A novel method of selective cell killing

Abstract

The present invention relates to a novel unconventional method of cell killing for selectively killing or / and inhibiting the growth of target cells. This unconventional method is to kill target cells by persistently over-accumulating organelle-localization sequence fusion antibodies (OLS fusion antibodies) in a key organelle of the target cells with the purpose of clogging or over-occupying the key organelle. The OLS fusion antibodies comprise a cell-binding domain and an organelle-localization sequence (OLS) without any cytotoxic payload. The cell-binding domain of the OLS fusion antibodies functions to selectively bind to a cell-surface antigen of the target cells. The OLS fragment of the OLS fusion antibodies functions to direct the OLS fusion antibodies to a specific organelle of the target cells. The present invention first demonstrates that selectively and persistently accumulating OLS fusion antibodies in a key organelle of the target cells can kill or / and inhibit the growth of the target cells.

Description

Technical Field

[0001] This invention relates to an unconventional novel cell killing method for selectively killing cells expressing specific cell surface antigens. Background Technology

[0002] Cancer is the second leading cause of death worldwide (Lancet 2016; oct 8; 388:1459-14544). Current cancer treatment faces at least four obstacles. The first obstacle is that existing targeted cancer therapies, in addition to killing cancer cells, also kill normal cells, often leading to serious side effects, sometimes even life-threatening ones. Commonly used targeted cancer drugs include: antibodies (Abs), antibody-drug conjugates (ADCs), bispecific T cell engagers (BiTEs), chimeric antigen receptor T cells (CAR-Ts), and tyrosine kinase inhibitors (TKIs). These drugs target both cancer cells and normal cells simultaneously because, in most cases, the antigens they target are expressed in both cancer cells and normal cells. The difference usually lies only in the level of expression: cancer cells highly express the target antigen, while normal cells express it at low levels (Leukemia volume 34, pages 985–1005 (2020); Mol Biol Int. 2014; 852748). Existing targeted cancer therapies (Ab, ADC, BiTE, CAR-T, or TKI) cannot effectively distinguish between normal cells expressing low levels of the target antigen and cancer cells expressing high levels of the target antigen.

[0003] Redundancy in cellular signaling pathways is the second major obstacle in current cancer treatment. Existing targeted cancer therapies often target only a single signaling pathway. However, cellular evolution has taken at least 3.8 billion years (The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sunderland (MA): Sinauer Associates; 2000). To survive, cells cannot rely on a single signaling pathway but have evolved redundant signaling pathway elements that can be substituted for each other to cope with changes in the survival environment. These redundant signaling pathway elements form new, intersecting signaling pathways, enabling cancer cells to overcome the therapeutic effects of drugs that target only a single specific signaling pathway (Cancer Res. 2015 Mar 1; 75(5): 808–812). Redundancy in cellular signaling pathways often leads to cancer patients developing resistance to current cancer drugs that target only a single signaling pathway in a very short time. For example, many cancer patients who received trastuzumab (a targeted drug that acts on the HER2 signaling pathway) showed initial efficacy, but developed resistance within just one year (N Engl J Med. 2001; 344:783-92; N Engl J Med. 2012; 366:109-19). The failure or weakening of a patient's normal immune function is the third obstacle currently facing cancer treatment. Many cancers develop and progress alongside the failure or weakening of the normal immune system in cancer patients (Nature Reviews Cancer Volume 21, pages 345–359 (2021)). Currently used targeted cancer therapies (such as Ab-, BiTE-, or CAR-T-based drugs) rely on the synergistic effect of the normal immune system to kill cancer cells. If a cancer patient's immune system is failed or weakened, Ab / BiTE / CAR-T-based targeted cancer drugs will not be able to effectively exert their therapeutic effect. The failure or weakening of normal immune function is one of the reasons why some cancer patients are unsuitable for targeted cancer drug therapy.

[0004] The presence of a suppressive tumor microenvironment is the fourth obstacle to current cancer treatment. It is one of the reasons why existing cancer therapies fail. The tumor microenvironment contains various types of immune cells, cancer-associated fibroblasts, endothelial cells, and various tissue-resident cells (Cancer Cell. 2023 Mar 13; 41(3):374-403). These diverse cell types in the tumor microenvironment interact, thus affecting the effectiveness of cancer drugs. Current clinical outcomes of cancer immunotherapy are both impressive and significantly limited: countless cancer patients have achieved good clinical results with existing therapies, but many more have only minimal or no effect from the same treatments. Increasing evidence suggests that the presence of a suppressive tumor microenvironment hinders the therapeutic effects of many existing targeted cancer drugs (Nat Med 24, 541–550 (2018); Jin, et al Signal TransductTarget Ther. 2020 Aug 25; 5(1):166).

[0005] Therefore, the medical community urgently needs a new type of cancer therapy that has the following characteristics: (1) it can selectively kill cells that highly express target antigens (cancer cells) while protecting cells that low express target antigens (normal cells); (2) it can avoid drug resistance caused by redundancy of cell signaling pathways (or minimize the possibility of such drug resistance); (3) its efficacy is not affected by the functional status of the patient's immune system (B cells, T cells, NK cells, antigen-presenting cells, etc.); (4) it can minimize the interference of the inhibitory tumor microenvironment on the treatment effect. Summary of the Invention

[0006] I. Technical Solution This invention provides a novel, unconventional cell-killing method that can meet the aforementioned unmet medical needs. The novel cell-killing method proposed in this invention inherently circumvents the four obstacles present in the aforementioned cancer treatments (which will be elaborated in detail in the following six paragraphs (I), (II), (III), (IV), (V), and (VI)).

[0007] (I) Excessive ion infiltration into cells can lead to cell death, a phenomenon known as oncosis (Am J Pathol. 1995 Jan; 146(1):3-15). A key characteristic of oncosis is the excessive influx of extracellular ions into the cell. This excessive ion influx causes the cell to swell to an abnormally large volume, ultimately leading to cell death.

[0008] (II) Based on the phenomenon of oncosis, the inventors of this invention hypothesize that: (1) if excessive antibodies are selectively transported from the extracellular space to a key organelle within the cell, these excessive antibodies will clog or over-occupy the organelle, thereby hindering various important cellular activities closely related to cell survival occurring in that key organelle; (2) the obstruction or stagnation of various important cellular activities occurring in the key organelle will ultimately lead to cell death. Therefore, the inventors of this invention have designed an OLS fusion antibody (organelle localization sequence fusion antibody) as a practical example to verify the above hypothesis. Unlike ADC drugs that rely on cytotoxic payloads (such as tubulin inhibitors) to kill target cells, the OLS fusion antibody of this invention can exert its cell-killing function without containing any cytotoxic payload. In the practical example provided by this invention, the OLS fusion antibody without any cytotoxic load consists of two parts: (1) a cell-binding domain, which is composed of a bispecific single-chain antibody with two epitopes; and (2) an organelle localization sequence (OLS), which includes a non-classical nuclear localization sequence (NLS) and a non-classical nucleolus localization sequence (NoLS). The cell-binding domain (bispecific single-chain antibody with two epitopes) of the OLS fusion antibody can simultaneously and specifically bind to two non-overlapping epitopes of the target cell surface antigen. This specific binding of the two epitopes aims to cross-link the target cell surface antigen through the two non-overlapping epitopes, thereby promoting the internalization of the OLS fusion antibody into the target cell through cell surface antigen (receptor)-mediated internalization. The NLS of the OLS fusion antibody is a nuclear localization sequence composed of amino acids. The NLS is used to guide the internalized OLS fusion antibody into the cell nucleus. The NoLS of this OLS fusion antibody is a nucleolar localization sequence composed of amino acids. The NoLS is used to guide the OLS fusion antibody, which has entered the cell nucleus, further into the nucleolus. The nucleolus has a highly ordered structure and is essential for cell division, growth, and survival (The Functional Nucleus. 2016 Apr 23:29–49; Biochimica et Biophysica Acta 1842 (2014) 798–801).Therefore, when an increasing number of extracellular OLS fusion antibodies selectively and persistently accumulate in the nucleolus of target cells via retrograde protein transport, various important cellular activities related to cell division, growth, and survival occurring in the nucleolus will be disrupted. This disruption of important intracellular activities will ultimately lead to cell death. Unlike many other anticancer drugs that kill cancer cells by targeting a specific cell signaling pathway (such as the HER2 or EGFR signaling pathway), OLS fusion antibodies kill cancer cells not by blocking a specific cell signaling pathway, but by selectively and persistently accumulating in key organelles of cancer cells, thereby blocking or over-occupying these key organelles to kill the cancer cells.

[0009] (III) NLS and NoLS can be divided into two categories: classical and non-classical. Classical NLS or classical NoLS contains a group of positively charged amino acids (e.g., arginine, lysine, histidine, arginine, lysine, histidine, abbreviated as R, K, H) (Lu, J. et al Cell Commun Signal 19, 60(2021); Nucleus.2015; 6(4)314-325). Numerous studies have shown that polypeptides composed of a group of positively charged amino acids can function as cell penetrating peptides (CPPs). Cell penetrating peptides (CPPs) can effectively, but non-specifically, penetrate the outer plasma membrane of various cell types (Chemistry & Biology, vol. 8, 943-948 August 2002; J Biol Chem. 2018 Sep 28; 293(39): 15221–15232). Although cell-penetrating peptides (CPPs) can mediate the entry of different molecules into cells with extremely high efficiency, their lack of cell specificity limits their widespread application (Mol Biotechnol. 65, 1387-1402). Therefore, to avoid the OLS portion of OLS fusion antibodies (containing NLS and NoLS) potentially possessing non-specific cell-penetrating peptide (CPP) function, and consequently leading to non-specific transmembrane penetration of the cell's outer plasma membrane, non-classical NLS types (NLS from HnRNPA1, NP-002127.1, aa 268-aa 305) and non-classical NoLS types (NoLS from NPM1, NP-002511.1, aa 258-aa 294) lacking positively charged amino acids (arginine, lysine, histidine) were selected for constructing the OLS fusion antibody of this invention. If the OLS structure of an OLS fusion antibody lacks a cell-penetrating peptide (CPP) function, the OLS fusion antibody can only bind to a specific surface antigen (receptor) of the target cell through its cell-binding domain, and then rely on receptor-mediated internalization to cross the outer plasma membrane of the target cell and enter the target cell. Therefore, this design of the OLS fusion antibody ensures that it can specifically act on the target cell.

[0010] (IV) The OLS fusion antibody (organelle localization sequence fusion antibody) of this invention comprises an antibody moiety (a bispecific single-chain antibody with two epitopes, i.e., a cell-binding domain) and a short peptide fragment (i.e., an OLS fragment). CPP fusion antibodies (cell-penetrating peptide fusion antibodies) reported in multiple publications also comprise an antibody moiety and a short peptide fragment (i.e., a CPP fragment) (Sci Rep. 2019 Dec 10; 9(1):18688; Theranostics. 2018 Jan 1; 8(2):549-562). Both the aforementioned OLS and CPP fragments are short peptides composed of several or tens of amino acids. Structurally, OLS and CPP fusion antibodies are identical or similar to each other. However, their mechanisms of action are completely different; they are two entirely different classes of short peptide fusion antibodies. The OLS fusion antibody of this invention relies on the specific binding of its cell-binding domain to a specific surface antigen (receptor) of the target cell to cross the cell membrane. The OLS fragment of an OLS fusion antibody does not have the ability to penetrate the cell membrane and does not play a role in the process of the OLS fusion antibody entering the target cell from the outside of the cell membrane. The OLS fragment of an OLS fusion antibody does not eliminate or reduce the selectivity of the OLS fusion antibody for the target cell. In contrast, the CPP fragment of a CPP fusion antibody has the ability to penetrate the cell membrane and plays a role in the process of the CPP fusion antibody entering the target cell from the outside of the cell membrane. The CPP fragment of a CPP fusion antibody eliminates or reduces the selectivity of the CPP fusion antibody for the target cell. Therefore, the OLS fusion antibody of the present invention is a completely different type of fusion antibody from the CPP fusion antibodies reported in many research publications.

[0011] (V) This novel cell-killing method performed by the OLS fusion antibody is an unconventional cell-killing method. Current cell-killing methods used in cancer treatment include: (1) antibodies killing cancer cells through the following mechanisms: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent phagocytosis (ADCP) (Nature reviews, Cancer 12, 278-287); (2) antibody-drug conjugates (ADCs) killing cancer cells through their carried cytotoxic payloads (such as microtubule inhibitors) (Front Pharmacol 2023;14:1274088); (3) bispecific T cell connectors (BiTEs) and chimeric antigen receptor T cells (CAR-Ts) killing cancer cells by activating specific T cells (J Hematol Oncol May 3;14, 75(2021); Blood Cancer J. 11, 69(2021)); (4) Immune checkpoint inhibitors (such as PD-1 antibodies) kill cancer cells by blocking proteins that regulate the function of the immune system (such as PD-1) (J Clin Med. 2023 Jun 27;12(13):4301); (5) Chemotherapy drugs or radiotherapy kill cancer cells by inducing DNA damage through chemical reagents or radioactive substances (Front. Oncol. 12:960317; MedComm (2020) Sep; 2(3); 315-340). In contrast, the cell-killing mechanism provided by this invention is to selectively and persistently accumulate OLS fusion antibodies outside the cell into the key organelles of the target cell, thereby killing the target cell by blocking or over-occupying the key organelles (analogously, the key organelles here are like a precision instrument, and the OLS fusion antibodies outside the cell are like debris, and when excessive debris enters the precision instrument, it will cause the instrument to be damaged). Therefore, the cell killing method provided by this invention is completely different from the conventional cell killing methods described above. This novel cell killing method provided by this invention is an unconventional cell killing method.

[0012] (VI) This unconventional novel cell-killing method performed by OLS fusion antibodies can circumvent the four obstacles mentioned above in current cancer treatment and meet the aforementioned unmet medical needs. (1) This unconventional novel cell-killing method can effectively distinguish between cells expressing high levels of cell surface antigens (cancer cells) and cells expressing low levels of cell surface antigens (normal cells). For this unconventional novel cell-killing method, the specific binding between the cell-binding domain and the cell surface antigen determines the cell internalization effect of the OLS fusion antibody and the subsequent cell-killing effect. Cells expressing high levels of cell surface antigens have the ability to transport sufficient amounts of external OLS fusion antibodies to key organelles. The accumulation of sufficient amounts of OLS fusion antibodies in key organelles will lead to cell death. Cells expressing low levels of cell surface antigens do not have the ability to transport sufficient amounts of external OLS fusion antibodies to key organelles. The inability to accumulate sufficient amounts of OLS fusion antibodies in key organelles will not lead to cell death. Therefore, this unconventional novel cell-killing method can selectively kill cells that highly express the target antigen (cancer cells) while protecting cells that low express the target antigen (normal cells). (2) This unconventional novel cell-killing method can largely avoid the drug resistance problem caused by redundancy in cell signaling pathways. The process of this novel cell-killing method is as follows: the external OLS fusion antibody binds to a specific cell surface antigen, the OLS fusion antibody is then internalized into the target cell, and the OLS fusion antibody continuously accumulates in the key organelles of the target cell through the OLS fragment. Excessive accumulation of OLS fusion antibody in the key organelles of the cell will eventually lead to cell death. For this unconventional novel cell-killing method, no matter how the target cell evolves to generate new redundant cell signaling pathways, as long as there is a sufficient amount of the specific antigen on the surface of the target cell and it can be bound by the OLS fusion antibody, the OLS fusion antibody can exert its cell-killing function after accumulating a sufficient amount in the key organelles of the target cell. Therefore, compared with existing cell-killing methods, this unconventional novel cell-killing method can better avoid the drug resistance problem caused by redundancy in cell signaling pathways. (3) This unconventional novel cell-killing method is not affected by the patient's immune cells and their functions (T cells, B cells, NK cells, antigen-presenting cells, etc.) being normal, because this novel cell-killing method relies on the continuous accumulation of external OLS fusion antibodies in the key organelles of the target cells, and it does not depend on any type of immune cells in the patient's body. (4) This unconventional novel cell-killing method is less or less affected by the suppressive tumor microenvironment, while current cancer treatments are significantly constrained by the suppressive tumor microenvironment (Cancer Lett 2017 Feb 28:387:61-68).For this unconventional novel cell-killing method, regardless of the manifestation or changes in the tumor microenvironment, as long as the target antigen exists in sufficient quantity on the cell surface and can be bound by the OLS fusion antibody, the OLS fusion antibody can exert its cell-killing function after sufficient accumulation in the key organelles of the target cell. Therefore, this novel cell-killing method relying on OLS fusion antibodies to kill cancer cells is currently the cell-killing method least affected by the suppressive tumor microenvironment.

[0013] In addition to being used to kill cancer cells, this novel cell killing method performed by the OLS fusion antibody of the present invention can also be applied to the following scenarios: (1) killing target cells infected by microorganisms (such as viruses, bacteria, fungi or parasites) as long as the infected cells can express antigens derived from the infecting microorganisms on their surface that can be recognized by the OLS fusion antibody, and the OLS fusion antibody can produce antigen (receptor)-mediated antibody internalization after binding with the antigen it recognizes; (2) killing target cells related to or associated with diseases (such as immune diseases or metabolic diseases) as long as the target cells can express specific antigens that can be recognized by the OLS fusion antibody on their surface, and the OLS fusion antibody can produce antigen (receptor)-mediated antibody internalization after binding with the antigen it recognizes.

[0014] II. Detailed Explanation of OLS Fusion Antibodies This invention provides a novel, unconventional method for cell killing. It involves selectively transporting an OLS fusion antibody (organelle localization sequence fusion antibody) from outside the target cell (i.e., outside the target cell's cytoplasmic membrane) to a key organelle closely linked to or related to cell growth and survival. The aim is to kill cells by blocking or over-occupying this key organelle. The OLS fusion antibody described in this invention comprises two components: a cell-binding domain and an organelle localization sequence (OLS).

[0015] 1. Cell-binding domain of OLS fusion antibody In one embodiment, the cell-binding domain of the OLS fusion antibody may be, but is not limited to, an intact antibody, an antigen-binding fragment, a protein scaffold, a cytokine, or a receptor ligand.

[0016] In one embodiment, the cell-binding domain of the OLS fusion antibody may be a single-specific cell-binding domain, preferably a single-specific antibody or a single-specific antigen-binding fragment, wherein the single-specific cell-binding domain is capable of binding to a specific cell surface antigen.

[0017] In one embodiment, the cell-binding domain of the OLS fusion antibody may be a bispecific cell-binding domain (i.e., composed of two binding domains), preferably a bispecific antibody or a bispecific antigen-binding fragment, wherein the two binding domains of the bispecific cell-binding domain are capable of: (1) specifically binding to different epitopes of the same cell surface antigen; or (2) specifically binding to two different cell surface antigens.

[0018] In one embodiment, the cell-binding domain of the OLS fusion antibody may be a bispecific cell-binding domain (i.e., composed of two binding domains), preferably a bispecific antibody or a bispecific antigen-binding fragment, wherein the first binding domain of the bispecific cell-binding domain can specifically bind to cell surface antigens, and the other binding domain can specifically bind to intracellular antigens. The OLS fusion antibody is internalized into the cell by specifically binding to the cell surface antigen through the first binding domain of its bispecific cell-binding domain, and the internalized OLS fusion antibody can specifically bind to the intracellular antigen through the other binding domain of its bispecific cell-binding domain.

[0019] In one embodiment, the cell-binding domain of the OLS fusion antibody may be a trispecific cell-binding domain (i.e., composed of three binding domains), preferably a trispecific antibody or a trispecific antigen-binding fragment, wherein the three parts of the trispecific cell-binding domain are capable of: (1) specifically binding to three different epitopes of the same cell surface antigen; or (2) specifically binding to three different cell surface antigens.

[0020] In one embodiment, the cell-binding domain of the OLS fusion antibody may be a trispecific cell-binding domain (i.e., composed of three binding domains), preferably a trispecific antibody or a trispecific antigen-binding fragment, wherein one or two of the three parts of the trispecific cell-binding domain specifically bind cell surface antigens (single or multiple), and the remaining parts specifically bind intracellular antigens (single or multiple); after internalization, the OLS fusion antibody gains the opportunity to contact intracellular antigens, and then can specifically bind to intracellular antigens (single or multiple) through its remaining parts (parts not bound to cell surface antigens).

[0021] In one embodiment, the cell-binding domain of the OLS fusion antibody may be a multispecific cell-binding domain (i.e. composed of multiple binding domains), preferably a multispecific antibody or a multispecific antigen-binding fragment, wherein the multiple portions of the multispecific cell-binding domain are capable of: (1) specifically binding to multiple different epitopes of the same cell surface antigen; or (2) specifically binding to multiple different cell surface antigens.

[0022] In one embodiment, the cell-binding domain of the OLS fusion antibody may be a multispecific cell-binding domain (i.e., composed of multiple binding domains), preferably a multispecific antibody or a multispecific antigen-binding fragment, wherein one or more portions of the multispecific cell-binding domain can specifically bind cell surface antigens (single or multiple), while the remaining portions can specifically bind intracellular antigens (single or multiple); after internalization, the OLS fusion antibody gains the opportunity to contact intracellular antigens, and then can specifically bind to intracellular antigens (single or multiple) through its remaining portions (the portions not bound to cell surface antigens).

[0023] In one embodiment, cell-killing effects can be achieved by targeting a single cell surface antigen with the cell-binding domain of the OLS fusion antibody. In another embodiment, cell-killing effects can be achieved by targeting two or more antigens with the cell-binding domain of the OLS fusion antibody, wherein the antigens may be entirely located on the cell surface, or partially located on the cell surface with the remainder located intracellularly. In yet another embodiment, the cell-killing effect can be enhanced by increasing the molecular size of the cell-binding domain of the OLS fusion antibody, because a larger cell-binding domain can more effectively block or over-occupy key organelles of the target cell than a smaller cell-binding domain.

[0024] In one embodiment, the cell-binding domain of the OLS fusion antibody may be an isotype of the antibody, including IgA, IgG, IgM, IgD, or IgE.

[0025] In one embodiment, the cell-binding domain of the OLS fusion antibody may be an antibody, wherein the antibody includes, but is not limited to: humanized antibodies, fully human antibodies, non-human antibodies and / or chimeric antibodies of human and non-human origin; and the antibody may be derived from mammals, birds, fish, amphibians or reptiles.

[0026] In one embodiment, the cell-binding domain of the OLS fusion antibody may be an antigen-binding fragment of the antibody, wherein the antigen-binding fragment includes, but is not limited to: humanized fragments, fully human fragments, non-human fragments and / or chimeric fragments of human and non-human origin; and the antigen-binding fragment may be derived from mammals, birds, fish, amphibians or reptiles.

[0027] In one embodiment, the cell-binding domain of the OLS fusion antibody may be a protein scaffold, wherein the protein scaffold may be, but is not limited to: humanized protein scaffolds, human protein scaffolds, non-human protein scaffolds and / or chimeric protein scaffolds of human and non-human origin; and the protein scaffold may be derived from mammals, birds, fish, amphibians or reptiles.

[0028] In one embodiment, the cell-binding domain of the OLS fusion antibody has an ultra-high affinity for the target antigen, wherein the affinity is preferably in the single-digit femto-molar range. In another preferred embodiment, after the OLS fusion antibody binds tightly to the target antigen through its cell-binding domain, it can drag the target antigen into a key organelle of the target cell, thereby achieving a stronger target cell killing effect by jointly blocking or over-occupying the key organelle with the target antigen.

[0029] In one embodiment, the specific binding of the cell-binding domain of the OLS fusion antibody to the target cell surface antigen must lead to the internalization of the OLS fusion antibody within the cell. In another embodiment, a suitable cell-binding domain is preferably selected or screened for constructing the OLS fusion antibody, which should have the function of inducing the internalization of the OLS fusion antibody after binding to the target cell surface antigen.

[0030] In one embodiment, the OLS fusion antibody must satisfy the following: (1) when the cell-binding domain of the OLS fusion antibody does not specifically bind to the target cell surface antigen, the OLS fusion antibody itself does not have the ability to pass through the target cell plasma membrane; (2) the OLS fusion antibody can only pass through the target cell plasma membrane through the internalization of the antigen-antibody complex after its cell-binding domain specifically binds to the target cell surface antigen.

[0031] In one embodiment, the cell-binding domain of the OLS fusion antibody specifically binds to cell surface antigens or receptors presented on the surface of target cells. In another embodiment, the cell-binding domain of the OLS fusion antibody specifically binds to cancer-associated antigens or cancer antigens presented on the surface of target cells. In yet another embodiment, the cell-binding domain of the OLS fusion antibody specifically binds to autoimmune disease-associated antigens or autoimmune disease antigens presented on the surface of target cells. In still another embodiment, the cell-binding domain of the OLS fusion antibody specifically binds to infectious disease-associated antigens or infectious disease antigens presented on the surface of target cells.

[0032] 2. Organelle Localization Sequence (OLS) of OLS Fusion Antibody In one embodiment, the key organelle targeted by the OLS fragment in the OLS fusion antibody may be, but is not limited to, the nucleolus, mitochondria, endoplasmic reticulum, nucleus, Golgi apparatus, or lysosome. In another preferred embodiment, the key organelle targeted by the OLS fragment in the OLS fusion antibody should have a highly ordered structure and be essential for cell survival. In yet another embodiment, after receptor-mediated cell internalization, the function of the OLS fragment of the OLS fusion antibody is to directionally deliver the OLS fusion antibody into specific organelles of the target cell.

[0033] In one embodiment, the OLS fragment in the OLS fusion antibody may be a nuclear localization sequence (NLS), provided that the NLS fragment does not possess cell-penetrating peptide (CPP) function, since CPP can nonselectively cross the cell membrane. In one embodiment, the OLS fragment in the OLS fusion antibody may be a nucleolus localization sequence (NoLS), provided that the NoLS fragment does not possess cell-penetrating peptide (CPP) function. In one embodiment, the OLS fragment in the OLS fusion antibody may be a combination of a nuclear localization sequence (NLS) and a nucleolus localization sequence (NoLS), provided that the combined sequence does not possess cell-penetrating peptide (CPP) function. In one embodiment, the OLS fragment in the OLS fusion antibody may be an endoplasmic reticulum localization sequence (ErLS), provided that the ErLS fragment does not possess cell-penetrating peptide (CPP) function. In one embodiment, the OLS fragment in the OLS fusion antibody may be a mitochondrial localization sequence (MLS), provided that the MLS fragment does not possess cell-penetrating peptide (CPP) function. In another embodiment, the OLS fragment in the OLS fusion antibody may be a lysosome localization sequence (LLS), provided that the LLS fragment does not possess cell-penetrating peptide (CPP) function. In yet another embodiment, the OLS fragment in the OLS fusion antibody may be a Golgi apparatus localization sequence (GaLS), provided that the GaLS fragment does not possess cell-penetrating peptide (CPP) function.

[0034] In one embodiment, the OLS fusion antibody comprises a cell-binding domain and an OLS fragment, wherein the OLS fragment may be located at: (1) the N-terminus of the cell-binding domain; (2) the C-terminus of the cell-binding domain; (3) both the N-terminus and C-terminus of the cell-binding domain (each with an OLS fragment); (4) the middle of the cell-binding domain; or (5) a random location within the cell-binding domain. This embodiment assumes that the location of the OLS fragment does not affect the function of the cell-binding domain (its function of binding to a specific target antigen) or the function of the OLS fragment itself (its function of guiding the OLS fusion antibody into key organelles).

[0035] In one embodiment, one, two, three or more organelle localization sequences (OLS) may be used when constructing a single OLS fusion antibody, wherein the one, two, three or more organelle localization sequences used may target the same key organelle or different key organelles.

[0036] In one embodiment, the OLS fragment of the OLS fusion antibody can be added to the cell-binding domain by genetic engineering or by chemical conjugation, provided that the conjugation process does not affect the organelle localization function of the OLS fragment or the target antigen binding function of the cell-binding domain.

[0037] In one embodiment, the OLS fragment of the OLS fusion antibody must not possess cell-penetrating peptide (CPP) function. In another embodiment, during the construction of the OLS fusion antibody, one or more point mutations are performed on the original OLS fragment to eliminate its inherent cell-penetrating peptide (CPP) function. In yet another embodiment, after eliminating the original cell-penetrating peptide function of the OLS fragment through point mutation, the constructed OLS fusion antibody can only enter the target cell through the specific binding of its cell-binding domain to the target cell surface antigen.

[0038] In one embodiment, the organelle localization sequence (OLS) is covalently linked to the cell-binding domain via a single linker peptide. In another embodiment, one or more linkers peptides can be used to link the OLS fragment to the cell-binding domain. Both embodiments are based on the premise that the linking method does not affect the organelle localization function of the OLS fragment or the target antigen binding function of the cell-binding domain.

[0039] In one implementation, an OLS fusion antibody can be constructed by linking organelle localization sequences (OLS) to cell-binding domains using a variety of known and established techniques, wherein the techniques can be based on standard chemical, biochemical, and / or molecular biological techniques.

[0040] 3. Drugs based on OLS fusion antibodies In one embodiment, the present invention provides a drug with an OLS fusion antibody as the active ingredient, wherein the OLS fusion antibody comprises a cell-binding domain and an organelle localization sequence (OLS), and the OLS fusion antibody achieves its cell-killing function by specifically blocking or over-occupying a key organelle of the target cell that is closely linked to or related to cell survival.

[0041] In one embodiment, a drug based on an OLS fusion antibody can be used to treat a disease or condition, provided that the disease or condition is related to or associated with a specific cell surface antigen recognized by the OLS fusion antibody, and the purpose of treatment is to eliminate specific cells expressing that specific cell surface antigen. In another embodiment, the disease or condition can be cancer, including but not limited to breast cancer, colorectal cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, or leukemia; in another embodiment, the disease or condition can be an infectious disease, including but not limited to viral infections, bacterial infections, fungal infections, or parasitic infections; in another embodiment, viral infections in the disease or condition include but are not limited to HBV infection, HPV infection, or HIV infection; in another embodiment, the disease or condition can be an autoimmune disease, including but not limited to rheumatoid arthritis, type 1 diabetes, multiple sclerosis, or psoriasis.

[0042] In one implementation, a drug containing an OLS fusion antibody can be used to treat a cultured cell population to reduce or completely eliminate a specific cell subpopulation. In a mixed culture system, when it is necessary to avoid interference from a specific cell subpopulation, the specific cell subpopulation present in the culture system can be eliminated by adding a drug containing an OLS fusion antibody. Similarly, this treatment method can also be used in clinical practice: a patient's blood is drawn to an extracorporeal environment (e.g., dialysis), treated with a drug containing an OLS fusion antibody to remove a specific cell subpopulation (e.g., a tumor cell subpopulation), and then the blood, free of that specific subtype of cells, is reinfused into the patient.

[0043] In one implementation, a drug based on an OLS fusion antibody can be used to selectively kill cells expressing high levels of cell surface antigens (e.g., tumor cells), while preserving cells expressing the same cell surface antigens at lower levels (e.g., normal cells).

[0044] In one embodiment, to more effectively kill target cells, the drug may be a combination of multiple OLS fusion antibodies that target different cell surface antigens, or a combination of multiple OLS fusion antibodies that target different epitopes of the same cell surface antigen. For example, for patients suspected of developing resistance due to the use of drugs containing only a single type of OLS fusion antibody, treatment can be given by administering a drug composition containing multiple types of OLS fusion antibodies, wherein the multiple types of OLS fusion antibodies target multiple different cell surface antigens or multiple different epitopes of the same cell surface antigen, thus avoiding resistance caused by mutations in a single cell surface antigen or a single epitope of a cell surface antigen.

[0045] In one embodiment, the drug may be a polynucleotide molecule capable of encoding an OLS fusion antibody, such as an mRNA molecule encoding an OLS fusion antibody. In another embodiment, the drug may be a DNA expression vector capable of expressing an OLS fusion antibody, such as the PCDNA3.1-OLS fusion antibody expression vector. In yet another embodiment, the drug may be a cell containing a polynucleotide encoding an OLS fusion antibody, such as T cells or B cells transfected with an mRNA molecule encoding an OLS fusion antibody.

[0046] 4. Production of OLS fusion antibodies The OLS fusion antibody described herein can be prepared using conventional and known genetic engineering techniques. For example, as needed, DNA encoding the cell-binding domain and DNA encoding the OLS sequence can be amplified first, then these DNA fragments can be ligated together by PCR amplification. Finally, the resulting DNA fragment is inserted into a gene expression vector and transfected into host cells. The OLS fusion antibody can then be prepared through protein expression in the host cells. As for the host cells used for expression, mammalian cells, yeast, animal cells, insect cells, plant cells, or bacteria can be used, with mammalian cells being preferred, and CHO cells or HEK293 cells being particularly preferred. Recombinant DNA, tissue culture, transformation, transfection, protein expression, and protein purification can all be performed using conventional and known standard techniques. Antibody expression and purification can be performed according to the manufacturer's instructions or conventional methods in the relevant field. These and related techniques and operations can generally be performed according to conventional methods known in the art. Attached Figure Description

[0047] Figure 1 The structures of various OLS fusion antibodies are shown. Figure 1 In this context, N represents the amino terminus of the cell-binding domain; C represents the carboxyl terminus of the cell-binding domain; and OLS (organelle localization sequence) represents the organelle localization sequence.

[0048] Figure 2 The structures of five anti-ROR1 scFv antibodies are shown, including AR1 antibody (anti-ROR1 single epitope single-specific scFv antibody 1), AR2 antibody (anti-ROR1 single epitope single-specific scFv antibody 2), AR bi-sccfv OLS fusion antibody (anti-ROR1 dual epitope dual-specific scFv organelle localization sequence fusion antibody), AR bi-sccfv antibody (anti-ROR1 dual epitope dual-specific scFv antibody), and AR bi-sccfv OLS GFP fusion antibody (anti-ROR1 dual epitope dual-specific scFv organelle localization sequence green fluorescent protein fusion antibody). Figure 2As shown, (1) the structure of AR1 consists of: 4A5 variable region of the light chain (4A5VL), amino acid linker peptide, 4A5 variable region of the heavy chain (4A5VH), and mouse CH2-CH3 domain (mCH2CH3, mouse IgG antibody second and third constant regions of the heavy chain); (2) the structure of AR2 consists of: 99961 variable region of the light chain (99961VL), amino acid linker peptide, 99961 variable region of the heavy chain (99961VH), and mouse CH2-CH3 domain (mCH2CH3); (3) AR bi-scfv OLS The structural composition of the fusion antibody: 4A5 light chain variable region (4A5VL), amino acid linker peptide, 99961 light chain variable region (99961VL), amino acid linker peptide, 99961 heavy chain variable region (99961VH), amino acid linker peptide, 4A5 heavy chain variable region (4A5VH), nuclear localization signal (NLS), human CH2-CH3 domain (hCH2CH3, human IgG antibody second and third constant regions of the heavy chain), nucleolar localization signal (NoLS); (4) The structural composition of the AR bi-scfv antibody: 4A5 light chain variable region (4A5VL), amino acid linker peptide, 99961 light chain variable region (99961VL), amino acid linker peptide, 99961 heavy chain variable region (99961VH), amino acid linker peptide, 4A5 heavy chain variable region (4A5VH), human CH2-CH3 domain (hCH2CH3); (5) AR bi-scfv The OLSGFP fusion antibody consists of the following structural components: 4A5 light chain variable region (4A5VL), amino acid linker peptide, 99961 light chain variable region (99961VL), amino acid linker peptide, 99961 heavy chain variable region (99961VH), amino acid linker peptide, 4A5 heavy chain variable region (4A5VH), nuclear localization signal (NLS), human CH2-CH3 domain (hCH2CH3), nucleolar localization signal (NoLS), and green fluorescent protein (GFP). Figure 2Zhong, 4A5, and 99961 are the names of two complete anti-ROR1 antibodies; NLS (nuclear localization sequence) represents the nuclear localization sequence; NoLS (nucleolus localization sequence) represents the nucleolus localization sequence; GFP (Green Fluorescent Protein) represents green fluorescent protein; AR is an abbreviation for anti-ROR1 scFv antibody; scFv antibody (single chain variable fragment antibody) represents single-chain variable fragment antibody.

[0049] Figure 3 (1) Line 1 shows the results of flow cytometry experiments of P815mock cells stained with PE-labeled mouse anti-human ROR1 monoclonal antibody; (2) Line 2 shows the results of flow cytometry experiments of P815ROR1 cells (clone 4C1) stained with PE-labeled mouse anti-human ROR1 monoclonal antibody; (3) Line 3 shows the results of flow cytometry experiments of P815ROR1 cells (clone 6B2) stained with PE-labeled mouse anti-human ROR1 monoclonal antibody.

[0050] Figure 4 (i) Line 1 (dashed line) shows the flow cytometry results of P815AR bi-scfv cells stained with negative control protein; (ii) Line 2 (solid line) shows the flow cytometry results of P815AR bi-scfv cells stained with human ROR1 protein. Note: Both the negative control protein and the human ROR1 protein are FITC-labeled proteins.

[0051] Figure 5 (1) Dashed lines show flow cytometry results of Jeko-1 cells stained with secondary antibody only; (2) Solid lines show flow cytometry results of Jeko-1 cells stained with AR1 or AR2 antibody first and then with secondary antibody.

[0052] Figure 6The results of four flow cytometry experiments are shown: (1) In Experiment 1, cells were directly stained with the AR bi-scfv OLS fusion antibody; (2) In Experiment 2, cells were first stained with AR1 and then stained with the AR bi-scfv OLS fusion antibody; (3) In Experiment 3, cells were first stained with AR2 and then stained with the AR bi-scfv OLS fusion antibody; (4) In Experiment 4, cells were first stained with both AR1 and AR2 and then stained with the AR bi-scfv OLS fusion antibody. Note: Dashed lines show the flow cytometry results of P815mock cells (ROR1 negative cells) stained with the corresponding antibodies; solid lines show the flow cytometry results of P815ROR1 cells (ROR1 positive cells) stained with the corresponding antibodies.

[0053] Figure 7 (Cytotoxicity Experiment 1), (1) In the left image, P815mock cells were treated with AR bi-scfv OLS fusion antibody for 12 days and then stained with trypan blue; (2) In the middle image, P815ROR1 cells (clone 4C1) were treated with AR bi-scfv OLS fusion antibody for 12 days and then stained with trypan blue; (3) In the right image, P815ROR1 cells (clone 6B2) were treated with AR bi-scfv OLS fusion antibody for 12 days and then stained with trypan blue. Note: P815ROR1 cells are ROR1 positive cells; P815mock cells are ROR1 negative cells.

[0054] Figure 8 (Cytotoxicity experiment 2), (1) In the left image, P815ROR1 cells were treated with AR bi-scfv antibody (antibody without OLS fragment) for 12 days and then stained with trypan blue; (2) In the right image, P815ROR1 cells were treated with AR bi-scfv OLS fusion antibody (antibody containing OLS fragment) for 12 days and then stained with trypan blue.

[0055] Figure 9 The results of the experiment, observed under a fluorescence microscope, show P815ROR1 cells after 12 days of treatment with the AR bi-scfv OLS GFP fusion antibody. Detailed Implementation

[0056] Before describing specific embodiments of the invention, those skilled in the art should understand that modifications can be made to the embodiments described herein without departing from the broad conception of the invention. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed herein. Unless otherwise defined, all technical and scientific terms used herein have the meanings consistent with the common understanding of those skilled in the art to which this invention pertains. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. These and other aspects of the invention will become apparent from the following detailed description and accompanying drawings. The embodiments listed herein are intended to provide those skilled in the art with a complete disclosure and description of how to implement and use the invention, and are not intended to limit the scope of the invention.

[0057] I. Definition The terms “a,” “an,” “the,” “some,” “a certain,” and “a certain” used in this specification are not intended to refer only to singular entities, but rather to include general categories that may be illustrated with specific examples, unless the context clearly specifies otherwise.

[0058] In this specification, unless the context clearly specifies otherwise, the word “comprising” and its variations such as “including” or “comprising” should be understood to mean that the said element or component is included, or a group of elements or components, but does not exclude any other unmentioned element, component or combination thereof.

[0059] In this specification, the term "or" means "and / or" and may be used interchangeably with "and / or" unless the context clearly indicates otherwise.

[0060] In this invention specification, the terms "specificity," "specific binding," and "specifically binding" all refer to the selective binding between the cell-binding domain of the OLS fusion antibody and the target antigen. Under specific conditions, the binding specificity can be tested by comparing the binding of the cell-binding domain to relevant and unrelated antigens.

[0061] In this invention specification, the term "cell" refers to the smallest functional unit capable of independent survival and constituting all living organisms and tissues. A mammalian cell comprises three main parts: the cell membrane, the nucleus, and the cytoplasm.

[0062] In this specification, the term "organelle" refers to a specific subunit within a cell that performs a specific function. Organelles perform one or more specific functions within a cell, and their mechanisms of action are similar to those of organs in the body. Examples of organelles include: the cell nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, nucleolus, and lysosomes.

[0063] In this specification, the term "binding domain" refers to a molecule or a portion thereof capable of binding to a target molecule. The term "cell-binding domain" specifically refers to a molecule or a portion thereof capable of binding to a target antigen expressed by a cell.

[0064] In this specification, the term "antigen" refers to a molecule that, when introduced into a host, can stimulate the host's immune system to produce a cellular and / or humoral antigen-specific immune response. The term "cell surface antigen" refers to an antigen expressed on the cell surface that can be recognized by cell-binding domains. In this specification, cell surface antigens include not only products encoded by the cell's own structural components but also products encoded by intracellular viruses, bacteria, fungi, or parasites.

[0065] In this specification, the term "antibody" is used in its broadest sense, explicitly encompassing both natural and engineered antibodies, provided they exhibit the desired biological activity. The term also includes Ig molecules composed solely of heavy chains (e.g., antibodies obtained from camels). Antibodies described in this specification can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), and can be derived from any suitable species.

[0066] In this specification, the term "antigen-binding fragment" refers to a polypeptide capable of specifically binding to a target antigen. Without limitation, the antigen-binding fragment may be a heavy chain complementarity-determining region (CDR), a light chain CDR, a heavy chain variable region (VH), a light chain variable region (VL), or a combination thereof. The antigen-binding fragment may be, but is not limited to, Fab fragments, Fv fragments, dAb fragments, F(ab')2 fragments, single-chain monospecific antibodies, single-chain bispecific antibodies, single-chain trispecific antibodies, single-chain tetraspecific antibodies, single-chain multispecific antibodies, cytokines, or receptor ligands, etc. The antigen-binding fragment may also be a protein scaffold, i.e., a protein structure having a structure similar to that of other antibodies besides antibodies and / or having the property of specifically recognizing and / or binding to a specific antigen. The antigen-binding fragment may also comprise any protein or portion thereof that retains the ability to specifically bind antigens, including non-immunoglobulins.

[0067] In this invention specification, the term "organelle localization sequence (OLS)" refers to an amino acid sequence that can introduce a protein into a target organelle. In this invention specification, the term "localization sequence" has the same meaning as "localization signal" commonly used in scientific literature and textbooks. In this invention specification, the term "nuclear localization sequence (NLS)" refers to an amino acid sequence that can introduce extracellular proteins into the cell nucleus; the term "nucleolar localization sequence (NoLS)" refers to an amino acid sequence that can introduce extracellular proteins into the cell nucleolus; the term "mitochondrial localization sequence (MLS)" refers to an amino acid sequence that can introduce extracellular proteins into the cell mitochondria; the term "Golgi localization sequence (GaLS)" refers to an amino acid sequence that can introduce extracellular proteins into the Golgi apparatus; the term "endoplasmic reticulum localization sequence (ErLS)" refers to an amino acid sequence that can introduce extracellular proteins into the cell endoplasmic reticulum; and the term "lysosome localization sequence (LLS)" refers to an amino acid sequence that can introduce extracellular proteins into the cell lysosome. In this specification, the term "OLS fusion antibody" refers to an organelle localization sequence fusion antibody. An OLS fusion antibody includes a cell-binding domain and an organelle localization sequence. The organelle localization sequence (OLS) described in this invention can be: nuclear localization sequence (NLS), nucleolar localization sequence (NoLS), mitochondrial localization sequence (MLS), Golgi localization sequence (GaLS), lysosomal localization sequence (LLS), or endoplasmic reticulum localization sequence (ErLS). The organelle localization sequence (OLS) described in this invention can also be a combination of two or more of the above-mentioned organelle localization sequences. In this specification, "OLS fusion antibody without any cytotoxic load" refers to an OLS fusion antibody that does not contain any tubulin inhibitors, topoisomerase I inhibitors, etc. In this specification, the term "non-classical nuclear localization sequence (NLS)" refers to an amino acid sequence that can guide proteins into the cell nucleus, but whose structural pattern differs from that of a typical classical nuclear localization sequence—which is usually composed of positively charged amino acid clusters (e.g., arginine clusters, lysine clusters, and histidine clusters, or various combinations of these positively charged amino acid clusters).

[0068] In this specification, the term "non-classical nucleolar positioning sequence (NoLS)" refers to an amino acid sequence that guides proteins into the cell nucleolus, but whose structural pattern differs from that of a typical classical nucleolar positioning sequence—which is usually composed of positively charged amino acid clusters (e.g., arginine clusters, lysine clusters, and histidine clusters, or various combinations of these positively charged amino acid clusters).

[0069] In this specification, the term "polynucleotide" refers to a combination of nucleotide monomers linked together by covalent bonds. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are examples of polynucleotides.

[0070] In this invention specification, the term "disease" refers to a medical condition with a definite cause; while "disorder" refers to an abnormality in health condition whose cause may be unknown or subjective. In this invention specification, the term "cancer" refers to a disease characterized by uncontrolled abnormal cell division that can invade adjacent tissues through the blood and lymphatic system, such as lung cancer, breast cancer, colon adenocarcinoma, rectal cancer, prostate cancer, and stomach cancer; the term "tumor" refers to localized swelling of the body caused by abnormal tissue proliferation, such as lipoma, leiomyoma, osteosarcoma, lymphoma, and melanoma; the term "autoimmune disease" refers to a disease in which the immune system mistakes its own tissues for foreign substances and launches an attack, such as rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, and psoriasis; and the term "infectious disease" refers to a disease caused by pathogens such as bacteria, viruses, fungi, or parasites, such as influenza, hepatitis B, AIDS, dengue fever, tuberculosis, onychomycosis, and malaria.

[0071] In this specification, the terms “treatment,” “therapeutic method,” and “treatment means” include therapeutic, preventive, or inhibitory measures taken against a disease or condition that are intended to produce any clinically beneficial effect, including but not limited to: relieving one or more symptoms, resolving the condition, or delaying or preventing the progression of the disease / condition.

[0072] II. Practical Application Cases of the Invention 1. Construction of OLS fusion antibody The OLS fusion antibody of this invention is composed of a cell-binding domain and organelle localization sequences (OLS). For example... Figure 1 As shown, based on the principle that the cell-binding domain and the OLS fragment do not interfere with each other's functions, when constructing an OLS fusion antibody, the OLS fragment can be added to: (1) the N-terminus of the cell-binding domain; (2) the C-terminus of the cell-binding domain; (3) the N-terminus and C-terminus of the cell-binding domain (each with an OLS fragment); (4) the middle of the cell-binding domain; (5) any site of the cell-binding domain. As a practical application example of the OLS fusion antibody, this invention designed and constructed an anti-ROR1 bispecific scFv OLS fusion antibody to verify a novel, unconventional cell-killing method.

[0073] 2. Preparation of anti-ROR1 monospecific scFv antibody (AR1 or AR2) This invention selects human ROR1 protein (NP_005003, a cell surface antigen / receptor) as the target antigen to test the cell-killing effect of OLS fusion antibody. The reasons for selecting ROR1 protein as the target antigen are: (1) ROR1 protein is a type I transmembrane protein with a molecular weight of 104 KD. It is a large molecular weight membrane protein composed of 937 amino acids. Its long extracellular domain can provide more available antibody cross-linking epitopes; (2) Multiple reports have shown that ROR1 protein has the functions of inhibiting cell apoptosis, enhancing EGFR signal transduction and promoting cell growth. This protein is highly expressed in a variety of cancer cells, but is not expressed or has extremely low expression levels in normal cells (Hematol Oncol 2011, 29: 17–21; Protein&Cell, Volume 5, issue 7, July 2014, 496–502).

[0074] To construct an anti-ROR1 bispecific scFv OLS fusion antibody (as an example of an OLS fusion antibody for testing novel cell-killing methods), this invention first prepared two anti-ROR1 monospecific scFv antibodies. The polynucleotide sequence of the first anti-ROR1 monospecific scFv antibody (named AR1) is derived from a patented full-length anti-ROR1 antibody: Antibody 4A5 (US Patent No. US20130273073A1). The polynucleotide sequence of the second anti-ROR1 monospecific scFv antibody (named AR2) is derived from another patented full-length anti-ROR1 antibody: Antibody 99961 (US Patent No. US9758591B2). These two published patents show that: (1) both parental full-length antibodies to AR1 and AR2 (antibody 4A5 and antibody 99961) can bind to the extracellular domain of the ROR1 protein, but target two non-overlapping epitopes; (2) after binding to the Ror1 protein, both parental full-length antibodies to AR1 and AR2 (antibody 4A5 and antibody 99961) can produce receptor-mediated antibody internalization. The structures of AR1 and AR2 are as follows: Figure 2 As shown: (1) 4A5VL and 4A5VH are the ROR1 binding domains of the AR1 antibody; (2) 99961VL and 99961VH are the ROR1 binding domains of the AR2 antibody; (3) The mCH2CH3 fragments in AR1 and AR2 are used as tags for antibody purification and detection.

[0075] The polynucleotide sequences encoding AR1 or AR2 were synthesized by Integrated DNA Technologies (IDT) and subcloned into the PCDNA3.1 vector (catalog number: V79020, Invitrogen) according to standard genetic engineering and molecular cloning techniques detailed in *Molecular Cloning: A Laboratory Manual* (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). The subcloned gene fragment (whose polynucleotide sequence includes the N-terminal signal peptide coding region and the C-terminal stop codon) was then verified by DNA sequencing (IDT). The resulting DNA constructs were named PCDNA3-AR1 or PCDNA3-AR2 plasmids. PCDNA3-AR1 and PCDNA3-AR2 plasmids were used to express two different anti-ROR1 monospecific scFv antibodies—AR1 and AR2, respectively.

[0076] AR1 and AR2 were expressed and produced in CHO cells (catalog number: R80007, Thermo Fisher Scientific) according to the manufacturer's instructions. The simplified procedure is as follows: CHO cells were first transfected with sequencing-validated plasmids (PCDNA3-AR1 and PCDNA3-AR2) using FreeStyle™ MAX (catalog number 16447100, Thermo Fisher Scientific). The transfected CHO cells were then cultured for 7 days in serum-free FreeStyle™ CHO expression medium (catalog number: 12651014, Thermo Fisher Scientific) at 37°C, 95% humidity, and 8% CO2 on a shaker at 135 rpm. Finally, the AR1 and AR2 antibodies were purified from the cell culture supernatant using Protein A / G agarose (catalog number 20421, Thermo Fisher Scientific). The purpose of preparing AR1 and AR2 is: (1) Before constructing the anti-ROR1 bispecific scFv OLS fusion antibody using AR1 and AR2, the functional verification of these two parental anti-ROR1 monospecific scFv antibodies (AR1 and AR2) must be completed first; (2) Monospecific AR1 and AR2 can be used as blocking reagents in flow cytometry experiments to verify whether the anti-ROR1 bispecific scFv OLS fusion antibody constructed from them can simultaneously bind two non-overlapping epitopes of the ROR1 protein.

[0077] 3. Preparation of anti-ROR1 biepitaxybispecific scFv OLS fusion antibody (an antibody containing an OLS fragment) When cell surface receptors (antigens) cross-link with their corresponding antibodies, an internalization of antibody-receptor (antigen) complexes mediated by cell surface receptors (antigens) can occur (J cheng et al, Antibodies 2020, 9(3),49). Based on this principle, this invention designs an anti-ROR1 bispecific scFv antibody (which can simultaneously and specifically bind to two extracellular non-overlapping epitopes of the ROR1 membrane protein), aiming to more effectively cross-link the ROR1 protein (a receptor membrane protein expressed on the cell surface) and thus transport more antibodies into target cells through antibody-receptor (antigen) internalization. In addition, in order to enable the designed anti-ROR1 bispecific scFv antibody to be further transported to a key organelle of the target cell, thereby killing the target cell by blocking or over-occupying the key organelle, this invention adds an organelle localization sequence (OLS) to the anti-ROR1 bispecific scFv antibody. The anti-ROR1 biepitaxy bispecific scFv antibody with added OLS fragments is named anti-ROR1 biepitaxy bispecific scFv OLS fusion antibody, or simply AR bi-scFv OLS. Figure 2 As shown, the anti-ROR1 biepisode bispecific scFv OLS fusion antibody consists of the following parts: a cell-binding domain (4A5VL-99961VL-99961VH-4A5VH), organelle localization sequences (OLS), and a human CH2CH3 fragment. The cell-binding domain of the anti-ROR1 biepisode bispecific scFv OLS fusion antibody is composed of two anti-ROR1 monospecific scFv antibodies (AR1 and AR2). The OLS portion of the anti-ROR1 biepisode bispecific scFv OLS fusion antibody includes non-canonical NLS and non-canonical NoLS. The non-canonical NLS is derived from the human HnRNPA1 protein (NP-002127.1, aa 268-aa 305). The non-canonical NoLS is derived from the human NPM1 protein (NP-002511.1, aa 258-aa 294). The hCH2CH3 fragment of the anti-ROR1 biepitaxybispecific scFv OLS fusion antibody was used as a tag for purification and detection.

[0078] The polynucleotide sequence encoding the cell-binding domain of the AR bi-scFv OLS fusion antibody was generated using overlap PCR with PCDNA3-AR1 and PCDNA3-AR2 plasmids as PCR templates. The polynucleotide sequences of the OLS fragment (containing NLS and NoLS) and hCH2CH3 fragment encoding the AR bi-scFv OLS fusion antibody were synthesized by IDT. The synthesized OLS and hCH2CH3 polynucleotide fragments were then ligated to the polynucleotide fragment encoding the cell-binding domain of the AR bi-scFv OLS fusion antibody using overlap PCR. Figure 2 As shown, the structural composition of the AR bi-scFv OLS fusion antibody is: 4A5VL-99961VL-99961VH-4A5VH-NLS-hCH2CH3-NoLS. All PCR primers were synthesized by IDT. The final PCR product was inserted into the PCDNA3.1 vector using DNA subcloning, and the sequence correctness of the subcloned fragment was verified by DNA sequencing. The sequence-verified DNA construct was named the PCDNA3-AR bi-scFv OLS plasmid. The PCDNA3-AR bi-scFv OLS plasmid was used to express the AR bi-scFv OLS fusion antibody, which is abbreviated as ARbi-scFv OLS.

[0079] The AR bi-scFv OLS fusion antibody was produced using the same process as AR1 and AR2, prepared via a CHO cell expression system. The fusion antibody was purified from cell culture supernatant using Protein A agarose gel (catalog number: 20333, Thermo Fisher), with the specific procedures strictly following the manufacturer's instructions. During the production of the AR bi-scFv OLS fusion antibody, the inventors observed that most of the fusion antibody remained in the cell nucleus, with only a portion being secreted into the cell culture supernatant. This is likely due to the presence of the OLS fragment (containing NLS and NoLS), which caused most of the fusion antibody expressed by CHO cells to remain in the cell nucleus.

[0080] 4. Preparation of anti-ROR1 biepitaxybispecific scFv antibody (an antibody without OLS fragments) To investigate the role of the OLS fragment in the AR bi-scFv OLS fusion antibody in the cell-killing effect of this fusion antibody, this invention also constructed an anti-ROR1 biepitaph bispecific scFv antibody without the OLS fragment, named AR bi-scFv antibody. Figure 2As shown, the structural composition of the AR bi-scFv antibody is: 4A5VL-99961VL-99961VH-4A5VH-hCH2CH3. The polynucleotide sequence encoding the AR bi-scFv antibody was amplified by PCR using the PCDNA3-AR bi-scFv OLS plasmid as a template. The obtained PCR product was placed into the PCDNA3.1 vector using DNA subcloning technology, and the sequence correctness of the subcloned fragment was verified by DNA sequencing. The final DNA vector was named PCDNA3-AR bi-scFv plasmid. The PCDNA3-AR bi-scFv plasmid is used to express the ROR1 bispecific scFv antibody (AR bi-scFv antibody, abbreviated as AR bi-scFv). The AR bi-scFv antibody was produced using the same process as the AR bi-scFv OLS fusion antibody, prepared through a CHO cell expression system.

[0081] 5. Preparation of anti-ROR1 biepitaxybispecific scFv OLS GFP fusion antibody To investigate the cell-killing mechanism of the AR bi-scFv OLS fusion antibody by detecting its localization in cells, this invention uses genetic engineering to add a GFP (green fluorescent protein) fragment to the C-terminus of the AR bi-scFv OLS fusion antibody, naming it the anti-ROR1 biepitaxybispecific scFv OLS GFP fusion antibody (abbreviated as AR bi-scFv OLS GFP). The structure of the AR bi-scFv OLS GFP fusion antibody is as follows. Figure 2 As shown. The polynucleotide sequence encoding this fusion antibody was obtained by overlapping PCR using PCDNA3-AR bi-scFv OLS plasmid and PCDNA3-GFP plasmid as templates. The PCR product was inserted into the PCDNA3.1 vector using DNA molecular subcloning technology, and the sequence correctness of the subcloned fragment was verified by DNA sequencing. The final DNA vector was named PCDNA3-AR bi-scFv OLS GFP plasmid. The PCDNA3-AR bi-scFv OLS GFP plasmid was used to express the anti-ROR1 bispecific scFv OLS GFP fusion antibody. The production of the ARbi-scFv OLS GFP fusion antibody followed the same process as the AR bi-scFv OLS fusion antibody, prepared using a CHO cell expression system.

[0082] 6. Preparation of P815ROR1 and P815mock cell lines To avoid the inherent functions of the ROR1 protein (such as inhibiting apoptosis or promoting cell growth) potentially affecting the cytotoxic effect of the anti-ROR1 bispecific scFv OLS fusion antibody, this invention constructed a stable ROR1-expressing cell line using the mouse-derived ROR1-negative cell line P815 (ATCC#TIB-64), named P815ROR1. P815ROR1 was selected as the target cell for cytotoxicity experiments in this invention.

[0083] The P815ROR1 cell line was constructed by transducing P815 cells with a lentivirus expressing the ROR1 protein. First, the ROR1 plasmid (Cat: HG13968-UT, Sino Biological, NM_005012.3, human ROR1 gene) was used as a PCR template to generate the ROR1 PCR product. This ROR1 PCR product was then inserted into the lentiviral vector PLEFT1a (catalog number: cDNA-pLV01, Biosettia, USA). The insertion procedure was strictly performed according to the manufacturer's instruction manual. The constructed DNA product was named PLEFT1a-ROR1 plasmid. All plasmids used for lentivirus preparation were purified using an endotoxin-free plasmid extraction kit (catalog number: 12362, QIAGEN, USA). After obtaining the PLEFT1a-ROR1 plasmid, human embryonic kidney cells 293T were transfected to package lentiviral particles expressing the ROR1 protein. 24 hours before plasmid transfection, 293T cells were injected with 12 × 10⁻⁶ cells per cell line. 6Viral cells were seeded at a density of [number] cells / flask in T150 cell culture flasks. The following day, 293T cells were co-transfected with 20 μg pVSVG, 20 μg pRSV REV, 20 μg pMDLg / p.RRE, and 60 μg of sequencing-verified PLEF1a-ROR1 plasmid. Lipofectamine 2000 (catalog number: 11668019, Invitrogen, USA) was used for transfection. Viral supernatants were collected at 24 and 48 hours post-transfection, and the two batches were combined for further concentration and purification. Lentiviral particles expressing the ROR1 protein were concentrated and purified from the collected viral supernatants by ultracentrifugation using a Beckman SW28 rotor (Beckman Coulter) at 28,000 rpm for 3 hours. Concentrated lentiviral particles expressing the ROR1 protein were transduced overnight into P815 cells at a virus-to-cell ratio of 3:1 in the presence of 8 μg / ml polybrene. During lentiviral transduction, P815 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (catalog number 11965092, Thermo Fisher Scientific, USA) at 37°C in a 5% CO2 incubator. Twenty-four hours after transduction, cells were selected using 2 μg / ml puromycin. Cells selected after transduction and puromycin were stained with PE-labeled mouse anti-human ROR1 monoclonal antibody (catalog number: 564474, BDPharmingen™, USA) according to the manufacturer's instructions, and then sorted into 96-well plates (single cell per well) by flow cytometry. Cells stably expressing the ROR1 protein after flow cytometry sorting were named P815ROR1 cells. Two P815ROR1 monoclonal cell lines (clones 4C1 and 6B2) with similar growth rates were selected as target cells for subsequent cell killing experiments after clonal expansion. To further confirm that ROR1 protein expression remained stable in both clone 4C1 and clone 6B2 cells after expansion, flow cytometry was used to re-examine ROR1 protein expression in these two monoclonal cell lines. The flow cytometry procedure was as follows: 1 × 10⁻⁶ cells were collected... 6P815ROR1 clone 4C1 cells, P815ROR1 clone 6B2 cells, or P815mock cells were stained with PE-labeled mouse anti-human ROR1 monoclonal antibody, washed three times with FACS buffer, and then analyzed by flow cytometry. The flow cytometry experiment was performed using a FACS-Calibur flow cytometer (BD BioSciences, USA), with CellQuest software (BD BioSciences, USA) used for data acquisition. The specific procedures for the flow cytometry experiment followed the flow cytometry protocol described in *Current Protocols in Immunology* (Coligan et al., Wiley-Interscience, 2002), including FACS staining and fluorescence intensity measurement. All flow cytometry data were analyzed using FlowJo software (TreeStar, USA). The P815mock cells described above were prepared by transducing P815 cells with lentiviral particles packaged in an empty PLEF1a vector containing no DNA insert fragments, and were named P815mock cells (ROR1 negative control cells). The preparation process of P815mock cells was completely similar to that of P815ROR1 cells. The experimental results of this flow cytometry experiment are as follows: Figure 3 As shown: (1) After staining with PE-labeled mouse anti-human ROR1 monoclonal antibody, the flow cytometry sorting histogram of the P815ROR1 clone population was more deflected than that of the P815mock cell population, indicating that both sorted monoclonal P815ROR1 cell lines (4C1 and 6B2) could stably express ROR1 protein; (2) After staining with PE-labeled mouse anti-human ROR1 monoclonal antibody, the flow cytometry sorting histogram of the 6B2 clone population was more deflected than that of the 4C1 clone population, indicating that the Ror1 protein expression level of the 6B2 clone cells was higher than that of the 4C1 clone cells.

[0084] 7. Preparation of P815AR bi-scFv cell line The AR bi-scFv OLS fusion antibody consists of an AR bi-scFv moiety (cell-binding domain) and an OLS moiety (organelle localization sequence). To rule out the possibility that the AR bi-scFv moiety itself might have a cytotoxic effect on P815 cells, this invention constructs a cell line stably expressing the AR bi-scFv antibody (named P815AR bi-scFv) based on the mouse cell line P815 to test whether the AR bi-scFv moiety alone would have a toxic effect on P815 cells. The basis of this experiment is that if P815 cells can stably express the AR bi-scFv antibody, then the AR bi-scFv moiety itself in the AR bi-scFv OLS fusion antibody will not have a toxic effect on P815 cells.

[0085] The construction process of P815AR bi-scFv cells is similar to that of P815ROR1 cells. Using pCDNA3-AR bi-scFv plasmid as a PCR template, PCR products for constructing the AR bi-scFv lentiviral expression vector were obtained. This PCR product (containing: 4A5VL-99961VL-99961VH-4A5VH-hCH2CH3) was then inserted into the lentiviral vector PLEF1a using DNA molecular cloning techniques, resulting in the DNA construct named PLEF1a-AR bi-scFv plasmid. PLEF1a-AR bi-scFv plasmid was used to produce AR bi-scFv lentiviral particles. The obtained AR bi-scFv lentiviral particles were used to transduce P815 cells. Twenty-four hours after transduction, cells were selected using 2 μg / ml puromycin. The selected cells were then analyzed by flow cytometry to determine if they stably expressed AR bi-scFv antibodies. The flow cytometry detection process is as follows: take 1×10 6 Cells that had undergone transduction and puromycin selection were first washed three times thoroughly with FACS buffer. Then, following the manufacturer's instructions, they were stained with 100 μL of FITC-labeled Ror1 protein (catalog number: RO1-HF253, ACROBiosystems) or negative control protein (catalog number: BC7-H5254, ACROBiosystems), at a concentration of 10 μg / mL. After washing three times with FACS buffer, flow cytometry was performed. The flow cytometry experiment was conducted using a FACS-Calibur flow cytometer. Data acquisition was performed using CellQuest software, and data analysis was performed using FlowJo software. The results of this flow cytometry experiment are as follows: Figure 4As shown, there is a significant flow cytometry histogram shift between the solid line (cells stained with Ror1 protein) and the dashed line (cells stained with negative control protein). Figure 4 The flow cytometry data shown confirms that P815 cells can stably express the AR bi-scFv antibody, indicating that the AR bi-scFv portion of the AR bi-scFv OLS fusion antibody itself has no toxic effect on P815 cells.

[0086] The OLS portion of the AR bi-scFv OLS fusion antibody comprises two components: NLS and NoLS. The NLS and NoLS sequences used in this invention (derived from human HnRNPA1 and NPM1 proteins, respectively) are completely conserved (identical) between the human and mouse species. Therefore, the OLS portion of the AR bi-scFv OLS fusion antibody will not produce toxic effects on mouse P815 cells.

[0087] The AR bi-scFv OLS fusion antibody consists of an AR bi-scFv moiety and an OLS moiety. The experimental results above indicate that neither AR bi-scFv alone nor OLS alone is toxic to P815 cells.

[0088] 8. Verify the binding function of AR1 or AR2 to the ROR1 protein. Before combining two anti-ROR1 monospecific scfv antibodies (AR1 and AR2) into an anti-ROR1 bispecific OLS fusion antibody (AR bi-scfv OLS), this invention first uses flow cytometry to verify whether AR1 or AR2 can bind to ROR1.

[0089] Jeko-1 cells (ATCC ID CRL-3006™) are human ROR1-positive cells (Blood 2010 Nov 25;116(22); 4532-4541). This is because AR1 and AR2 contain mouse CH2CH3 fragments (e.g., [missing information]) used as tags for purification and detection. Figure 2 As shown in the figure, the secondary antibody used in this flow cytometry experiment is a goat anti-mouse secondary antibody. To avoid staining interference from the secondary antibody (the goat anti-mouse secondary antibody may bind to mouse-derived cells), this flow cytometry experiment uses human ROR1-positive Jeko-1 cells (instead of mouse ROR1-positive P815 ROR1 cells) as test cells to verify the binding function of AR1 and AR2 to ROR1.

[0090] The parental full-length antibodies for AR1 and AR2 (Antibody 4A5 and Antibody 99961) are two published and well-identified anti-ROR1 antibodies (US Patent Nos.: US20130273073A1 and US9758591B2). Therefore, in verifying the ROR1 binding function of AR1 or AR2 by flow cytometry, this invention uses only Jeko-1 cells (ROR1-positive cells) as test cells. Unlike other flow cytometry experiments that use ROR1-negative cells as negative controls, this flow cytometry experiment uses Jeko-1 cells stained only with the secondary antibody as a negative control for testing ROR1 binding function.

[0091] Take 1×10 6 Jeko-1 cells were stained with 100 ng AR1 or AR2 for 30 minutes, washed three times with FACS buffer, and then stained with FITC-labeled goat anti-mouse IgG secondary antibody (R&D Systems, Inc., Catalog No.: F0103B) according to the manufacturer's instructions. After washing three times with FACS buffer, flow cytometry was performed. Flow cytometry experiments were performed using a FACS-Calibur flow cytometer, data were acquired using CellQuest software, and analyzed using FlowJo software. Figure 5 As shown, there is a significant flow cytometry histogram shift between the solid line (cells stained with AR1 or AR2 first, then with secondary antibody) and the dashed line (cells stained with secondary antibody only), which indicates that: (1) both AR1 and AR2 can bind to Ror1 protein; and (2) both AR1 and AR2 are functional anti-ROR1 monospecific scfv antibodies.

[0092] 9. Verify the binding function of the AR bi-scFv OLS fusion antibody to the ROR1 protein. The anti-ROR1 bispecific scFv OLS fusion antibody (AR bi-scFv OLS fusion antibody) is constructed from two anti-ROR1 monospecific scFv antibodies (AR1 and AR2). These two anti-ROR1 monospecific scFv antibodies (AR1 and AR2) bind to two non-overlapping epitopes in the extracellular domain of the ROR1 protein, respectively (US Patent Nos.: US20130273073A1 and US9758591B2). To verify that the AR bi-scFv OLS fusion antibody derived from AR1 and AR2 can simultaneously bind to two non-overlapping epitopes in the extracellular domain of the ROR1 protein, the present invention performed the following... Figure 6The four flow cytometry experiments for detecting ROR1 protein binding function are shown below: (1) Experiment 1: P815 ROR1 cells (solid line, ROR1 positive cells) or P815 mock cells (dashed line, ROR1 negative cells) were stained with 100 ng AR bi-scFv OLS fusion antibody. After washing three times with FACS buffer, they were stained with FITC-labeled mouse anti-human IgG Fc secondary antibody (catalog number 9040-02, Southern Biotech) according to the manufacturer's instructions. After washing three times with FACS buffer, they were then stained with FITC-labeled mouse anti-human IgG Fc secondary antibody (catalog number 9040-02, Southern Biotech). After washing three times with FACS buffer, they were then stained with FITC-labeled mouse anti-human IgG Fc secondary antibody. (2) Experiment 2: P815 ROR1 cells (solid line) or P815 mock cells (dashed line) were first co-incubated with excess AR1 (10 μg / reaction system) to block the extracellular domain epitopes of ROR1 protein recognized by AR1. After washing three times with FACS buffer, they were stained with 100 ng AR bi-scFv OLS fusion antibody. After washing three times with FACS buffer, they were then stained with FITC-labeled mouse anti-human IgG Fc secondary antibody. (3) Experiment 3: P815 ROR1 cells (solid line) or P815mock cells (dashed line) were first co-incubated with excess AR2 (10 μg / reaction system) to block the extracellular epitopes of ROR1 protein recognized by AR2. After washing three times with FACS buffer, they were stained with 100 ng AR bi-scFv OLS fusion antibody, washed three times with FACS buffer, stained with FITC-labeled mouse anti-human IgG Fc secondary antibody, washed three times with FACS buffer, and finally detected by flow cytometry. (4) Experiment 4: P815 ROR1 cells (solid line) or P815mock cells (dashed line) were first co-incubated with excess AR1 and AR2 (10 μg / reaction system) to simultaneously block the two non-overlapping extracellular epitopes of ROR1 protein recognized by AR1 and AR2. After washing three times with FACS buffer, they were stained with 100 ng AR bi-scFv OLS fusion antibody, washed three times with FACS buffer, and finally detected by flow cytometry. Staining with bi-scFv OLS fusion antibody, followed by washing three times with FACS buffer, then staining with FITC-labeled mouse anti-human IgG Fc secondary antibody, and washing three times with FACS buffer, was performed before flow cytometry analysis. To avoid immediate internalization of AR1, AR2, and AR bi-scFv OLS fusion antibodies upon binding to the ROR1 protein on the cell surface, which could affect subsequent detection experiments, all flow cytometry procedures were performed on ice, and all antibody staining times were strictly controlled to 20 minutes. The results of the four flow cytometry assays are as follows: Figure 6As shown: (1) In Experiment 1, there was a significant flow cytometry histogram shift between the solid and dashed lines, indicating that the ARbi-scFv OLS fusion antibody could bind to the ROR1 protein; (2) In Experiment 2, there was a significant flow cytometry histogram shift between the solid and dashed lines, indicating that although the amount of AR1 used was excessive (10 μg / reaction), AR1 alone could not block the specific binding of the ARbi-scFv OLS fusion antibody to the ROR1 protein; (3) In Experiment 3, there was a significant flow cytometry histogram shift between the solid and dashed lines, indicating that although the amount of AR2 used was excessive (10 μg / reaction), AR2 alone could not block the specific binding of the ARbi-scFv OLS fusion antibody to the ROR1 protein; (4) In Experiment 4, there was no significant flow cytometry histogram shift between the solid and dashed lines, indicating that the combined action of AR1 and AR2 could basically block the specific binding of the ARbi-scFv OLS fusion antibody to the ROR1 protein. Figure 6 The data shown indicate that the AR bi-scFv OLS fusion antibody has two functional ROR1 binding domains and can simultaneously bind two non-overlapping ROR1 extracellular epitopes. This AR bi-scFv OLS fusion antibody is a functional anti-ROR1 dual epitope bispecific scFv antibody.

[0093] 10. Cell killing assay using AR bi-scFv OLS fusion antibody (an antibody containing an OLS fragment). After confirming that the bi-scFv OLS fusion antibody can simultaneously bind to two non-overlapping ROR1 extracellular domain epitopes, this invention uses the fusion antibody to treat P815ROR1 cells or P815 mock cells to detect its cytotoxic effect.

[0094] Considering that the serum concentration in the cell culture medium can affect the sensitivity of cell killing experiments (high-concentration serum culture medium can provide sufficient cell growth factors, and cells cultured in high-concentration serum culture medium will show higher survival rates due to the excessive supply of cell growth factors), for the purpose of proof of concept, this invention cultured P815ROR1 cells and P815mock cells in low-serum culture medium (2% fetal bovine serum) instead of the conventional 10% serum culture medium when conducting cell killing experiments.

[0095] On the first day of the experiment, 15 ml of cell culture medium containing 2% fetal bovine serum (FBS) and 200 ng / mL AR bi-scFv OLS fusion antibody was added to a T75 cell culture flask. Each cell culture flask was seeded with 2 × 10⁶ cells / ml. 4P815ROR1 cells or P815mock cells were cultured in a 37°C, 5% CO2 incubator. Every 48 hours, 5 ml of fresh cell culture medium containing 2% fetal bovine serum (FBS) with 1 μg of AR bi-scFv OLS fusion antibody was added to the initial 15 ml of cell culture medium. Cells were cultured in T75 cell culture flasks for 12 days without changing the old culture medium or transferring the cells to new flasks. Every 48 hours, 5 ml of fresh culture medium containing AR bi-scFv OLS fusion antibody (containing 2% FBS) was added to the original flask to replenish the antibody. On day 12, cell viability was assessed using trypan blue staining (catalog number: 15250061, Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The cell viability assay was performed as follows: 0.4% trypan blue solution (dissolved in phosphate buffer at pH 7.2) was mixed with the cell culture medium at a 1:1 ratio, and the cells were immediately photographed under a low-power microscope. Cells stained with trypan blue were considered dead. The results of the cell killing assay were as follows: Figure 7 As shown: (1) Figure 7 All three images show that after 12 days of treatment with the AR bi-scFv OLS fusion antibody, P815ROR1 cells (including clone 4C1 cells and clone 6B2 cells) showed extensive trypan blue staining, while P815mock cells were hardly stained with trypan blue. This indicates that the AR bi-scFv OLS fusion antibody can kill P815ROR1 cells (ROR1 positive cells) after 12 days of co-incubation with target cells, but has almost no killing effect on P815mock cells (ROR1 negative cells); (2) Figure 7 The images in the middle and right show that after 12 days of treatment with the AR bi-scFv OLS fusion antibody, the residual cell count of clone 6B2 was significantly lower than that of clone 4C1. This indicates that the AR bi-scFv OLS fusion antibody is effective against ROR1 protein-highly expressing cell lines (such as...). Figure 3 As shown, clone 6B2 cells (expressing higher levels of ROR1 protein than clone 4C1 cells) exhibited more significant cell-killing and / or cell growth-inhibiting effects. In summary, Figure 7The data show that: (1) After 12 days of long-term treatment, the AR bi-scFv OLS fusion antibody can selectively kill ROR1 positive cells, but has no significant effect on ROR1 negative cells; (2) The cell-killing and / or growth-inhibiting effects of the AR bi-scFv OLS fusion antibody depend on the presence of ROR1 protein; (3) The cell-killing and / or growth-inhibiting efficiency of the AR bi-scFv OLS fusion antibody is positively correlated with the expression level of ROR1 protein; (4) Since P815 cells themselves do not express ROR1 protein, P815ROR1 cells are genetically engineered cells that express exogenous ROR1 protein. Therefore, the killing and / or growth-inhibiting effects of the AR bi-scFv OLS fusion antibody on P815ROR1 cells are unrelated to the original biological functions of ROR1 protein (such as inhibiting apoptosis or promoting cell proliferation).

[0096] 11. Cell killing assay using AR bi-scFv antibody (an antibody without OLS fragments). After confirming that the AR bi-scFv OLS fusion antibody can selectively kill ROR1-positive cells, this invention prepared an AR bi-scFv antibody (an anti-ROR1 biepitaxy bispecific scFv antibody without the OLS fragment) to investigate whether the AR bi-scFv portion of the AR bi-scFv OLS fusion antibody alone has the function of killing ROR1-positive cells. This invention was then used to test its killing effect on P815ROR1 cells. In this experiment, the AR bi-scFv OLS fusion antibody (an anti-ROR1 biepitaxy bispecific scFv antibody containing the OLS fragment) was used as a positive control.

[0097] On the first day of the experiment, 2×10 4Two T75 cell culture flasks containing P815ROR1 cells were seeded, with 15 mL of cell culture medium containing 2% fetal bovine serum (FBS) and 200 ng / mL AR bi-scFv antibody or AR bi-scFv OLS fusion antibody added. The flasks were then incubated at 37°C in a 5% CO2 incubator. Two T75 cell culture flasks containing P815ROR1 cells were prepared for this experiment, with AR bi-scFv antibody or AR bi-scFv OLS fusion antibody added to each flask. Every 48 hours, 1 μg of the corresponding antibody was added to the initial 15 mL of cell culture medium (diluted with 5 mL of freshly prepared culture medium containing 2% FBS). The cells were cultured in the T75 flasks for 12 days without changing the old culture medium or transferring the cells to the new flasks. Only 5 mL of fresh 2% FBS culture medium containing the corresponding antibody was added to the original flasks to maintain the antibody concentration. On day 12 of the experiment, cell viability was assessed using trypan blue staining, following the manufacturer's instructions. The results are as follows: Figure 8 As shown: After 12 days of culture, P815ROR1 cells treated with AR bi-scFv OLS fusion antibody showed extensive trypan blue staining, while P815ROR1 cells treated with AR bi-scFv antibody (the same anti-ROR1 bi-epitope bispecific scFv antibody but lacking the OLS fragment) showed almost no trypan blue staining. The above data indicate that: (1) AR bi-scFv OLS fusion antibody (an antibody containing the OLS fragment) has a specific killing effect on P815ROR1 cells, while AR bi-scFv antibody (an antibody lacking the OLS fragment) has no such effect on P815ROR1 cells; (2) The cell-killing function of the anti-ROR1 bi-epitope bispecific scFv antibody depends on the presence of the OLS fragment.

[0098] 12. Mechanism of action of AR bi-scFv OLS fusion antibody To investigate how AR bi-scFv OLS fusion antibodies kill target cells, this invention utilizes genetic engineering to add a green fluorescent protein (GFP) fragment to the C-terminus of the AR bi-scFv OLS fusion antibody, constructing a GFP-tagged AR bi-scFv OLS GFP fusion antibody. The GFP-tagged AR bi-scFv OLS fusion antibody can be used to directly observe its intracellular localization after binding to the cell surface protein ROR1, thereby contributing to the investigation of the mechanism of action of the AR bi-scFv OLS fusion antibody.

[0099] On the first day of the experiment, take 2×10 4 P815ROR1 cells were seeded in T75 cell culture flasks, and 15 mL of culture medium containing 2% fetal bovine serum (FBS) and 200 ng / mL AR bi-scFv OLS GFP fusion antibody was added. The flasks were then incubated at 37°C in a 5% CO2 incubator. Every 48 hours, 1 μg of AR bi-scFv OLS GFP fusion antibody (pre-diluted in 5 mL of fresh culture medium containing 2% FBS) was added to the initial 15 mL of cell culture medium. Cells were cultured in T75 flasks for 12 days without changing the old culture medium or transferring the cells to new flasks; antibody replenishment was achieved solely by adding 5 mL of fresh 2% FBS culture medium containing AR bi-scFv OLS GFP fusion antibody to the original flask. On day 12, surviving P815ROR1 cells were observed under a fluorescence microscope. Results are as follows: Figure 9 As shown, the fluorescence signal of the AR bi-scFv OLS GFP fusion antibody is significantly distributed in the cell membrane region (exhibiting a characteristic membrane staining pattern), and is also enriched in the nucleolar structure (exhibiting a clear nucleolar staining feature).

[0100] Based on the above experimental data, the mechanism of action of AR bi-scFv OLS fusion antibody on P815ROR1 cells can be explained as follows: the nucleolus (a key organelle of the cell) of P815ROR1 cells becomes blocked or over-occupied due to the continuous accumulation of AR bi-scFv OLS fusion antibody (P815ROR1 cells were continuously treated with AR bi-scFv OLS fusion antibody for 12 days), which in turn hinders important cellular activities related to cell survival that occur in the nucleolus. The obstruction of important cellular activities ultimately leads to cell death and / or cell growth inhibition.

[0101] III. Conclusion Extracellular OLS fusion antibodies (organelle localization sequence fusion antibodies) selectively kill and / or inhibit the growth of target cells by persistently and selectively accumulating in a key organelle of the target cell (which should have a highly organized structure and be essential for cell survival, such as the nucleolus and mitochondria). The cytotoxic and / or growth-inhibiting effects of OLS fusion antibodies depend on the presence of cell surface antigens that can be recognized by the OLS fusion antibody. The cytotoxic and / or growth-inhibiting effects of OLS fusion antibodies are related to the expression level of the cell surface antigens recognized by the OLS fusion antibody. Moreover, the cytotoxic and / or growth-inhibiting effects of OLS fusion antibodies are independent of any original biological function of the cell surface antigens recognized by the OLS fusion antibody (such as anti-apoptosis, promotion of cell proliferation, cell signaling, etc.).

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Claims

1. A method for selectively killing target cells and / or inhibiting target cell growth, characterized in that, This includes contacting the target cells with an OLS fusion antibody, wherein the OLS fusion antibody accumulates within the organelles of the target cells, achieving selective killing and / or growth inhibition by blocking or over-occupying the organelles; wherein the OLS fusion antibody comprises: (i) a cell-binding domain that specifically binds to cell surface antigens of the target cell; and (ii) an organelle localization sequence (OLS) that guides the OLS fusion antibody into the organelles of the target cell.

2. The method according to claim 1, characterized in that, The cell-binding domain is selected from the group consisting of: antibodies, antigen-binding fragments of antibodies, protein scaffolds, cytokines, receptor ligands, and combinations thereof.

3. The method according to claim 1 or 2, characterized in that, The organelle localization sequences (OLS) are selected from the group consisting of: nuclear localization sequences, nucleolar localization sequences, endoplasmic reticulum localization sequences, mitochondrial localization sequences, lysosomal localization sequences, Golgi apparatus localization sequences, and any combination of two or more of the above sequences.

4. The method according to any one of claims 1 to 3, characterized in that, The target cells are cells that express specific cell surface antigens, selected from the following group: cancer cells, tumor cells, immune cells, virus-infected cells, bacterial-infected cells, fungal-infected cells, parasite-infected cells, and combinations thereof.

5. A pharmaceutical composition for treating a disease, characterized in that, The active ingredient comprises an OLS fusion antibody as defined in claim 1, and a pharmaceutically acceptable carrier or excipient. The disease is associated with a cell surface antigen recognized by the OLS fusion antibody, and the treatment is achieved by the accumulation, blockage, or over-occupation of the OLS fusion antibody within the target organelle.

6. The pharmaceutical composition according to claim 5, characterized in that, The diseases mentioned are selected from the following group: cancer, tumors, autoimmune diseases, infectious diseases, and combinations thereof.

7. A pharmaceutical composition for treating a disease, characterized in that, The active ingredient comprises a polynucleotide encoding the OLS fusion antibody as defined in claim 1, and a pharmaceutically acceptable carrier or excipient. The disease is associated with a cell surface antigen recognized by the OLS fusion antibody, and the treatment is achieved by the accumulation, blockage, or over-occupation of the OLS fusion antibody generated by the polynucleotide expression within the target organelle.

8. The pharmaceutical composition according to claim 7, characterized in that, The diseases mentioned are selected from the following group: cancer, tumors, autoimmune diseases, infectious diseases, and combinations thereof.

9. The pharmaceutical composition according to any one of claims 5 to 8, characterized in that, The pharmaceutical composition is formulated for administration via a route selected from the group consisting of: intravenous injection, subcutaneous injection, intramuscular injection, intratumoral injection, local administration, and combinations thereof.

10. An OLS fusion antibody, characterized in that, Include: (i) a cell-binding domain that specifically binds to cell surface antigens of the target cell; and (ii) an organelle localization sequence (OLS) that guides the OLS fusion antibody into the organelles of the target cell; The OLS fusion antibody is used to accumulate within the organelles to selectively kill the target cells and / or inhibit their growth by blocking or over-occupying the organelles.

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