been found to be refractory to cancer immunotherapy.

[0029] In another embodiment of the present invention, it is preferable to further include the step of administering an additional antitumor therapy to the subject, wherein the antitumor therapy is preferably, but not limited to, chemotherapy, immunotherapy, treatment by biology or small molecules, vaccination, or cell therapy.

[0030] Methods for preparing the trispecific binding molecule of the present invention and administering it to a subject in need are well known to those skilled in the art or are readily determined by those skilled in the art. The route of administration of the binding molecule may be, for example, oral, parenteral, inhaled, or topical. As used herein, the term parenteral includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. However, in other methods compatible with the teachings of this invention, the binding molecule may be delivered directly to a site of harmful cell populations, which may increase the exposure of the lesioned tissue to the therapeutic agent.

[0031] The binding molecule may be administered in a pharmaceutically effective amount for the treatment of diseases such as certain types of cancer. The pharmaceutical composition may include, for example, a pharmaceutically acceptable carrier comprising water, an ion exchanger, a protein, a buffering material, and a salt. Preservatives and other additives may also be present. The carrier may be a solvent or a dispersion medium. Suitable formulations for use in the therapeutic methods disclosed herein are described in the literature (see: Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980)).

[0032] In any case, a sterile injectable solution may be prepared by incorporating the binding molecule(s) alone or together with another activator in an effective amount into a suitable solvent and then filtration sterilizing. The formulation may also be packaged and sold in kit form. Such manufactured articles may have a label or package insert indicating that the relevant composition is useful for treating subjects suffering from or predisposed to a disease or disorder.

[0033] Parenteral formulations may be a single bolus dose, an infusion, or a maintenance dose following a loading bolus dose. These compositions may be administered at specific fixed or variable intervals, for example, once a week or once a month, or on a "as needed" basis.

[0034] The above composition may be administered as a single dose, multiple doses, or over an established period of infusion. Additionally, the dosage regimen may be adjusted to provide an optimal desired response (e.g., therapeutic or prophylactic response).

[0035] The therapeutically effective dose of a composition for treating such diseases depends on a number of different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, other agents administered, and whether the treatment is prophylactic or therapeutic. Generally, the patient is human, but non-human mammals, including genetically modified mammals, may also be treated. The therapeutic dose may be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

[0036] The amount of at least one binding molecule administered can be easily determined by a person skilled in the art without excessive experimentation. Factors affecting the mode of administration and the respective amount of at least one binding molecule include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the subject receiving treatment. Similarly, the amount of the binding molecule administered will vary depending on the mode of administration and whether the subject receives a single dose or multiple doses of this agent.

[0037] The binding molecule may also be used in the manufacture of drugs for treating types of cancer, including, for example, the cancers listed above.

[0038] A subject treated with the composition described herein may be treatment naive or may be pretreated with one or more other therapies (e.g., at least one other anticancer therapy) before receiving a drug containing a binding molecule. The subject does not need to be a responder to the prior therapy or pretreatment by the therapies. Accordingly, a subject receiving a drug containing a binding molecule may have responded to, responded poorly to, initially responded to, or subsequently failed to respond to, or failed to respond to, one or more prior therapies in which the pretreatment by the prior therapy or the pretreatment consisted of multiple therapies. Accordingly, the present disclosure provides a method for treating a patient who is a poor responder or non-responder to another therapy, comprising the step of administering the binding molecule described herein. A method for overcoming or preventing resistance to cancer therapy, or preventing or delaying recurrence, is also provided, comprising the step of administering the binding molecule as disclosed herein or the composition as described herein.

[0039] Even if a patient has previously been treated with an anticancer agent, a person skilled in the art can determine whether the person did not respond to the agent or was refractory. For example, non-response to an anticancer agent may be reflected in increased cancer / tumor growth and / or increased tumor size, increased formation (or increase) of metastases, or increased number or size of metastases. Non-response may also be the occurrence of tumors or metastases, for example, a shortened time to disease progression after tumor resection, or, for example, in neoadjuvant therapy, an increase in (a) tumor(s) size and / or (a) metastases. Based on these parameters or other parameters known in the art, a group of patients who do not respond to treatment with an anticancer agent may be identified, and patients in this group may then be treated with the binding molecule described herein.

[0040] The binding molecules of the present invention and compositions containing the binding molecules may also be used to treat, for example, patients who are poor responders or non-responders to other therapies. As used herein, the term "non-responder" may refer to an individual / patient / subject who is unlikely to respond to treatment using anticancer agents. As used herein, "low likelihood of response" refers to a reduced likelihood of a pathological complete response occurring in a patient treated with an anticancer agent. In some aspects, the patient may be a good responder initially, and resistance to treatment may develop during treatment with such anticancer agents, leading to a poor response or non-response to treatment.

[0041] The term “good responder” as used herein refers to an individual in whom the tumor does not show growth, metastasis, increase in the number or size of metastases, etc. during or after treatment with anticancer drugs, for example, an individual in whom the tumor does not experience growth, metastasis, increase in the number or size of metastases, etc. over a certain period (e.g., about 1 year after initial diagnosis) based on a series of imaging studies, and / or an individual in whom the tumor does not experience growth, metastasis, increase in the number or size of metastases, etc., and / or an individual in whom the tumor does not experience a certain lifespan (e.g., about 2 years or more after initial diagnosis).

[0042] As used herein, the term "poor responder" refers to an individual in whom, for example, during or immediately after standard treatment using anticancer drugs, the tumor grows, metastasizes, or experiences adverse clinical effects due to the tumor. The term "poor responder" also includes individuals who transitioned from a "good responder" to a "poor responder" during treatment using anticancer drugs.

[0043] If a subject is evaluated as a "non-responder," a "poor responder," or "lowly likely to respond" (e.g., based on the presence of specific biomarkers in cancer cells), the subject may be treated with the binding molecules disclosed herein.

[0044] A method for co-administering a binding molecule as described herein and at least one other therapy is also provided. The binding molecule and at least one other therapy may be co-administered together as a single composition, or co-administered together as separate compositions at the same time or at overlapping times. In some aspects, the binding molecule may be used as an adjuvant therapy.

[0045] The binding molecule can also be used in the manufacture of a drug for treating a subject suffering from cancer, wherein the binding molecule is administered before the subject is treated with at least one other therapy.

[0046] Binding molecules may also be used in a method to prevent or reduce the risk of cancer in a subject by administering an effective amount of binding protein or a composition containing binding protein to the subject. The cancer may be pancreatic tumor, liver cancer, lung cancer, esophageal cancer, brain cancer, gastric cancer, and glioblastoma (GBM), and the patient may be remission from previously diagnosed and / or previously treated cancer. The patient may be considered at risk of cancer due to environmental exposure, tobacco use or exposure, genetic mutations, or a family history of cancer.

[0047] The present invention will be described below.

[0048] This invention sought to approach the method of enhancing tumor targeting using the concept of avidity rather than affinity.

[0049] Most of the tumor antigens we encounter are tumor-associated antigens (TAAs). Therefore, the key consideration for enhancing tumor selectivity by distinguishing between tumors and normal tissues based on TAAs is that the density of TAAs is significantly higher than that of normal cells. When a tumor targeting ligand binds as a monomer to TAAs, which show low expression in normal tissues, it exhibits low affinity; however, when it binds multivalently to tumor cells with high TAA density, multimeric affinity accumulates, leading to an increase in avidity. Ultimately, this serves as the distinguishing factor for tumor cells compared to normal tissues.

[0050] In contrast to existing bispecific antibodies that combine a tumor targeting ligand and an effector in a 1:1 ratio, the present invention adopts an approach that increases avidity by adding a tumor co-targeting ligand to the tumor targeting ligand. Thus, a total of three elements are loaded, including three components: a tumor targeting ligand, a co-targeting ligand, and an effector molecule. Ultimately, the key to avidity-enhanced tumor targeting is that the avidity—the binding strength between the tumor targeting and the co-targeting ligand—must be stronger than the affinity of the effector molecule.

[0051] The hypothesis is that avidity-enhanced tumor targeting increases target selectivity, which ultimately reduces on-target off-tumor effects, and that this increased therapeutic index acts as enhanced clinical efficacy. To achieve this approach, a substance with three functions was incorporated and named TRION, a Tri-specific Immuno-Oncology substance (Fig. 1).

[0052] The inventors anticipate that the biodistribution of TRION will involve increased selective accumulation in tumors due to its enhanced tumor binding affinity compared to existing bispecific antibodies. Furthermore, avidity-enhanced tumor targeting will result in an improved therapeutic index proportional to the increased avidity. Therefore, the distinguishing feature of TRION is that, in the case of I / O substances such as IL-2, where sufficient clinical doses cannot be used due to systemic toxicity, higher doses can be administered in proportion to the increase in the therapeutic index, thereby leading to enhanced clinical efficacy.

[0053] Furthermore, how to repolarize the immunosuppressive tumor microenvironment (TME) and overcome tumor heterogeneity is an approach related to myeloid-derived suppressor cells (MDSCs), which constitute the largest portion of the tumor environment.

[0054] In general, in solid tumors, MDSCs account for ~80%, and among them, tumor-associated macrophages (TAMs) account for 70~80%.

[0055] If tumor-infiltrating immune cells are isolated and examined from day 5 to day 21 after tumor injection, CD4 cells necessary for anti-cancer immunity + & CD8 + In the case of T cells, their numbers increase until the 12th, but from the 15th onwards, they actually show a decreasing trend. Conversely, TAMs increase exponentially. In other words, as the solid tumor grows, the proportion of TAMs relative to T cells increases dramatically overall.

[0056] At this time, the characteristics of TAM exhibit an M2 phenotype that inhibits T cell proliferation. Therefore, while the anti-tumor antibody shows high anticancer efficacy when administered before day 10, if administered on day 15 or 19, the anti-tumor antibody is almost ineffective due to the strong immunosuppressive function caused by TAM. However, if TAM is removed with clodronate, the anticancer efficacy of the anti-tumor antibody is restored. Ultimately, removing TAM, or further repolarizing its characteristics in a direction that enhances T cell function, will lead to an increase in anticancer efficacy.

[0057] Next, regarding tumor heterogeneity, it may be possible to overcome it by inducing T cell immunity capable of recognizing and eliminating even very small amounts of tumor cells. By increasing phagocytosis by dendritic cells (DCs) as well as macrophages through the repolarization of TAMs, new tumor-specific T cells are induced through in situ T cell priming against cancer antigens, thereby ultimately expanding the breadth of anti-tumor immunity and serving as a method to overcome tumor heterogeneity.

[0058] In this context, the target selected in the present invention is CD47.

[0059] Whether macrophage phagocytosis occurs is determined by the balance between pro-phagocytic and anti-phagocytic signals. Among these, CD47 is widely distributed in normal tissue as an anti-phagocytic signal called the 'don't eat me' signal. However, because this expression is overexpressed in tumors, it acts as an innate immune checkpoint molecule mediated by the tumor.

[0060] Blocking CD47 signaling increases phagocytosis by macrophages, leading to an increase in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, it increases phagocytosis not only in macrophages but also in dendritic cells (DCs). Although DC phagocytosis contributes less to cancer cell elimination compared to macrophages, a more significant point is that DCs are responsible for antigen cross-presentation and in situ T-cell priming, which are crucial for T-cell priming. Consequently, blocking CD47 innate immune checkpoint signals acts as a bridge connecting in situ T-cell priming within the tumor by DCs to anti-tumor adaptive immunity.

[0061] Pancreatic tumors, considered the most challenging type of cancer for treatment, also have a very low clinical response rate to immune checkpoint blockade, which is known to be attributed to immunosuppressive TME.

[0062] The expression pattern of CD47 in pancreatic tumors is closely linked to the expression of TAMs. To investigate the mechanism of TME repolarization—more specifically, TAM repolarization—when blocked with anti-CD47, single-cell RNA sequencing was performed using tumor-infiltrating immune cells. The results showed that while the control group exhibited the M2 phenotype in macrophages, treatment with anti-CD47 caused a shift to the M1 phenotype; similarly, lymphoid cells were found to shift to a more activated phenotype. In conclusion, anti-CD47 alone converts the phenotypes of lymphoid and myeloid tumor-infiltrating immune cells toward anti-tumor immunity, and also [addresses] the T cells present in tumor-infiltrating lymphocytes (TILs). reg It reduces the inhibitory effect even on regulatory T cells. In addition, this TME repolarizing function leads to a synergistic increase in anti-tumor immunity when combined with immune checkpoint inhibitors that act on TdLNs (tumor-draining lymph nodes).

[0063] To determine MoA, when comparing DCs present in the tumor draining lymph node with DCs present in the tumor, CD8s present in the tumor due to DCs present in the tumor + The results showed that T cells are primed. In other words, while the site of T cell reinvigoration for the efficacy of existing immune checkpoint inhibitors is known to be the tumor draining lymph node, this suggests that anti-CD47 functions within the tumor meridian. Therefore, by presenting the conclusion that blocking CD47 signals is more effective when targeting the tumor rather than the TdLN, this supports the justification for selecting CD47 as a candidate target for tumor-targeting TRION.

[0064] It is currently being developed as an anti-CD47 monotherapy approach related to anti-CD47 blockade.

[0065] In contrast, the approach of the present invention is distinguished in that it seeks synergy with T cell stimulating effector substances along with tumor targeting of anti-CD47.

[0066] Next, in addition to the function of anti-CD47, IL-2 is additionally selected in the present invention.

[0067] The triple-specific immunotumor therapeutic agent of the present invention maximizes the therapeutic index through avidity-driven high tumor selectivity, enhances anti-tumor efficacy in cold tumors through TME repolarization and T cell priming, and due to these characteristics, will be the best combination partner for PD-1 blockade,

[0068] Given that it can be applied to patients refractory to existing immunotherapy and patients with recurrent cancer, and that its application can be expanded to various solid tumors by replacing cancer antigens, the triple-specific immuno-oncology therapeutic agent of the present invention will become a safe and highly effective next-generation I / O anticancer treatment if it leads to an increase in the therapeutic index in clinical practice.

[0069] Figure 1 is a conceptual diagram of TRION, a tri-specific immuno-oncology substance.

[0070] Figure 2 is a schematic diagram and expression cassette of three types of TRION antibodies (TRION-101', TRION-102', TRION-103'),

[0071] Figure 3 shows a schematic diagram and expression cassette of four types of control antibodies (TRION-C1, TRION-C2, Cantuzumab, Magrolimab),

[0072] Figure 4 shows the SDS-PAGE analysis results of three types of TRION antibodies (TRION-101', TRION-102', TRION-103') and four types of control antibodies (TRION-C1, TRION-C2, Cantuzumab, Magrolimab),

[0073] Figure 5 shows the results of the CD47-GST animal cell expression cassette and SDS-PAGE analysis,

[0074] Figure 6 confirms the CD47 binding affinity of TRION antibodies via ELISA.

[0075] Figure 7 confirms the IL-2Rβ binding affinity of TRION antibodies via ELISA.

[0076] Figure 8 is a figure confirming the binding affinity of TRION antibodies to MUC-1 via ELISA, showing that TRION antibodies selectively recognize hypoglycosylated tumor-type MUC-1.

[0077] Figures 9 to 11 illustrate that Capan2 is expressed for both MUC-1 and CD47, where Fig. 9) affinity of α-MUC-1 for Capan2 (Triplicate), Fig. 10) affinity of α-CD47 for Capan2 (Triplicate), and Fig. 11) affinity of TRION-103' for Capan2, where y-axis: normalized / 100%: largest value in each data point / 0%: smallest value in each data point, and Capan2-TRION-103' no triplicate.

[0078] Figures 12 to 14 show that SNU407 expresses only CD47 (Triplicate).

[0079] Fig. 12) Affinity of a-MUC-1 for SNU407 Fig. 13) Affinity of a-CD47 for SNU407 Fig. 14) Affinity of TRION-103' for SNU407, y-axis: normalized / 100%: largest value in each data point / 0%: smallest value in each data point,

[0080] Figures 15 to 17 are figures showing that Caov3 expresses only CD47 (Triplicate),

[0081] Fig. 15) Affinity of a-MUC-1 for Caov3, Fig. 16) Affinity of a-CD47 for Caov3, Fig. 17) Affinity of TRION-103' for Caov3, y-axis: normalized / 100%: largest value in each data point / 0%: smallest value in each data point,

[0082] Figures 18 to 20 illustrate that Colo205 is expressed (triplicated) for both MUC-1 and CD47, with Figure 18) affinity of α-MUC-1 for Colo205, Figure 19) affinity of α-CD47 for Colo205, and Figure 20) affinity of TRION-103' for Colo205. All cell-based binding assay data were analyzed by flow cytometry. y-axis: normalized / 100%: largest value in each data point / 0%: smallest value in each data point.

[0083] Figures 21 and 22 show the dependence of TRION on MUC-1. From the results showing a decrease in binding affinity when Colo205 (Figure 21) and Capan2 (Figure 22) tumor cell lines were pretreated with blocking for MUC-1 and CD47, respectively, it can be seen that the binding strength to MUC-1 and CD47 accumulates and increases. From the results showing that the decrease in binding strength is greater in the case of MUC-1 blocking pretreatment compared to CD47 blocking pretreatment, it can be seen that MUC-1 binding contributes more significantly to the total accumulated binding strength.

[0084] FIGS. 23 to 25 illustrate the binding affinity of TRION-101' to each PBMC cell at different concentrations (based on concentration).

[0085] FIGS. 26 to 30 illustrate the binding affinity of TRION-101' at different concentrations according to each PBMC cell type (based on cell type).

[0086] FIGS. 31 and 32 are MUC-1 + CD47 + Capan2, MUC-1 + CD47 + This is a figure indirectly comparing the binding affinity of TRION-101' to the Colo205 tumor cell line.

[0087] Figure 33 illustrates the higher affinity of TRION-101' for tumor cells. As can be seen in Figure 33, TRION-101' binds to tumor cells (Capan-2, COLO205) with a KD of less than nanomolar (~0.2 nM), whereas its affinity for immune cells is 10 to 30 times lower (KD ~2.7–7.2 nM). This demonstrates superior tumor selectivity based on differential ligand expression and affinity balance.

[0088] FIGS. 34 and 35 illustrate the target selectivity of the TRIONs of the present invention in a mixture of RBCs and tumor cells, wherein TRION-101′ of the present invention has a binding affinity (K) approximately 100 times stronger for tumor cells compared to RBCs. D(RBC) / K D(Capan2 The values ​​were approximately 200 for Cantuzumab (aMUC-1), approximately 1 for Magrolimab (aCD47), and approximately 100 for TRION-101'(aMUC-1xaCD47xIL2v), confirming a tumor-selective targeting profile.

[0089] FIGS. 36 to 39 are PBMCs of TRIONS of the present invention Act As shown in the figure illustrating target selectivity in the mixture of & COLO205, the present invention confirmed that TRION-101′ had a binding affinity for tumor cells that was 40 to 90 times higher than that of immune cells, and confirmed tumor-selective targeting characteristics.

[0090] Sequential Engagement in the diagram: Conditional immunotherapy, if MUC-1 (tumor target), CD47 binding (TME reprogramming) and IL-2 action,

[0091] In conclusion, while Cantuzumab in Fig. 36 showed a pattern of specifically binding only to tumor cells, Magrolimab in Fig. 37 exhibited broad binding characteristics, binding nonspecifically to both tumor cells and immune cells. In contrast, TRION-101′ in Fig. 38 demonstrated a sequential binding mechanism in which it first binds to tumor cells and then induces bridging with immune cells. This supports the concept that TRION-101′ is a conditional immunotherapeutic agent that activates only intratumoral anticancer cells (in-vivo TILs), binding to CD47 and secreting IL-2 only when targeted at tumor tissue.

[0092] The present invention will be described in more detail below through non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention shall not be interpreted as being limited by the following examples.

[0093] Example 1: Cloning of TRION antibody and control antibody

[0094] Three types of TRION antibodies, which are multi-target antibody therapeutics, were designed by combining cantuzumab targeting human MUC-1, magrolimab scFv targeting human CD47, and human IL-2v, an IL-2 variant that induces T-cell activation (Fig. 2). Magrolimab scFv was connected to the light chain end of cantuzumab using three GGGGS linkers (1': pMAZ-cantuzumab LC-magrolimab scFv), and two heavy chains of TRION-101' (2: pMAZ-cantuzumab HC Knob, 3: pMAZ-cantuzumab HC Hole-IL2v) were designed to be conjugable during antibody production using Knob into Hole technology.

[0095] As controls, control antibodies (TRION-C1, TRION-C2) of TRION-101' and TRION-102' that did not contain Magrolimab scFv, and IgG-form antibodies for Cantuzumab and Magrolimab were designed (Fig. 3). Although Magrolimab is an IgG4 antibody, it was constructed by introducing the same IgG1 Fc as the TRION antibody to exclude the influence of Fc. All heavy and light chain genes were synthesized using primer assembly and ligated into the pMAZ-IgL-GlycoT vector using BssHII and XbaI restriction enzyme cleavage sites to be expressed in animal cells (Figs. 2, 3, Table 1).

[0096] Antibody Name Antibody Part Sequence (Amino Acid) TRION-101'HCKnob Sequence Number 1QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHoleSEQ ID NO.2QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTLC 서열번호3DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKTRION-102'HC 서열번호4QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTLC 서열번호5DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKTRION-103'HC 서열번호 6QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLC 서열번호7DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKTRION-C1HCKnob서열번호 8QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHole서열번호9QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTLC 서열번호 10DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECTRION-C2HC 서열번호11QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTLC 서열번호 12DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECMagrolimabHC 서열번호13QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLC 서열번호 14DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECCantuzumabHC 서열번호15QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMGWIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYNWYFDVWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLC sequence number 16DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGTK LELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0097] Table 1 shows the amino acid sequences of three types of TRION antibodies (TRION-101', TRION-102', TRION-103') and four types of control antibodies.

[0098]

[0099] Example 2: Antibody production in animal cells

[0100] The three previously prepared TRION antibodies and the four control antibodies' heavy and light chain animal cell expression plasmids were mixed in a 1:1 ratio, and PEI-MAX ®(Polysciences) and the antibody gene were mixed in a 4:1 ratio and reacted at room temperature for 20 minutes. The previous day 2 × 10 6 Expi293F animal cells, subcultured at a density of cells / ml, were transfected and incubated in a shaking CO2 incubator at 37°C, 125 rpm, and 8% CO2 for 7 days. After incubation, the supernatant was separated by centrifugation, 12.5 ml of 25× PBS was added to equilibrate, and the solution was filtered through a 0.2 μm bottle-top filter (Merck Millipore). ProA was added to the filtered culture medium. TM 1 ml of rProtein A resin (Amicogen) was added and stirred at 4°C for 16 hours, after which the resin was recovered using an open column. Subsequently, the solution was washed with 5 ml of 1× PBS, and the protein was eluted using 3 ml of 100 mM glycine (pH 2.7). The solution was then neutralized with 1 ml of 1 M Tris-HCl (pH 8.0), and buffer exchange was performed with PBS using a centrifugal filter unit (30K; Merck Millipore). The size and purity of the purified antibody proteins were analyzed by 12% SDS-PAGE under non-reducing and reducing conditions (Fig. 4).

[0101] Example 3: CD47-GST Antigen Cloning and Production

[0102] The CD47 (amino acids 19-141) antigen was synthesized using primer assembly and fused with GST via assembly PCR to construct a dimeric antigen. Subsequently, it was ligated into the pMAZ-IgL-GlycoT vector using BssHII and XbaI restriction enzyme cleavage sites and designed to be expressed in animal cells. PEI-MAX ®After mixing the CD47-GST animal cell expression plasmid constructed with (Polysciences) in a 4:1 ratio and reacting at room temperature for 20 minutes, the previous day 2 × 10 6 Transfection was performed on 300 ml of Expi293F animal cells subcultured at a density of cells / ml, and the cells were cultured in a shaking CO2 incubator at 37°C, 125 rpm, and 8% CO2 for 7 days. After separating only the supernatant by centrifugation, 12.5 ml of 25× PBS was added to equilibrate, and the mixture was filtered through a 0.2 μm filter using a bottle top filter (Merck Millipore). 1 ml of glutathione agarose 4B resin (Becton Dickinson Diagnostic Systems, Sparks) was added to the filtered culture medium and stirred at 4°C for 16 hours, after which the resin was recovered using an open column. Subsequently, the sample was washed with 5 ml of 1× PBS, eluted with 2.5 ml of 50 mM Tris-HCl (pH 8.0) and 10 mM reduced glutathione (SIGMA), and buffer exchange was performed with PBS using a centrifugal filter unit (3K; Merck Millipore). The size and purity of the purified CD47-GST antigen protein were analyzed by SDS-PAGE under non-reducing (NR) and reducing (R) conditions (Fig. 5).

[0103] Example 4: Analysis of CD47 binding affinity of TRION antibodies via ELISA

[0104] The prepared dimeric form of CD47-GST was diluted to a concentration of 4 μg / ml with 0.05 M Na2CO3 (pH 9.6), dispensed in 50 μl aliquots into Flat Bottom Polystyrene High Bind 96-well microplates (Costar), and immobilized at 4°C for 16 hours. Subsequently, the plates were blocked for 1 hour at room temperature using 100 μl of 4% skim milk (0.05% PBST, pH 7.4), followed by 4 washes with 150 μl of 0.05% PBST. Six antibodies (TRION-101', TRION-102', TRION-103', TRION-C1, TRION-C2, Magrolimab) were serially diluted starting from 200 nM, dispensed in 50 μl aliquots into each well, and incubated at room temperature for 1 hour. 50 μl of Protein A-HRP (1:20,000; GenScript) was added and reacted at room temperature for 1 hour, after which 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to induce color development. The reaction was terminated by adding 50 μl of 2 M H2SO4, and the binding affinity to CD47 was analyzed by measuring the absorbance at 450 nm using an Epoch Microplate Spectrophotometer (BioTek) (Fig. 6).

[0105] Example 5: Analysis of IL-2Rβ binding affinity of TRION antibodies via ELISA

[0106] Recombinant Human IL-2R beta Protein (R&D Systems; Cat. No.: 224-2B-025) was diluted to a concentration of 4 μg / ml with 0.05 M Na2CO3 (pH 9.6), 50 μl was dispensed into Flat Bottom Polystyrene High Bind 96-well microplates (Costar), and immobilized at 4°C for 16 hours. Afterward, the plates were blocked at room temperature for 1 hour using 100 μl of 4% skim milk (0.05% PBST, pH 7.4), followed by 4 washes with 150 μl of 0.05% PBST. Seven types of antibodies (TRION-101', TRION-102', TRION-103', TRION-C1, TRION-C2, Cantuzumab, Magrolimab) were serially diluted starting from a concentration of 200 nM, 50 μl was dispensed into each well, and the reaction was carried out at room temperature for 1 hour. After adding 50 μl of Protein A-HRP (1:20,000; GenScript) and reacting at room temperature for 1 hour, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to induce color development. The reaction was terminated by adding 50 μl of 2 M H2SO4, and the binding affinity to IL-2Rβ was analyzed by measuring the absorbance at 450 nm using an Epoch Microplate Spectrophotometer (BioTek) (Fig. 7).

[0107] Example 6: Analysis of the binding affinity of TRION antibodies against MUC-1

[0108] Recombinant human MUC-1 Fc (R&D Systems; Cat. No.: 10332-MU-050) was diluted to a concentration of 4 μg / ml with 0.05 M Na2CO3 (pH 9.6), dispensed into 50 μl aliquots onto a Flat Bottom Polystyrene High Bind 96-well microplate (Costar), and immobilized at 4°C for 16 hours. Subsequently, the plates were blocked at room temperature for 1 hour using 100 μl of 4% skim milk (0.05% PBST, pH 7.4), followed by 4 washes with 150 μl of 0.05% PBST. Six types of antibodies (TRION-101', TRION-102', TRION-103', TRION-C1, TRION-C2, Cantuzumab) were serially diluted starting from a concentration of 200 nM, 50 μl was dispensed into each well, and the reaction was carried out at room temperature for 1 hour. After adding 50 μl of HRP Anti-Kappa light chain antibody (1:5000; Abcam) and reacting at room temperature for 1 hour, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to induce color development. The reaction was terminated by adding 50 μl of 2 M H2SO4, and the binding affinity to MUC-1 was analyzed by measuring the absorbance at 450 nm using an Epoch Microplate Spectrophotometer (BioTek) (Fig. 8).

[0109]

[0110] Example 7: Cell-based binding assay

[0111] Cell lines, antibodies, and reagents

[0112] All cell lines were purchased from the Korea Cell Line Bank. (Capan-2, Colo205, Caov3, SNU407)

[0113] FACs buffer was prepared by mixing DPBS (WELGENE) and 5% FBS (Cytiva).

[0114] anti-MUC1(huC242), anti-CD47(Magrolimab), TRION-103', and TRION-101' were prepared as in the above-mentioned embodiment of the present invention.

[0115] Propidium Iodide (PI) was purchased from eBioscience.

[0116] Alexa Fluor® 647 AffiniPure™ Goat Anti-Human IgG (cat#: 109-605-008) was purchased from Jackson ImmunoResearch.

[0117] Cell-based binding assay

[0118] Cells were washed twice with cold FACs buffer and seeded into 96-well plates at a density of 3 x 10^4 cells / well. After centrifugation at 1750 rpm at 4°C for 3 minutes, the supernatant was removed.

[0119] 100 µl / well of primary antibody was incubated at 4°C for 30 minutes. The primary antibody was diluted 1 / 3 and incubated at several points.

[0120] After 30 minutes, 100 µl of FACs buffer was added, and the cells were centrifuged at 1750 rpm at 4°C for 3 minutes. The cells were inoculated with a secondary fluorescent antibody for 30 minutes in a light-free environment at 4°C. After 30 minutes, the cells were centrifuged at 1750 rpm at 4°C for 3 minutes after adding 100 µl of FACs buffer, and then resuspended. PI was treated at a 1000:1 ratio, and the data were verified via flow cytometry.

[0121]

[0122] The results of the cell-based binding assay above are summarized in Figures 9 to 20 and Tables 2 to 3.

[0123] a-MUC1a-CD47TRION103'SNU 407x0.1222(0.2056)6.811(11.459)Caov-3x0.5133(0.2056)3.856(1.544)Capan-20.0540.20560.2030Colo 2050.12180.15540.2667

[0124] Table 2 shows the estimated binding affinity of TRION-103' by correcting the binding affinities of SNU 407 and Caov-3 for CD47 binding affinity to Capan-2. Table 2 demonstrates that compared to SNU 407 and Caov-3 cell lines expressing only CD47, Capan-2 and Colo205 cell lines expressing both MUC-1 and CD47 show higher binding affinity of TRION-103' (6.8 nM, 3.9 nM vs. 0.20 nM, 0.27 nM). When the affinity for anti-CD47 is corrected by the values ​​of Capan-2 (0.12nM → 0.21nM; 0.51nM → 0.21nM) and the binding strength of Caov-3 to TRION-103' is estimated at 11.46nM and 1.54nM, it can be seen that when MUC-1 binding is combined with CD47 binding, the values ​​increase by 56 times and 8 times, respectively (Table 2).

[0125] a-MUC1a-CD47TRION103'SNU 407x0.1222(0.1554)6.811(8.66)Caov-3x0.5133(0.1554)3.856(1.167)Capan-20.0540.20560.2030Colo 2050.12180.15540.2667

[0126] Table 3 shows the estimated binding strength of TRION-103' by correcting the binding strengths of SNU 407 and Caov-3 with the CD47 binding strength to Colo205,

[0127] Using the same method applied in Table 2, it can be estimated that the total cumulative binding strength increases by 32 times and 4 times, respectively, with the addition of MUC-1 binding strength when corrected for CD47 binding strength to Colo205 (Table 3).

[0128] From the above results, it appears that the binding of TRION depends heavily on MUC1, as the addition of MUC-1 significantly increases the binding of TRION.

[0129] Pretreatment blocking assay

[0130]

[0131] Cells were washed twice with cold FACs buffer and seeded into 96-well plates at a density of 5 x 10^4 cells / well. After centrifugation at 1750 rpm at 4°C for 3 minutes, the supernatant was removed. Cells were either pretreated with an overconcentration of antibody for 15 minutes at 4°C or were not pretreated.

[0132] Subsequently, the supernatant was removed after centrifugation at 4°C and 1750 rpm for 3 minutes. Fluorescent TRION-101' was diluted to various concentrations and applied. After incubation at 4°C for 25 minutes, the samples were washed three times with cold FACs buffer. After resuspension with FACs buffer, PI was added at a 1000:1 ratio, and the data were analyzed via flow cytometry.

[0133] The results are shown in Figures 21 to 22.

[0134] Figures 21 and 22 show the dependence of TRION on MUC-1. From the results showing a decrease in binding affinity when a) Col205 and b) Capan2 tumor cell lines were pretreated with blocking for MUC-1 and CD47, respectively, it can be seen that the binding strength to MUC-1 and CD47 accumulates and increases. From the results showing that the decrease in binding strength is greater in the case of MUC-1 blocking pretreatment compared to CD47 blocking pretreatment, it can be seen that the cumulative binding strength of MUC-1 is more important.

[0135] Example 8: PBMC binding study_FACS

[0136] Cell thawing and staining

[0137] After thawing the frozen PBMC and performing cell counting, the cells were resuspended using 10% FBS RPMI-1640 media at a concentration of 1 x 10^6 PBMCs / ml.

[0138] 2 x 10^6 PBMCs were transferred to a FACS tube, centrifuged (400G, 5 min), and resuspended in 80 µl of PBS.

[0139] antibody staining

[0140] The Ab mixture was calculated to be 20 µl of BV buffer + 2 µl of L / D + 1–2 µl of FACS antibody per sample tube. The FACS panel is as shown in Table 4 below, and the Live / Dead cell staining dye was used after diluting it with BV buffer at a ratio of 1:19.

[0141] Trion surfaceViolet Laser(405nm)Blue laser (488nm)450 / 50525 / 50610 / 20660 / 20710 / 50780 / 60530 / 30695 / 40BV421BV510BV605BV650BV711BV786FITCPerCP-Cy5.5AntibodyCD14CD4CD567AADCompanyCloneCatalogue No.Etc.ERERHWTrion surfaceYellow-Green (561nM)Red laser (640 nm)586 / 15610 / 20670 / 30780 / 60660 / 20730 / 45780 / 60PEPE-TRPE-Cy5PE-Cy7APCA700APC-H7AntibodyCD3CD19CD8secondaryCD16CompanyBDClone1D3Catalogue No.562291Etc.SYSYHW

[0142] The pre-prepared FACS antibody mixture was added to the PBMC and stained at RT for 15 minutes. After washing with 2 ml of FACS buffer, centrifugation (400 G, 5 min) was performed, followed by resuspension with 100 µl of FACS buffer.

[0143] TRION-101' binding / staining

[0144] PBMC samples were treated with TRION-101 at concentrations of 100 nM / 50 nM / 10 nM / 1 nM / 0.33 nM / 0.1 nM / 0.01 nM. The samples were incubated on ice for 30 minutes. After washing with 2 ml of FACS buffer and centrifuging (400 g, 5 min), the samples were resuspended with 200 µl of FACS buffer, treated with APC-human IgG secondary antibody, incubated on ice for 30 minutes, washed with 2 ml of FACS buffer, centrifuged (400 g, 5 min), and resuspended with 300 µl of FACS buffer before analysis by flow cytometry.

[0145] The binding affinity of TRION-101' at different concentrations according to each PBMC cell type is shown in Figures 23 to 25. As can be seen in Figures 23 to 25, significant binding was observed only at concentrations of 3.3 nM or higher, regardless of the cell type, and no binding was observed at 1 nM or lower.

[0146] In addition, the binding affinity of TRION-101' at different concentrations according to each PBMC cell type is shown in Figures 26 to 30. As can be seen in Figures 26 to 30, significant binding was observed only at concentrations of 3.3 nM or higher, regardless of the cell type, and no binding was observed at 1 nM or lower.

[0147] Also MUC-1 + CD47 + Capan2, MUC-1 + CD47 + The binding affinity of TRION-101' to Colo205 tumor cell lines was indirectly compared in Figures 31 to 32.

[0148] For reference, TRION-103' is a substance without IL-2 from 101', as can be seen in Figure 2, and has the same tumor targeting performance as TRION-101'.

[0149] As can be seen in FIGS. 31 and 32, the degree of binding of TRION-103' to each tumor cell line at a concentration of 1 nM is estimated to be 85% for Capan2 and 82% for Colo205, and

[0150] While TRION-101' does not bind to each immune cell of PBMC at 1 nM, MUC-1 + CD47 + Binding to tumor cell lines is estimated to be over 80% at 1 nM. Based on these results, it is inferred that TRION-101' is more advantageous for tumor targeting as its affinity balance is skewed toward tumor cells.

Claims

1. A binding protein comprising a first binding domain, a second binding domain and a third binding domain, The first binding domain above is a domain that specifically binds to a tumor-associated antigen (TAA); The second binding domain is an auxiliary domain that specifically binds to TAA, and at the same time, a domain that specifically binds to an immune checkpoint protein for in situ T cell priming while repolarizing the tumor microenvironment (TME) from tolerogenic to pro-inflammatory; and The above-mentioned third binding domain is a binding protein that is an effector molecule for inducing T cell response activation and in situ T cell priming within the tumor microenvironment.

2. A binding protein according to claim 1, wherein the first binding domain, the second binding domain, and the third binding domain comprise an immunoglobulin binding domain.

3. A binding protein according to paragraph 2, wherein the immunoglobulin binding domain is a human immunoglobulin binding domain.

4. A binding protein according to claim 1, wherein the tumor-associated antigen (TAA) is one or more tumor-associated antigens selected from the group consisting of Mucin 1, cell surface associated (MUC1), mesothelin (MSLN), ganglioside (GD2), glypican-3 (GPC3), human epidermal growth factor receptor 2 (HER2), carcinoembryonic antigen (CEA), epidermal growth factor receptor and its variants (EGFR / EGFRvIII), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and claudin 18.

2.

5. In claim 1, the immune checkpoint protein is a binding protein that is CD47, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, OX40, B-7 series ligand, or a combination thereof.

6. In claim 1, the effector molecule is a binding protein that is IL-21, IL-2, IL-12, IL-15, CD40L, 4-1BBL, or a combination thereof.

7. In claim 1, the binding protein comprises the heavy chain amino acid sequence of SEQ ID NO. 1 or 2 and the light chain amino acid sequence of SEQ ID NO. 3, or Comprising the heavy chain amino acid sequence of SEQ ID NO. 4 and the light chain amino acid sequence of SEQ ID NO. 5, or A binding protein comprising the heavy chain amino acid sequence of SEQ ID NO. 6 and the light chain amino acid sequence of SEQ ID NO.

7.

8. A pharmaceutical composition comprising a binding protein according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. In claim 8, the above composition is a composition for treating cancer.

10. A method for treating cancer in a subject, comprising the step of administering a binding protein according to any one of claims 1 to 7 or a composition according to claim 8 to a subject in need thereof.

11. A method according to claim 10, further comprising the step of administering an antitumor agent to the subject.

12. The method according to claims 10 and 11, wherein the cancer is selected from the group consisting of pancreatic tumor, liver cancer, lung cancer, esophageal cancer, brain cancer, gastric cancer, and glioblastoma (GBM).

13. The method according to claim 10 or 11, wherein the subject has previously been treated with cancer immunotherapy or has been found to be refractory to cancer immunotherapy.

14. A method according to claim 10, further comprising the step of administering additional antitumor therapy to the subject.

15. A method according to claim 14, wherein the antitumor therapy is chemotherapy, immunotherapy, treatment by biology or small molecules, vaccination, or cell therapy.

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