Medicaments, methods and uses thereof

Abstract

The present invention relates to methods and conjugates for improving the permeability of a biological substance into the tumor microenvironment (TME) for therapeutic purposes, suitably comprising at least: i. A therapeutic domain; ii. A cleavable domain; and iii. A stabilizing domain.

Description

Technical Field

[0001] This invention relates to methods and conjugates for improving the permeability of biological substances into the tumor microenvironment (TME) for therapeutic purposes. Background Technology

[0002] Cancer is a leading cause of the global disease burden, and projections show that the global cancer burden will continue to increase. Cancer is a broad term referring to a large group of diseases that can affect any part of the body. The defining characteristic of cancer is the rapid production of unwanted cells that grow beyond their normal boundaries and can then invade adjacent parts of the body and spread to other organs.

[0003] Antibody-based therapies have revolutionized treatment, particularly in oncology. Engineered antigen-binding domains can bind to specific tumor markers and can activate or inactivate signaling pathways. This leads to a slowdown or cessation of cell proliferation and tumor growth, or tumor shrinkage (Zahavi D, Weiner L (2020). Monoclonal Antibodies in Cancer Therapy. Antibodies (Basel). 9(3):34). Engineered antigen-binding domains are also used in immunotherapy, where they can activate or inactivate immune cells within the tumor to halt or slow tumor proliferation and growth.

[0004] The success of these antibody-based therapies is due to the high specificity of the antigen-binding domain to its tumor targets. This mitigates non-specific binding to similar antigens, resulting in better targeting. Antibody-based therapies can be multivalent, so a single antibody can bind to its target multiple times, which produces additional efficacy that small molecules cannot provide (Imai K, Takaoka A (2006). Comparing antibody and small-molecule therapies for cancer. Nat Rev Cancer. 6(9):714-27).

[0005] Antibodies also contain a fragment crystallizable (Fc) region. This region has several functions, one of which is to prolong the antibody's half-life. This is achieved in part by increasing the antibody's size through the Fc region, allowing it to overcome the glomerular filtration barrier (GFB) threshold.

[0006] The main problem in this field remains the penetration of antibody-based therapies into the tumor microenvironment (TME). It is estimated that only 0.001%–7% of injected antibodies are able to penetrate the core of the tumor (Khongorzul et al. (2020). Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res. 18(1):3–19). This low penetration of biologics into the TME is associated with poor efficacy. This poor efficacy leads to the use of higher doses of the administered biologic to achieve therapeutic effects; however, higher doses may be harmful and / or toxic to patients.

[0007] One contributing factor to lower penetration is thought to be larger antibody size, and therefore, much effort has been focused on generating smaller antibody fragments to achieve better efficacy. Modeling experiments have also shown that smaller antibody fragments have better tissue penetration (Thurber et al. (2008). Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearance. Adv Drug Deliv Rev. 60(12):1421-34).

[0008] Small antibody fragments often lack the Fc region, which reduces the antibody's half-life (Li et al., 2019) and leads to lower efficacy.

[0009] In healthy vascularized tissue, cells are only a few micrometers (μm) away from blood vessels, so organ / cell penetration is not a problem. However, the large cell clusters acquired during tumor formation lead to an increased distance between the nearest blood vessel and the cells. This makes the accessibility and delivery of therapies to cells problematic.

[0010] This also results in unusual and significant characteristics within the tumor. For example, the distance from blood vessels leads to hypoxia within the tumor; low oxygen concentrations and elevated carbon dioxide levels result in a lower pH level. Furthermore, this environment promotes carcinogenesis. To achieve this, specific proteases are expressed, allowing rogue cells to escape into the bloodstream, invade local tissues, and eventually metastasize. Therefore, aberrantly expressed proteases are found in the tumor mesenchyme (TME) during tumor development. The expression of different protease combinations varies depending on the tumor type and location. These proteases have been used to specifically target antibody activity in vivo, such as WO2013192546A1 and WO2015066279A2.

[0011] Some protease cleavage sites are well known in the scientific literature, and cleavable domains containing such cleavage sites can be easily constructed using well-established genetic engineering techniques and / or chemical synthesis techniques known in the art. However, other protease cleavage sites are not yet known, and therefore more experiments are needed to design such cleavable domains.

[0012] Although these methods have been used to improve antibody specificity, a problem remains: antibodies have poor permeability and therefore poor efficacy. This invention aims to address this problem. Summary of the Invention

[0013] This invention is based on the use of tumor-specific proteases to enhance antibody penetration by specifically cleaving the antibody at certain engineered sites upon arrival at the TME. This invention is based on the discovery that such sequences around the antibody hinge region do not affect antibody expression or function.

[0014] The invention is also based on cutting the therapeutic agent and thus reducing its size at the tumor site or in the vicinity of unwanted cells or tumors, thereby significantly improving the specificity and permeability of the therapeutic agent and thus improving efficacy.

[0015] Therefore, in one aspect, the present invention provides an engineered pharmaceutical agent comprising at least: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilizing domain, wherein the cleavable domain is positioned between the therapeutic domain and the stabilizing domain.

[0016] The present invention introduces a cleavable domain between the therapeutic domain and the stable domain, the cleavage site of which selectively cleaves in the region near unwanted cells, the therapeutic domain being released from the stable domain in the region near unwanted cells, and being able to bind to the unwanted cells or to antigens in the unwanted cells.

[0017] This invention provides a means of improving the permeability of a drug to a tumor by using size and diffusion, so as to penetrate deeper into the hypoxic parts of the tumor, thereby achieving enhanced drug efficacy. Attached Figure Description

[0018] Figure 1 - A schematic diagram showing that the therapeutic domain is Fab and the cleavage site is located in the upper or lower part of the antibody's hinge region. After entering the TME, conditions allow for the antibody to be cleaved into three domains: the Fab domain (x2) and the Fc domain (x1).

[0019] Figure 2- A schematic diagram showing that the therapeutic domain is Fab and the cleavable domain is located after the hinge region of the antibody. After entering the TME, conditions allow the antibody to be cleaved into two domains: the F(ab')2 domain (x1) and the Fc domain (x1).

[0020] Figure 3 - A schematic diagram showing that the treatment domain is not Fab and the slicable domain exists before the hinge region containing the Fc entity. Upon entering the TME, conditions allow the agent to be sliced ​​into 3 domains: the treatment domain (x2) and the Fc domain (x1).

[0021] Figure 4 - A schematic diagram showing that the treatment domain is not Fab and the slicable domain exists after the hinge region containing the Fc entity. Upon entering the TME, conditions allow the agent to be sliced ​​into two domains: the treatment domain (x1) and the Fc domain (x1).

[0022] Figure 5 - An example of a cleavable structural domain existing at a junction structural domain that separates the therapeutic structural domain of a biological agent from its stable structural domain.

[0023] Figure 6 – Schematic diagram of different locations of the cuttable structural domain within the hinge region. (A) Designs D1-D4 – Cuttable site located within the upper hinge region; (B) Designs D6-D7 – Cuttable site located within the lower hinge region; (C) Design D5 engineered with glycosylation sites and used as a negative control. The location and length of the connector vary in different designs. The cuttable site may also be located within the middle hinge region (not shown).

[0024] Figure 7 – Antibody concentration was assessed using Protein A Octet.

[0025] Figure 8 – Showing a gel image of protein quality analysis (SDS-PAGE).

[0026] Figure 9 – Antibody concentration was repeatedly assessed using Protein A Octet.

[0027] Figure 10 – Showing a repeat gel image from a protein quality analysis (SDS-PAGE). The box indicates the antibody heavy chain (approximately 50 kDa).

[0028] Figure 11 –Chromatographic evaluation of candidate expression and purification. (A) Trastuzumab (WT, also known as mAb) control and (B) D1.

[0029] Figure 12– Showing gel images of (A) non-reduced protein quality analysis (SDS-PAGE) and (B) reduced protein quality analysis (SDS-PAGE). Channels: 1 = Molecular weight marker, in kilodaltons (kDa); 2 = Trastuzumab mAb; 3 = Trastuzumab D1; 4 = Trastuzumab D2; 5 = Trastuzumab D3; 6 = Trastuzumab D4; 7 = Trastuzumab D5; 8 = Trastuzumab D6; 9 = Trastuzumab D7.

[0030] Figure 13 – The combination of trastuzumab mAb and engineered design with hHER2.

[0031] Figure 14 –12-point dilution kinetics measurements. (A) Trastuzumab mAb and (B) D1.

[0032] Figure 15 – Gel images showing the release of the uPA protease from control mAb, D1, and D2 antibodies, including the F(ab) of the D1-D5 fragment and the F(ab') of the D6-D7 fragment. 1 = Molecular weight marker; 2 = mAb + 0 nM protease; 3 = mAb + 23.5 nM protease; 4 = mAb + 235 nM protease; 5 = D1 + 0 nM protease; 6 = D1 + 23.5 nM protease; 7 = D1 + 235 nM protease; 8 = D2 0 nM protease; 9 = D2 + 23.5 nM protease; 10 = D2 + 235 nM protease; 11 = D3 + 0 nM protease; 12 = D3 + 23.5 nM protease; 13 = mAb + 235 nM protease; 14 = D4 + 0 nM protease; 15 = D4 + 23.5 nM protease; 16 = D4 + 235 nM protease; 17 = D5 + 0 nM protease; 18 = D5 + 23.5 nM protease; 19 = D5 + 235 nM protease; 20 = D6 + 0 nM protease; 21 = D6 + 23.5 nM protease; 22 = D6 + 235 nM protease; 23 = D7 + 0 nM protease; 24 = D7 + 23.5 nM protease; 25 = D7 + 235 nM protease. Fc is the middle row of bands, and F(ab) and F(ab')2+ / light chain are the bottom row of bands. F(ab) and F(ab')2 appear to be the same size on the gel because DTT reduces the released F(ab)'2 to 2 Fabs.

[0033] Figure 16 –UPA treatment followed by trastuzumab and engineered antibody binding to HER2 –Fab assay.

[0034] Figure 17 – Binding of trastuzumab and engineered antibodies to the FCγ receptor. (A) FCγ receptor Ia; (B) FCγ receptor IIa; (C) FCγ receptor IIb; (D) FCγ receptor IIIa.

[0035] Figure 18 –Large-scale 20 mg expression of (A) trastuzumab mAb 98.5% monomer and (B) trastuzumab D3 94.3% monomer.

[0036] Figure 19 – SDS-PAGE gel electrophoresis shows the expression and purity of trastuzumab-mAb and D3. 1 = trastuzumab-mAb and 2 = trastuzumab D3. NR = non-reducing and R = reducing. PageRuler Plus pre-stained gradient was used.

[0037] Figure 20 –By ELISA, large-scale preparations of trastuzumab mAb and D3 were shown to bind to hHER2.

[0038] Figure 21 – Expression of trastuzumab-mAb and trastuzumab D1 in the CHO-K1 cell line.

[0039] Figure 22 – Comparison of expressions from CHO-K1 and Expi-CHO.

[0040] Figure 23 – SDS-PAGE gel electrophoresis shows the expression and purity of trastuzumab engineered antibodies at different cleavage sites. 1. Molecular weight markers, 2. Empty channels, 3. Trastuzumab D9 (with ADAM10 site).

[0041] Figure 24 –Trastuzumab mAb and the binding of trastuzumab D3 and D9 to hHER2.

[0042] Figure 25 – The following are non-reducing SDS-PAGE gels showing the expression and purity of: 1. Molecular weight marker, 2. Bevacizumab mAb, 3. Bevacizumab D13, 4. Cetuximab mAb, 5. Cetuximab D12, 6. M5A mAb, 7. M5A D15, 8. Pembrolizumab mAb, 9. Pembrolizumab D14a, 10. Pembrolizumab D14b, 11. Ipilimumab mAb, 12. Ipilimumab D16.

[0043] Figure 26 –(A) Binding of cetuximab mAb and cetuximab D12 to hEGFR; (B) Binding of bevacizumab mAb and bevacizumab D13 to hVEGF.

[0044] Figure 27 – shows the SDS-PAGE gels of the following protease digests: (A) 1. Molecular weight marker, 2. Bevacizumab mAb + 0 nM protease, 3. Bevacizumab mAb + 23.5 nM protease, 4. Bevacizumab mAb + 235 nM protease, 5. Bevacizumab D13 + 0 nM protease, 6. Bevacizumab D13 + 23.5 nM protease, 7. Bevacizumab D13 + 235 nM protease, 8. 9. Cetuximab mAb + 0 nM protease, 10. Cetuximab mAb + 23.5 nM protease, 11. Cetuximab mAb + 235 nM protease, 12. Cetuximab D12 + 0 nM protease, 13. Cetuximab D12 + 23.5 nM protease; (B) 1. Molecular weight marker, 2. M5AmAb + 0 nM protease, 3. M5A 4. M5AmAb + 23.5 nM protease, 5. M5A D15 + 0 nM protease, 6. M5A D15 + 23.5 nM protease, 7. M5A D15 + 235 nM protease; (C) 1. Molecular weight marker, 2. Pembrolizumab mAb, 3. Pembrolizumab D14a + 500 μM protease, 4. Pembrolizumab D14b + 500 μM; (D) 1. Molecular weight marker, 2. Trastuzumab D3, 3. Trastuzumab D9. Protease was added at a 1:1 molar ratio of antibody to protease.

[0045] Figure 28 – Binding of different antibody-derived fragments to their corresponding targets: (A) Binding of pembrolizumab, D14a, and D14b to hPD1-HIS; (B) Binding of M5A and D15 to hCEACAM-5; (C) Binding of ipilimumab and D16 to hCTLA-4. Insertion of cleavage sites does not significantly affect the ability of engineered antibodies to bind to their targets.

[0046] Figure 29 – Schematic diagrams of different types of experimentally tested therapeutic domains. (A) A drug in which the therapeutic domain is Fab; (B) A drug in which the therapeutic domain is VHH; (C) A drug in which the therapeutic domain is ScFv. In (A), (B), and (C), the cleavable domain is located at the upper hinge, and the stable domain is the Fc region.

[0047] Figure 30 – SDS-PAGE gel, showing: (A) Expression and purity of various engineered agents containing VHH or ScFv as therapeutic domains. Channels: 1 = Molecular weight marker; 2 = Anti-EGFR VHH-FC (D17); 3 = Anti-EGFR VHH control (7D12-FC); 4 = Anti-Her2 ScFv-FC (D18); 5 = Anti-Her2 ScFv-FC control (trastuzumab Vl-Vh long linker); (B) Proteolytic digestion of VHH and ScFv-FC constructs with uPA. Channels: 1. Molecular weight marker; Channel 2. D17, containing 250 μM uPA; 3 = Anti-EGFR VHH control, containing 250 μM uPA; 4 = D18, containing 250 μM uPA; 5 = Anti-Her2 ScFv-FC control, containing 250 μM uPA. All proteases were added at a 1:1 antibody-protease molar concentration; (C) The binding of anti-EGFR VHH-FC (D17) to hEGFR was assessed by ELISA; (D) The binding of anti-Her2 ScFv-FC (D18) to hHER2 was assessed by ELISA.

[0048] Figure 31 – Binding of tumor samples to human Her2 using Vk light chain assay. (A) In the mouse study, five “Group 2” (G2) mice were injected with trastuzumab mAb, and five “Group 3” (G3) mice were injected with trastuzumab D3. Compared with the accumulation of trastuzumab mAb in G2 mice, G3 mice showed increased accumulation of trastuzumab D3 in tumors. (B) Combined data from the mouse study in (A) showed that 15.6 pM of trastuzumab D3 was present in G3 mice and 7.8 pM of trastuzumab mAb was present in G2 mice. Detailed Implementation

[0049] The agent can be a therapeutic agent. By "therapeutic agent," we mean that it has beneficial or desired results (including, and preferably, beneficial or desired clinical results), i.e., a therapeutic agent (curative agent) or "preventative agent" administered to treat and / or prevent disease. Our term "agent" includes the meaning of biological agents, including proteins, oligopeptides, polypeptides, enzymes, antibodies and portions thereof, vaccines, nucleotides, antibody analogs, antibody mimics, immunoglobulins, immunomodulators, blood, blood components, cells, allergens, genes, viruses, toxins, venoms, or combinations thereof.

[0050] In one embodiment, the agent is an engineered agent. By "engineered agent," we mean that the agent is not naturally occurring but synthetic. In one embodiment, the stabilizing and therapeutic domains are naturally occurring (such as in antibodies); while the cleavable domain is engineered into the hinge region.

[0051] The term "therapeutic domain" as we use it encompasses any domain or entity that has a therapeutically beneficial effect on the subject. A therapeutic domain can bind to an endogenous target, such as a tumor antigen that is endogenous to the subject.

[0052] The term "cleavable domain" as used here includes any cleavable domain or entity at the tumor site that is readily cleaved by another domain or entity. Cleavage can occur at one or more locations within the cleavable domain. Such sites within the cleavable domain are called cleavage sites. Preferably, the cleavable domain is a peptide containing a substrate for an enzyme.

[0053] Antibodies contain naturally occurring cleavage sites for non-tumor-specific proteases and / or bacterial enzymes (Brezski and Jordan. 2010. Cleavage of IgGs by proteases associated with invasive diseases. mAbs 2:3, 212-220). In one embodiment, the pharmaceutical agent of the present invention does not contain naturally occurring cleavage sites. Therefore, in one embodiment, the pharmaceutical agent of the present invention does not contain any cleavable domains susceptible to the following enzymes: papain, pepsin, gamma-glutamyl endopeptidase I (GluV8), Streptococcus pyogenes immunoglobulin degrading enzyme (IdeS), streptococcal protease (SpeB), pseudolysin, mirabilysin, and trepolisin.

[0054] In one embodiment, the cleavable domain is artificially introduced into a naturally occurring pharmaceutical agent. The phrase "the cleavable domain is artificially introduced" means that the cleavable domain is not present in the naturally occurring pharmaceutical agent. The agent is engineered such that the cleavable domain is artificially introduced.

[0055] As used herein, the term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, fragments generated from Fab expression libraries, and multispecific (e.g., bispecific) antibodies. Such fragments include fragments of the complete antibody that retain its binding activity to the target, Fv, F(ab'), and F(ab')2 fragments, as well as single-chain antibodies (scFv), fusion proteins, and other synthetic proteins containing the antigen-binding site of the antibody. Containing only a portion of the antibody's targeting portion may be advantageous due to optimized blood clearance and a lower likelihood of nonspecific binding due to the presence of the Fc region. Also included are domain antibodies (dAb), double-chain antibodies, and nanobodies (such as camel antibodies, engineered camel antibodies, shark antibodies, or llama antibodies). The advantages of using antibody fragments instead of the entire antibody are multifaceted. The smaller size of the fragment may result in improved pharmacological properties, such as better penetration into solid tissues. Furthermore, antigen-binding fragments (such as Fab, Fv, ScFv, and dAb antibody fragments) can be expressed in E. coli or secreted from E. coli or yeast, thus allowing for easy production in the laboratory and economical production on a commercial scale.

[0056] Antibodies can belong to any of the IgG, IgE, IgA, IgM, and IgD classes and can be derived from any species. If the antibody is IgG, it can be any of IgG1, IgG2, IgG3, or IgG4. However, it is preferred that the antibody, or at least its constant region, is derived from a specific host when the agent is to be administered to that host. Antibodies can be human antibodies, i.e., they have the amino acid sequence of human antibodies specific to the selected antigen. Alternatively, they can be mouse antibodies, chimeric antibodies, or humanized antibodies. For example, when the agent is to be administered to humans, the antibody is preferably a human antibody or a humanized antibody, and so on.

[0057] Technicians can use well-established techniques in the art to prepare suitable antibodies that bind to specific antigens expressed by unwanted cells. Methods for preparing monoclonal antibodies and antibody fragments are well known in the art and include hybridoma techniques.

[0058] The term "stabilizing domain" as we use it includes any domain or entity that can stabilize or prolong the half-life of a drug in a biological system by reducing or inhibiting degradation and / or reducing or inhibiting the clearance of the drug, either whole or in part.

[0059] In the embodiments, the agent comprises amino acids. Preferably, the agent is a protein, peptide, bicyclic peptide, tricyclic peptide, or polypeptide. As used herein, the term "protein" is used in its conventional sense, referring to a plurality of amino acids linked together by peptide bonds to form a polypeptide polymer chain. In additional embodiments, the agent comprises non-natural isomers or amino acids.

[0060] Polynucleotides encoding suitable therapeutic domains are known in the art, or can be easily designed from known sequences (such as sequences of proteins known to interact with surface markers expressed on unwanted cells, or sequences of proteins contained in nucleotide sequence databases such as GenBank, EMBL, and dbEST). Polynucleotides encoding suitable stable domains are known in the art, or can be easily designed and fabricated from known sequences. Polynucleotides encoding suitable cleavable domains are known in the art, or can be easily designed and fabricated from known sequences. Those skilled in the art are able to fabricate such agents, which are typically developed based on known methods such as chemical synthesis techniques and / or genetic engineering techniques.

[0061] In genetic engineering techniques, nucleic acids are expressed in a suitable host to produce the engineered agents of the present invention. Therefore, nucleic acids encoding the agents of the present invention can be used according to known techniques (with appropriate modifications in view of the teachings contained herein) to construct expression vectors, which are then used to transform suitable host cells to express and produce the agents of the present invention.

[0062] It should be understood that the nucleic acid encoding the agent of the present invention can be linked with a variety of other nucleic acid sequences to be introduced into a suitable host. The accompanying nucleic acid will depend on the nature of the host, the manner in which the nucleic acid is introduced into the host, and whether an episome is required for maintenance or integration, as is well known in the art.

[0063] The amino acid residues described herein are generally in their natural "L" isomer form. However, in some cases, residues in the "D" isomer form may be substituted with L amino acid residues, provided that the pharmaceutical agents of the present invention retain their function. Unless otherwise explicitly stated, this definition also includes chemically modified amino acids, including amino acid analogs (such as penicillamine, 3-mercapto-D-valine), naturally occurring non-protein-producing amino acids (such as ortholeucine), β-amino acids, azide peptides, N-methylated amino acids, and chemically synthesized compounds having the characteristic properties of amino acids known in the art. The term "protein-producing" indicates that the amino acid can be integrated into proteins in the cell via well-known metabolic pathways. This definition also includes amino acids whose functional side groups have been chemically derivatized. Such derivatized molecules include, for example, those in which the free amino group has been derivatized to form amine hydrochloride, p-toluenesulfonyl, benzyloxycarbonyl, tert-butoxycarbonyl, chloroacetyl, or formyl. The free carboxyl group may be derivatized to form salts, methyl esters and ethyl esters, or other types of esters or hydrazides. The free hydroxyl group may be derivatized to form O-acyl or O-alkyl derivatives. Peptide portions of one or more naturally occurring amino acid derivatives containing twenty standard amino acids are also included as derivatives.

[0064] It should be understood that the peptide portion of the pharmaceutical agent of the present invention may be a peptide "mimic," that is, a peptide mimic that imitates the structural features of a peptide comprising or composed of the amino acid sequences described herein. Peptide mimics may have advantages in terms of therapeutic use, resistance to degradation, permeability, or possible oral administration.

[0065] Non-protein entities (such as PEG molecules) can be fused using methods well known in the art. Covalent chemical conjugation techniques may include, but are not limited to: (i) adding a C-terminal cysteine ​​residue and conjugating it via maleimide chemistry; (ii) conjugating via lysine; and (iii) conjugating using a His tag.

[0066] As is well known, the glomerular filtration barrier (GFB) is a highly specialized blood filtration interface that exhibits high conductivity for small and medium-sized solutes in plasma but remains relatively impermeable to large molecules. Thus, the barrier enables renal elimination of small and medium-sized solutes but prevents renal elimination of large molecules, acting as a sieve that typically allows only water and small solutes to pass through for removal by the kidneys. In one embodiment, the size of a pharmaceutical agent prevents it from penetrating the glomerular filtration barrier. Suitable agents exceeding a certain size will not pass through the GFB and therefore will not be eliminated by the kidneys. Those skilled in the art will understand that the GFB threshold size will vary by animal species and may even vary by subject. Furthermore, in disease, glomerular selective permeability may change, making the GFB “more permeable,” allowing large molecules (e.g., albumin) to be eliminated by the kidneys. Other properties of the pharmaceutical agent may also influence prevention of agent penetration into the GFB, such as charge, composition, or surface modifications.

[0067] In some embodiments, the agent has dimensions of at least 6 nanometers (nm), at least 6.5 nm, at least 7 nm, at least 7.5 nm, at least 8 nm, at least 8.5 nm, at least 9 nm, at least 9.5 nm, and at least 10 nm. Our use of "dimensions" includes the meaning of the hydrodynamic diameter of the agent. Those skilled in the art will be able to select an appropriate measurement method to measure the size of the agent. For example, dynamic light scattering (DLS) or any standard technique in the art can be used to measure the hydrodynamic diameter.

[0068] In another embodiment, the agent has a concentration of at least 40 kilodaltons (kDa), at least 41 kDa, at least 42 kDa, at least 43 kDa, at least 44 kDa, at least 45 kDa, at least 46 kDa, at least 47 kDa, at least 48 kDa, at least 49 kDa, at least 50 kDa, at least 51 kDa, at least 52 kDa, at least 53 kDa, at least 54 kDa, at least 55 kDa, at least 56 kDa, at least 57 kDa, at least 58 kDa, at least 59 kDa, at least 60 kDa, and so on. Molecular weights of at least 61 kDa, at least 62 kDa, at least 63 kDa, at least 64 kDa, at least 65 kDa, at least 66 kDa, at least 67 kDa, at least 68 kDa, at least 69 kDa, at least 70 kDa, at least 71 kDa, at least 72 kDa, at least 73 kDa, at least 74 kDa, at least 75 kDa, at least 76 kDa, at least 77 kDa, at least 78 kDa, at least 79 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, and at least 150 kDa.

[0069] The molecular weight (also known as molar mass, M) of a pharmaceutical substance is the mass of one mole of that substance, expressed in M ​​grams. The molecular weight of a pharmaceutical substance can be measured or calculated using standard techniques known in the art, such as mass spectrometry, viscosity- and light-scattering-based methods, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0070] In some embodiments, the half-life of the agent in a biological system is at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least 24 hours, at least 26 hours, at least 28 hours, at least 30 hours, at least 32 hours, at least 36 hours, at least 38 hours, at least 40 hours, at least 42 hours, at least 44 hours, at least 46 hours, at least 48 hours, at least one week, at least two weeks, at least three weeks, at least four weeks, at least one month, or at least two months.

[0071] In some embodiments, the half-life in a biological system is measured between about 35°C and about 40°C, between about 36°C and about 39°C, and between about 36.5°C and about 37.5°C.

[0072] "half-life" ("t") 1 / 2 "Pharmacokinetics (PK)" is well known in the art and refers to the time required for the amount of an active pharmaceutical ingredient in the body to decrease by 50%. Those skilled in the art will be able to select an appropriate assay (e.g., enzyme-linked immunosorbent assay (ELISA)) to periodically measure the amount of the pharmaceutical ingredient in serum over time.

[0073] A longer half-life drug requires a longer time to be eliminated from the biological system, and therefore the amount of drug exposed to the TME increases. In some embodiments, the half-life of the drug in the biological system is at least 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or at least 15 times longer than the half-life of the therapeutic domain in the biological system when it is separated from the cleavable domain and / or the stable domain (i.e., not conjugated to them).

[0074] Treatment domain

[0075] In some embodiments, the therapeutic domain is a domain in a biological system having a half-life of less than 10 hours, less than 9.5 hours, less than 9 hours, less than 8.5 hours, less than 8 hours, less than 7.5 hours, less than 7 hours, less than 6.5 hours, less than 6 hours, less than 5.5 hours, less than 5 hours, less than 4.5 hours, less than 4 hours, less than 3.5 hours, less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1.5 hours, less than 1 hour, less than 60 minutes, less than 40 minutes, less than 20 minutes, less than 10 minutes, less than 5 minutes, or less than 2 minutes when it exists separately from the cleavable domain and the stable domain.

[0076] The term "penetrating solid tumor" as we use it includes the following meanings: the drug or therapeutic domain is taken up by the solid tumor, for example, through diffusion, intracellular transport (e.g., transcytosis), or paracellular transport. Preferably, the drug or therapeutic domain is taken up by the solid tumor through diffusion. Those skilled in the art will be able to select appropriate methods to measure the ability of a drug to penetrate a solid tumor, for example, by immunostaining or immunofluorescence analysis of the drug in in vivo tumors, organoids or tumor bodies, xenograft mouse models, or in vitro tumor models.

[0077] Our use of the term "solid tumor" encompasses atypical aggregates of various cell types, including, for example, cancer cells, cancer stem cells, connective tissue cells, and immune cells. In this embodiment, the solid tumor is malignant. In some embodiments, a solid tumor may be carcinoma, sarcoma, lymphoma, or melanoma.

[0078] In some embodiments, when the therapeutic domain exists separately from the cleavable and / or stable domains (i.e., not coupled to them), its ability to penetrate solid tumors is increased relative to the ability of the agent.

[0079] In some embodiments, the increase is at least 1.5 times, at least 2 times, at least 2.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 35 times.

[0080] "Tumor uptake level" refers to the amount of the applied therapeutic agent actually taken up by the tumor. In some embodiments, the tumor uptake level when the therapeutic domain is cleaved from the agent is at least 8% ID / g, preferably at least 9% ID / g, and more preferably 10% ID / g. Our use of "% ID / g" includes the meaning of the average concentration of total antibodies (bound + free) in the tumor (Schmidt and Wittrup, 2009). Those skilled in the art will be able to select appropriate methods to measure the tumor uptake level, such as by immunostaining, immunofluorescence, or radiolabeling of the agent in in vivo tumors, xenograft mouse models, or in vitro tumor models.

[0081] In some embodiments, the increased permeability and / or tumor uptake levels are sustained. In another embodiment, the increased permeability and / or tumor uptake levels are temporary. Our use of "sustainable" includes the following meanings: the increased agent level is maintained and / or continues to permeate the solid tumor over time. Our use of "maintained over time" includes the following meanings: the increased level is maintained for at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 24 hours, at least 48 hours, or at least 36 hours. The term “temporary” as we use it includes the following meanings: the increased drug level fails to be maintained and / or reduced over time (e.g., after 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, 0.5 minutes or less).

[0082] In some embodiments, after the therapeutic domain is cleaved from the agent, the tumor uptake level of the therapeutic domain is increased relative to the tumor uptake level of the agent. In some embodiments, this increase is at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 35 times.

[0083] Preferably, the therapeutic domain is selected from any one or more of the following: i. an antigen-binding domain; ii. a Fab region; iii. an F(ab')2 region; iv. a scFv region; v. a tandem scFv region; vi. a domain antibody, preferably a single-domain antibody (sdAb); vii. a nanobody; viiii. a monoclonal antibody; ix. a polyclonal antibody; x. a double-chain antibody; xi. a triple-chain antibody; xii. a tetra-chain antibody; xiii. a penta-chain antibody; xiv. a hexa-chain antibody; xv. an antibody-drug conjugate; xvi. a bispecific peptide, such as a bispecific antibody; xvii. a multispecific peptide, such as a multispecific antibody; xx. a bicyclic peptide; xxi. a tricyclic peptide. Most preferably, the therapeutic domain is any one of the following: i. an antigen-binding domain; ii. a Fab region; iii. an F(ab')2 region; iv. a scFv region; v. a tandem scFv region. In one embodiment, the therapeutic domain is not a single-domain antibody (sdAb).

[0084] Preferably, the therapeutic domain is or is derived from the Fab region, F(ab')2 region, or scFv region.

[0085] In one embodiment, the therapeutic domain includes no more than one binding domain. In a further embodiment, the therapeutic domain does indeed act as a cross-linking agent between the tumor and the T cell.

[0086] In some embodiments, the treatment domain further includes a hinge region.

[0087] Our use of "antigen-binding domain" encompasses domains that can bind to antigens. These domains include both antibody and non-antibody portions. Our use of "antibody portion" includes antibody fragments (such as Fab, Fv, ScFv, dAb, and nanobodies). Our use of "non-antibody portion" includes non-antibody scaffold types such as affibody, affilin, anticalcin, atrimer, DARPin, FN3 scaffolds (such as adnectin and centyrin), fynomer, kunitz domain, pronectin, obody, inhibitory cystine knots (also known as cys knots or knottin), thyrodoxin repeat sequences, fibronectin domains, and lipid transport proteins.

[0088] The fragment antigen-binding (Fab) region is the area in an antibody that binds to the antigen. The Fab region consists of a constant domain and a variable domain from each of the heavy and light chains. Figure 1 The Fab region contains monovalent epitope binding sites.

[0089] The bivalent antibody (F(ab')2) region is a Fab region with additional amino acids linked together. Figure 2 The F(ab')2 region may include the entire hinge region that connects the two heavy chains together.

[0090] The single-chain variable (scFv) region is an antibody heavy chain (V) H ) and light chains (V L ) A fusion protein with a variable region. The two chains are linked by short linker peptides. The scFv region can be V-shaped. L -V H or V H -V L Orientation. The scFv region can be monovalent (scFv) or polyvalent, such as bivalent (e.g., tandem scFv, di-scFv or bi-scFv, double-chain antibody), trivalent (e.g., triple-chain antibody, tri-antibody or tri-scFv), or tetravalent (e.g., tetra-chain antibody), pentavalent (e.g., penta-chain antibody), or hexavalent (e.g., hexa-chain antibody).

[0091] Antibody-drug conjugates (ADCs) are molecules containing an antibody linked to a biologically active cytotoxic drug. When used to prevent or treat conditions characterized by the presence of unwanted cells (such as cancer), the antibody is specific to an antigen expressed on the unwanted cells and delivers the cytotoxic drug to the desired location in the body. Examples of ADCs include, but are not limited to, trastuzumab emtansine (also known as Kadcyla – manufactured by Genentech or Roche), ventromuzumab venumab (also known as Padcev – manufactured by Astellas or Seattle Genetics), trastuzumab destrautuzumab (also known as Enhertu – manufactured by AstraZeneca / Daiichi Sankyo), trodelvy (also known as Trodelvy – manufactured by Immunomedics), babetoumab (also known as Blenrep – manufactured by GlaxoSmithKline), and vetisol ventromuzumab (also known as Tivdak – manufactured by Seagen Inc.).

[0092] Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments composed of a single monomeric variable antibody domain. They can be derived from V... H Structural domains (e.g., V from camels and other camelids) H H (or VHH) fragments, V from cartilaginous fish NAR (fragment), V L Antibodies with structural domains or human structural domains.

[0093] Monoclonal antibodies (mAbs) are antibodies produced from cell lineages created by cloning unique cells, or by humanized mice, or by recombinant in vitro techniques (such as phage display, yeast display, or mammalian display) or based on in vitro display (such as ribosome display, mRNA display, cis display). Monoclonal antibodies can have monovalent affinity, binding only to the same epitope (monospecific). In contrast, polyclonal antibodies bind to multiple epitopes (multispecific) and are typically made from several different antibody-secreting plasma cell lineages. In some embodiments, monoclonal antibodies can be engineered to be multispecific antibodies to increase the number of epitopes they bind to.

[0094] The therapeutic domain can be single-specific or multi-specific, such as bispecific or trispecific. By "multi-specific," we mean that the peptide (such as an antibody or antibody fragment) is specific to two or more antigens (e.g., bispecific tandem di-scFv). In one embodiment, the therapeutic domain is not multi-specific.

[0095] Bicyclic and tricyclic peptides are synthetic short peptides that are constrained to form two or three rings using chemical linking compounds called scaffolds, thereby stabilizing their geometry. Examples of bicyclic peptides include, but are not limited to, BT8009 (anti-Nectin4), BT1718 (anti-MT1-MMP), and BT5528 (anti-EphA2).

[0096] In some embodiments, the therapeutic domain has a molecular weight of less than or equal to 1 kDa, less than or equal to 2 kDa, less than or equal to 3 kDa, less than or equal to 4 kDa, less than or equal to 5 kDa, less than or equal to 6 kDa, less than or equal to 7 kDa, less than or equal to 8 kDa, less than or equal to 9 kDa, less than or equal to 10 kDa, less than or equal to 15 kDa, less than or equal to 20 kDa, less than or equal to 25 kDa, less than or equal to 27 kDa, less than or equal to 30 kDa, less than or equal to 35 kDa, less than or equal to 40 kDa, less than or equal to 45 kDa, less than or equal to 50 kDa, less than or equal to 55 kDa, less than or equal to 60 kDa, less than or equal to 65 kDa, less than or equal to 70 kDa, less than or equal to 75 kDa, less than or equal to 80 kDa, less than or equal to 85 kDa, less than or equal to 90 kDa, less than or equal to 95 kDa, or less than or equal to 100 kDa.

[0097] In one embodiment, the cleaved therapeutic domain has a molecular weight of no more than 60 kDa, no more than 55 kDa, no more than 50 kDa, no more than 40 kDa, no more than 30 kDa, no more than 25 kDa, no more than 20 kDa, and no more than 15 kDa.

[0098] Preferably, the pharmaceutical agent is selected from the group consisting of: i. monoclonal antibodies; ii. polyclonal antibodies; iii. double-chain antibodies; iv. triple-chain antibodies; v. tetra-chain antibodies; vi. penta-chain antibodies; vii. hexa-chain antibodies; viiii. antibody-drug conjugates; ix. bispecific peptides, such as bispecific antibodies; and / or x. multispecific peptides, such as multispecific antibodies.

[0099] Particularly preferred agents and / or therapeutic domains include the following antibodies: antibodies derived from anti-epidermal growth factor receptor (EGFR) antibodies, such as cetuximab-derived fragments, panitumumab-derived fragments, and zalumumab-derived fragments; antibodies derived from anti-Her2 antibodies, such as trastuzumab-derived fragments and pertuzumab-derived fragments; antibodies derived from anti-CD20 antibodies, such as rituximab-derived fragments; antibodies derived from anti-CD22 antibodies, such as intuzumab-derived fragments; antibodies derived from anti-CD70 antibodies; antibodies derived from anti-CD33 antibodies, such as hp67.6-derived fragments and gemtuzumab-derived fragments; and antibodies derived from anti-MUC1 antibodies, such as GP1.4-derived fragments and SM3-derived fragments. Derived from anti-CD40 antibodies, derived from anti-CD74 antibodies, derived from anti-P-cadherin antibodies, derived from anti-EpCAM antibodies; derived from anti-CD138 antibodies; derived from anti-E-cadherin antibodies; derived from anti-CEA antibodies, such as M5A; derived from anti-FGFR3 antibodies; derived from anti-PSMA antibodies, such as J591; derived from anti-CTLA4 antibodies, such as ipilimumab-derived fragments and tesimumab-derived fragments; derived from anti-PD1 antibodies, such as pembrolizumab-derived fragments and nivolumab-derived fragments; and derived from anti-PDL1 antibodies, such as atezolizumab-derived fragments; and derived from anti-VEGF (vascular endothelial growth factor) antibodies, such as bevacizumab.

[0100] Our terms "antibody derived from" and "antibody-derived fragment" include the following meanings: an antibody or antibody fragment contains a portion or modified portion of the parent antibody from which it is derived. For example, an antibody-derived fragment may have the same or similar sequence as portions of the parent antibody. In some embodiments, the antibody-derived fragment has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% sequence identity with portions of the antibody sequence.

[0101] In some embodiments, the uncut drug provided herein comprises a trastuzumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of:

[0102] The cuttable structural domains and cut points of the following engineering designs D1-D9 and D12-D19 are indicated by bold and underline, respectively.

[0103] D1: Engineered trastuzumab antibody design 1 (SEQ ID NO:1)

[0104]

[0105] D2: Engineered trastuzumab antibody design 2 (SEQ ID NO:2)

[0106]

[0107]

[0108] D3: Engineered trastuzumab antibody design 3 (SEQ ID NO:3)

[0109]

[0110] D4: Engineered trastuzumab antibody design 4 (SEQ ID NO:4)

[0111]

[0112] D5: Engineered trastuzumab antibody design 5 (SEQ ID NO:5)

[0113]

[0114] D6: Engineered trastuzumab antibody design 6 (SEQ ID NO:6)

[0115]

[0116]

[0117] D7: Engineered trastuzumab antibody design 7 (SEQ ID NO:7)

[0118]

[0119] D9: Engineered trastuzumab antibody design 9 [ADAM10] (SEQ ID NO:8)

[0120]

[0121] D17: Engineered anti-EGFR antibody design 17[VHH 7D12-FC](SEQ ID NO:9)

[0122]

[0123] D18: Engineered trastuzumab antibody design 18 [anti-Her2 ScFv-FC] (SEQ ID NO:10)

[0124]

[0125]

[0126] In some embodiments, the uncut drug provided herein comprises a cetuximab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following sequence:

[0127] D12: Engineered cetuximab antibody design 12 (SEQ ID NO:11):

[0128]

[0129] In some embodiments, the uncut drug provided herein comprises a bevacizumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following sequences:

[0130] D13: Engineered bevacizumab antibody design 13 (SEQ ID NO:12):

[0131]

[0132] In some embodiments, the uncuttered drug provided herein comprises a pembrolizumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of:

[0133] D14a: Engineered pembrolizumab antibody design 14a (SEQ ID NO:13):

[0134]

[0135] D14b: Engineered pembrolizumab antibody design 14b (SEQ ID NO:14):

[0136]

[0137] In some embodiments, the uncuttered pharmaceutical agent provided herein comprises an M5A-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following sequences:

[0138] D15: Engineered M5A antibody design 15 (SEQ ID NO:15):

[0139]

[0140] In some embodiments, the uncut drug provided herein comprises an ipilimumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following sequences:

[0141] D16: Engineered Ipilimumab antibody design 16 (SEQ ID NO:16):

[0142]

[0143] In some embodiments, the therapeutic domain is monovalent, bivalent, trivalent, or multivalent (e.g., tetravalent). A monovalent therapeutic domain may have an affinity for one epitope, antigen, or microbial strain; while a multivalent (e.g., bivalent, trivalent, or tetravalent) therapeutic domain may have an affinity for multiple epitopes, antigens, or microbial strains. In one embodiment, the therapeutic domain is not multivalent.

[0144] In one embodiment, the therapeutic domain targets unwanted cells or directs an agent to unwanted cells. Our use of "therapeutic domain targeting an agent" includes the following meanings: the therapeutic domain may be a specific binding partner of an entity expressed by or associated with unwanted cells.

[0145] In one embodiment, the therapeutic domain is a specific binding partner of an entity expressed or associated with a target cell or tissue. Typically, the expressed entity is selectively expressed on unwanted cells. For example, the abundance of an entity expressed on unwanted cells is typically 10, 100, 500, 1,000, 5,000, or 10,000 higher than the abundance of an entity expressed on other cells in the body to be treated. However, as described below, the cleavage site provides additional specificity for the release location of the therapeutic domain, thus allowing the binding partner to bind to entities expressed similarly or insufficiently on unwanted cells relative to other cells in the body.

[0146] However, most preferably, the therapeutic domain is a specific binding partner of an entity expressed by or associated with an unwanted cell (rather than any other cell).

[0147] The term "binding mate" as we use it encompasses molecules that bind to a target entity expressed by a specific cell. Preferably, the binding mate selectively binds to that entity. An antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to that target with greater affinity, stronger binding force, and / or longer binding time compared to other unrelated targets or molecules.

[0148] It should be understood that the specificity of an antibody to its target can be determined and / or defined based on affinity measurements. Affinity (K) D The equilibrium constant for the dissociation between antigen and antibody, K, is a measure of the binding strength between the epitope and the antigen-binding site on the antibody: K D The smaller the value, the stronger the binding strength between antigen-binding molecules (alternatively, affinity can also be expressed as the affinity constant (K)). A ), that is, 1 / K D It will be apparent to those skilled in the art that affinity can be determined by any method known in the art and described herein. 1×10 -6 M any K D The value is generally considered to indicate nonspecific binding.

[0149] For example, preferably, the conjugate has a K value relative to the target. D Value, the K D The affinity is at least five or ten times lower than that of at least one other entity expressed by another cell type (e.g., normal cell type) (i.e., higher affinity), and preferably more than 100 or 500 times lower. More preferably, the binding partner of the entity has a K value more than 1000 or 5000 times lower than that of at least one other entity expressed by another cell type (e.g., normal cell type). D The binding specificity of the binding molecule can be determined experimentally using methods known in the art. Such methods include, but are not limited to, biophysical biolayer interferometry (BLI), isothermal titration calorimetry (ITC), Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradioassay (IRMA), enzyme immunoassay (EIA), and surface plasmon resonance (SPR).

[0150] Typically, a binding partner is a binding partner that binds to an entity present (or in contact with) the unwanted cell at a significantly higher concentration than in any normal cells of the host. For example, a binding partner can bind to a tumor-associated antigen expressed on the cell membrane. In another embodiment, a binding partner can bind to an entity expressed similarly or insufficiently relative to other cells in the body on unwanted cells. In one embodiment, the entity is naturally occurring or endogenously expressed.

[0151] Preferably, the target entity is an unwanted cell surface antigen, i.e., a cell surface marker. The diameter of a solid tumor can range from small (e.g., 1 cm) to large (e.g., greater than 10 cm). Solid tumors are 3D structures with peripheral and internal portions. The target entity may be located at the periphery (e.g., the surface) and / or the interior (e.g., the center) of the tumor. The center of the lesion is considered to be the internal portion inside the tumor and may be at least 0.5 cm, at least 1 cm, at least 2 cm, at least 3 cm, at least 4 cm, at least 5 cm, at least 6 cm, or at least 7 cm from the tumor surface (Sopik and Narod, 2018. The relationship between tumor size, nodal status and distant metastases: on the origins of breast cancer. Breast Cancer Res Treat. 2018; 170(3):647–656).

[0152] "Unwanted cells" include cells that the host or patient does not want to be present, such as tumor cells or other pathogenic cells.

[0153] In some embodiments, the therapeutic domain can be any compound or portion that specifically binds to (in a non-immune sense) an entity expressed by the unwanted cell or otherwise is associated with the unwanted cell. Thus, the therapeutic domain can be any of the following: i. a T cell receptor (TCR) domain; or ii. a receptor domain; or iii. a receptor-mimicking domain; or iv. a cytokine; or v. a hormone; or vi. a growth factor; or vii. a peptide; or viiii. a peptide derivative.

[0154] In one embodiment, the therapeutic domain may bind to an intracellular target. In another embodiment, the therapeutic domain may bind to an extracellular target. In some embodiments, the therapeutic domain may bind to both intracellular and extracellular targets.

[0155] In another embodiment, the therapeutic domain does not target the agent to unwanted cells.

[0156] Particularly useful therapeutic domains may include peptides or peptide derivatives, such as MYC (also known as c-Myc) inhibitors (e.g., H1 peptide, OmoMYC) and HOX (homeobox) inhibitors (e.g., HRX9, HTL-001 (HOX Therapeutics Ltd.)).

[0157] Particularly useful therapeutic domains may include cytokines such as IGF (insulin-like growth factor), EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), IL (interleukin)-2, IL-6, IL-4, or HGF (hepatocyte growth factor, scattering factor, SF, hepatocyte-poietin A).

[0158] Insulin-like growth factors (IGF-1 and IGF-11) are preferentially taken up by malignant cells and can therefore be used to target tumor cells. Similarly, EGF can be used to target malignant cells that upregulate EGF receptors. Furthermore, tumor-associated angiogenesis overexpresses VEGF receptors and can therefore be targeted by the VEGF growth factor family. Myeloma cells express IL-6 receptors and secrete IL-6, which stimulates cell proliferation in an autocrine manner. Therefore, IL-6 can be used as a therapeutic domain for myeloma. In another example, the therapeutic domain is melanoma-promoting hormone (MSH), which binds to the MSH receptor, which is highly expressed in melanoma cells.

[0159] In an embodiment, the therapeutic domain binds to an antigen expressed by unwanted cells, selected from the list including: CEA (anti-carcinoembryonic antigen); Her2 / Neu; CD22 (sialic acid-binding immunoglobulin-like lectin 2, SIGLEC2, SIGLEC-2, B lymphocyte adhesion molecule, BL-CAM, Leu-14); EPCAM (epithelial cell adhesion molecule, tumor-associated calcium signaling protein 1, TACSTD1, gastrointestinal tumor-associated protein 2, GA733-2, epithelial glycoprotein 2, EGP-2, epithelial cell adhesion molecule, Ep-CAM, KSA). KS1 / 4 antigen, M4S, tumor antigen 17-1A, EpCAM, CD326); EGFR (epidermal growth factor receptor, receptor tyrosine protein kinase erbB-1, ERBB1, HER1, HER-1, ERBB); PMSA; CTLA-4 (cytotoxic T lymphocyte-associated antigen 4, CTLA4, CD152); CD30; CD20; CD33 (sialic acid-binding immunoglobulin-like lectin 3, SIGLEC3, SIGLEC-3, gpG7, p67); CD80 (B7-1, CD28LG1); CD86 (B 7-2, CD28LG2); CD2; CA125; carbonic anhydrase IX; CD70 (tumor necrosis factor superfamily member 7, TNFSF7, CD27LG, CD27L); CD74 (major histocompatibility class II constant chain, MH2); CD56; CD40 (tumor necrosis factor receptor superfamily member 5, TNFRSF5, p50); CD19; c-met / HGFR; TRAIL-R1; DR5; PD-1; PD1L; IGF-1R; VEGF; VEGF-R2; prostate stem cell antigen (PSCA); MUC1 salivary gland Liquid acidified carbohydrates, tumor-associated antigens (CA242, cancer antigen 242); CanAg; mesothelin; P-cadherin; myostatin (GDF8); Cripto (TDGF1); ACVRL1 / ALK1; MUC5AC; CEACAM (carcinoembryonic antigen-associated cell adhesion molecule); CD137; CXCR4; neurociliacin; phosphatidylinositol proteoglycan (Glypican); HER3 / EGFR; PDGFRa (platelet-derived growth factor receptor α subunit, PDGFR2, CD140a); EphA2; CD138. Preferably, the therapeutic domain binds to an antigen selected from the list including: Her2, VEGF, PD-1, PMSA, CEA, CTLA-4, or EGFR.

[0160] In some embodiments, the therapeutic domain provided herein comprises a trastuzumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of:

[0161] Engineered trastuzumab with therapeutic domains 1-5 and 8-10 (SEQ ID NO:17)

[0162] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0163] Engineered trastuzumab with therapeutic domains 6 and 7 (SEQ ID NO:18)

[0164] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0165] Engineered trastuzumab design for the therapeutic domain 17 (SEQ ID NO:19)

[0166] QVQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSS

[0167] Engineered trastuzumab design with therapeutic domain 18 (SEQ ID NO:20)

[0168] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

[0169] In some embodiments, the therapeutic domain provided herein comprises a cetuximab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following sequences:

[0170] Engineered cetuximab design 12 therapeutic domain (SEQ ID NO:21)

[0171] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQ GTLVTVSAASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0172] In some embodiments, the therapeutic domain provided herein comprises a bevacizumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following sequences:

[0173] Engineered bevacizumab design 13 therapeutic domain (SEQ ID NO:22)

[0174] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0175] In some embodiments, the therapeutic domain provided herein comprises a pembrolizumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following sequences:

[0176] Engineered pembrolizumab with therapeutic domains 14a and 14b (SEQ ID NO:23)

[0177] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY

[0178] In some embodiments, the therapeutic domain provided herein comprises an M5A-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following sequences:

[0179] Therapeutic domain of engineered M5A design 15 (SEQ ID NO:24)

[0180] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWG QGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0181] In some embodiments, the therapeutic domain provided herein comprises an ipilimumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following sequences:

[0182] Engineered ipilimumab design 16 therapeutic domain (SEQ ID NO:25)

[0183] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

[0184] Suitablely, the cleavable domain does not interfere with the binding of the agent to its binding partner. It should be understood that the location of the cleavable domain within the agent ensures that the ability of the agent to bind to its binding partner is unaffected by the cleavable domain, whether in a cleaved state (therapeutic domain) or an uncleaved state (the agent). Those skilled in the art will be able to select appropriate methods to measure the ability of the agent to bind to its binding partner, such as by ELISA or BIAcore.

[0185] In another embodiment, the therapeutic domain binds to its binding partner before the cleavable domain has been cleaved. In other words, the therapeutic domain binds to its binding partner without being released from the agent. In one embodiment, the therapeutic domain is not masked. "Masked" means that the therapeutic domain is blocked from binding to its binding partner. After the cleavable domain is cleaved, the masked therapeutic domain becomes unmasked. In another embodiment, the agent does not contain a masking domain.

[0186] In a further embodiment, once the cleavable domain is cleaved, the therapeutic domain retains its ability to bind to its binding partner. It should be understood that the release of the stabilizing domain from the therapeutic domain does not negatively reduce the binding of the therapeutic domain to its target.

[0187] In one embodiment, the therapeutic domain is at a half-maximal inhibitory concentration (IC50) of 0.2 to 1.4 nanomolars (nM) relative to its target. 50 Combined with its target, such as approximately 0.2, 0.205, 0.210, 0.215, 0.220, 0.225, 0.230, 0.235, 0.240, 0.245, 0.250, 0.255, 0.260, 0.265, 0.270, 0.275, 0.276, 0.277, 0.278, 0.279, or 0.280n M is approximately 0.260, 0.265, 0.270, 0.275, 0.280, 0.285, 0.290, 0.295, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.325, 1.350, 1.375, 1.380, 1.385, 1.39, 1.395, or 1.4. IC 50 IC indicates the efficacy of a therapeutic domain in inhibiting a specific biological or biochemical function. It is a quantitative measure, indicating how much of a specific inhibitory substance is needed to inhibit 50% of a given biological process or component. Any suitable means can be used to measure IC. 50 For example, through functional assays or competitive binding assays (such as enzyme-linked immunosorbent assay (ELISA)).

[0188] In one embodiment, the therapeutic domain has a molecular weight of 1.5 to 2.25 nM, such as about 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1 .70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89 or 1.90 to approximately 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00 The dissociation constants (K) of 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, or 2.25. D K D The dissociation constant indicates the tendency of a binding domain to dissociate from its target (i.e., the binding domain of a therapeutic domain from its binding partner (e.g., a tumor antigen)). A smaller dissociation constant indicates a tighter binding of the binding domain, or a higher affinity between the binding domain and its target. K can be measured using any suitable method. D For example, through functional testing, competitive combination testing, or live or real-time combination (such as BIAcore).

[0189] In one embodiment, the cleavable domain does not interfere with the thermal stability of the agent. It should be understood that the location of the cleavable domain within the agent ensures that the thermal stability of the agent is not significantly affected by the cleavable domain, whether in a cleaved state (therapeutic domain) or an uncleaved state (agent). Those skilled in the art will be able to select appropriate methods to measure thermal stability, such as fluorescence, static light scattering (SLS), and dynamic light scattering (DLS).

[0190] In one embodiment, the agent has a temperature of 65 degrees Celsius (°C) to 75 degrees Celsius, such as 65.1, 65.2, 65.3, 65.4, 65.5, 65.6, 65.7, 65.8, 65.9, 66.0, 66.1, 66.2, 66.3, 66.4, 66.5, 66.6, 66.7, 66.8, 66.9, 67.0, 67.1, 67.2, 67.3, 67.4, 67.5, 67.6, and 67. 7, 67.8, 67.9, 68.0, 68.1, 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 69.0, 69.1, 69.2, 69.3, 69.4, 69.5, 69.6, 69.7, 69.8, 69.9 or 70.0℃ to approximately 68.0, 68.1, 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 6 8.8, 68.9, 69.0, 69.1, 69.2, 69.3, 69.4, 69.5, 69.6, 69.7, 69.8, 69.9, 70.0, 70.1, 70.2, 70.3, 70.4, 70.5, 70.6, 70.7, 70.8, 70.9, 71.0, 71.1, 71.2, 71.3, 71.4, 71.5, 71.6, 71.7, 71.8, 71.9, 7 Melting temperatures (T) of 2.0, 72.1, 72.2, 72.3, 72.4, 72.5, 72.6, 72.7, 72.8, 72.9, 73.0, 73.1, 73.2, 73.3, 73.4, 73.5, 73.6, 73.7, 73.8, 73.9, 74.0, 74.1, 74.2, 74.3, 74.4, 74.5, 74.6, 74.7, 74.8, 74.9, and 75.0℃. m T protein m This is related to the results of protein denaturation. Methods for measuring melting temperature are well known in the art and include differential scanning calorimetry (DSC), differential scanning fluorescence (DSF), and other well-known thermal displacement determinations.

[0191] In one embodiment, the agent has a temperature of 65°C to 80°C, such as 65.1, 65.2, 65.3, 65.4, 65.5, 65.6, 65.7, 65.8, 65.9, 66.0, 66.1, 66.2, 66.3, 66.4, 66.5, 66.6, 66.7, 66.8, 66.9, 67.0, 67.1, 67.2. 67.3, 67.4, 67.5, 67.6, 67.7, 67.8, 67.9, 68.0, 68.1, 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 69.0, 69.1, 69.2, 69.3, 69.4, 69.5, 69.6, 69.7, 69.8, 6 9.9, 70.0, 70.1, 70.2, 70.3, 70.4, 70.5, 70.6, 70.7, 70.8, 70.9, 71.0, 71.1, 71.2, 71.3, 71.4, 71.5, 72.0, 72.5, 73.0, 73.5, 74.0, 74.5 or 75.0, 75.5, 76.0, 76 The bulking temperature (T) is approximately 73.0, 73.5, 74.0, 74.5, 75.0, 75.5, 76.0, 76.5, 77.0, 77.5, 78.0, 78.5, 79.0, 79.1, 79.2, 79.3, 79.4, 79.5, 79.6, 79.7, 79.8, 79.9, or 80.0 °C. agg ). What we mean by "T" agg "This includes the temperature at which aggregation begins or the temperature at which molecules tend to aggregate together. Methods for measuring thermal stability are well known in the art and include dynamic light scattering (DLS), static light scattering (SLS), and / or fluorescence."

[0192] DLS measures the hydrodynamic size and size distribution of particles in solution and can be plotted over time and temperature. Typically, proteins are stable at low temperatures and show reproducible size (and scattering intensity) measurements, while at higher temperatures (T0...), the results are more consistent. agg Under these conditions, protein molecules will exhibit a tendency to aggregate.

[0193] In one embodiment, the cleavable domain does not interfere with the binding of the stable domain to the Fcγ receptor (FcR), optionally wherein the Fcγ receptor is Fcγ receptor Ia and / or Fcγ receptor IIa. FcRs are membrane proteins expressed by several hematopoietic cells that recognize Fc regions of several immunoglobulin classes and subclasses. The Fc region of an antibody can bind to Fc receptors (FcγRI, FcγRII, FcγRIII) expressed on the surface of immune cells, complement (C1q), and FcRn (neonatal FcR) in the blood, thereby activating the immune system. Interactions mediated by the antibody Fc domain can significantly influence the functional outcomes of antibody therapy through interactions between the Fc domain and Fc receptors on different cell types, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Notably, the combination of IgG and neonatal FcR (FcRn) can protect the antibody from degradation, thereby prolonging its half-life.

[0194] In another embodiment, the cleavable domain may interfere with the binding of the stable domain to the Fcγ receptor.

[0195] It should be understood that the localization of the cleavable domain in the drug ensures that the binding of the Fcγ receptor is not significantly affected by the cleavable domain, whether in the cleaved state (stable domain) or the uncleaved state (drug). Those skilled in the art will be able to select appropriate methods to measure binding, such as by functional assays or competitive binding assays (e.g., enzyme-linked immunosorbent assay (ELISA)).

[0196] In one embodiment, the therapeutic domain reduces or inhibits the proliferation of unwanted cells or the growth of a tumor.

[0197] In one embodiment, the therapeutic domain stimulates immune cells, such as immune checkpoint inhibitors (ICIs), immunomodulators, and cytokines.

[0198] It should be understood that immune cells can include any of the following: lymphocytes (B cells and T cells), antigen-presenting cells (APCs), natural killer (NK) cells, macrophages, monocytes, and dendritic cells. T cells recognize peptide antigens derived from proteins degraded within the cell, and these peptide antigens are loaded onto MHC molecules on the cell surface—a process known as antigen presentation (APC).

[0199] The term "stimulation" as we use it includes the activation and proliferation of immune cells (such as T cells). In some embodiments, stimulation also includes co-stimulation to prevent the recruitment of inhibitory effector cells and to prevent T cell depletion.

[0200] In another embodiment, the therapeutic domain blocks immune cells. The terms "block" and "inhibit" are used interchangeably and encompass both partial and complete inhibition / blockage. In one embodiment, the blocked immune cells are regulatory T cells (Tregs). Tregs are a special subset of T cells that play a key role in preventing autoimmunity by inhibiting T cell proliferation and cytokine production.

[0201] As discussed herein, the primary function of a therapeutic domain is to exert a clinically or therapeutically beneficial effect on a target tissue. Optionally, a therapeutic domain may have a secondary function, wherein the therapeutic domain targets an agent to the target tissue. For example, the therapeutic domain according to the claimed invention is an antibody-drug conjugate (ADC) that comprises a Fab domain (which targets the ADC to unwanted cells) and a cytotoxic drug (also known as a payload) chemically linked to the Fab region. Thus, a therapeutic domain can serve to: (i) exert a clinically or therapeutically beneficial effect, and (ii) target itself to a target tissue.

[0202] Stable structural domain

[0203] In some embodiments, the stabilizing domain is a protein-based domain or polymer. For example, a protein-based domain may comprise a structured peptide (e.g., IgG Fc region, HSA), an elastin-like peptide (ELP-modified), an inert peptide such as XTEN (also known as recombinant PEG or "rPEG"), a homoamino acid polymer (HAP-modified), or a proline-alanine-serine polymer (PAS-modified). In another embodiment, the stabilizing domain is not a protein, but rather a chemical motif (e.g., polyethylene glycol-modified or hyaluronic acid).

[0204] In one embodiment, the stable domain targets the therapeutic domain to unwanted cells. Our use of "stable domain targeting the therapeutic domain" includes the following meanings: the stable domain may be a specific binding partner of an entity expressed by or associated with the unwanted cell. In another embodiment, the stable domain does not target the therapeutic domain to unwanted cells.

[0205] In some embodiments, the stable domain is: i. an Fc region, optionally wherein the Fc region is an IgG, IgE, IgM, IgD, or IgA family Fc region or a bispecific Fc region; or; or ii. a PEGylated domain; or iii. a PAS-modified domain; or iv. an XTEN-modified domain; or v. a HES-modified domain; or v. a lipid-modified domain; or vii. a glycosylated domain; or viii. human serum albumin (HSA) protein or a fragment thereof; or ix. an HSA-binding protein; or x. a protein that binds to circulating blood cells; or xi. a Fab domain, optionally wherein the Fab domain is targeted at HSA or at an antigen identical to the antigen-binding domain. Preferably, the stable domain is: i. an Fc region, optionally wherein the Fc region is an IgG, IgE, IgM, IgD, or IgA family Fc region or a bispecific Fc region. In one embodiment, the stable domain does not include a Fab domain.

[0206] The Fc region and / or hinge region can be selected from any type of immunoglobulin, including IgM, IgG, IgA, IgD, and IgE. Each isotype also includes different subtypes. For example, IgG subtypes include IgG1, IgG2, IgG3, and IgG4, and IgA subtypes include IgA1 and IgA2. It should be understood that different isotypes and subtypes exist in different species.

[0207] Other serum proteins that can serve as stabilizing domains may include albumin, fibrinogen, fibronectin, hemoglobin, transferrin, and immunoglobulin domains.

[0208] Suitable stable domains can display one or more N-glycosylation motifs. N-glycosylation is the process of attaching a glycan oligosaccharide to the amide nitrogen of an asparagine (N) residue in a protein. N-glycosylation motifs can be found in the YNSTY (SEQ ID NO:26) sequence of CH2 in the Fc region, for example, N297.

[0209] The Fc region contains constant regions (CH)CH2 and CH3 from the heavy chain. The antibody is generally Y-shaped, with a flexible hinge (interdomain) region at its center. The flexibility of this interdomain hinge region is crucial for bivalent binding of the antibody, allowing two binding pockets to interact with the antigen site at different distances. The Fc region contains constant regions (CH)CH2 and CH3 from the heavy chain. The Fc region can be composed of homoimmunoglobulin molecules (preferably homoIgG molecules). In one embodiment, the stable domain is not a heteroIgG molecule.

[0210] In some embodiments, the stable structural domain comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of:

[0211] Engineered trastuzumab design for stable domains at D1-6, D9, D12, D13, D15, and D18 (SEQ ID NO:27)

[0212] ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0213] Stable domain of engineered trastuzumab design 7 (SEQ ID NO:28)

[0214] FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0215] Engineered pembrolizumab design for stable domains of D14a and D14b (SEQ ID NO:29)

[0216] EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0217] As discussed herein, the primary function of a stabilizing domain is to stabilize or prolong the half-life of a drug in a biological system. Optionally, the stabilizing domain may have secondary functions, wherein the stabilizing domain has clinically or therapeutically beneficial effects. For example, the drug according to the claimed invention is an antibody-drug conjugate (ADC) that will comprise a therapeutic domain (which targets the ADC to unwanted cells), a cleavable domain, and a stabilizing domain. The stabilizing domain may comprise a cytotoxic drug (also known as a payload) chemically linked to the Fc region. Thus, stabilization can serve to: (i) stabilize or prolong the half-life of the drug, and (ii) exert clinically or therapeutically beneficial effects.

[0218] Cuttable structural domain

[0219] The cleavable domain is positioned between the therapeutic domain and the stable domain, connecting the two in the drug substance. Selective cleavage of the cleavable domain releases the therapeutic domain from the stable domain, allowing it to perform its therapeutic function. Typically, the stable domain is subsequently degraded or released and cleared by the GFB. It should be understood that when the drug substance is cleaved, some of the cleavable domain may remain attached to the therapeutic domain, and some may remain attached to the stable domain.

[0220] The cleavable domain contains at least one cleavage site. A "cleavage site" refers to a site in the amino acid sequence that serves as a substrate for an enzyme (such as an extracellular enzyme). Examples include cleavage sites for enzymes such as urokinase plasminogen activator (uPA), fibroblast activator protein (FAP), podocyte protein, or MT-SP1. A cleavage site can be a site that can be cleaved by an enzyme (such as any of a protease, nuclease, lipase, lyase, phosphatase, or carbohydrate enzyme), which may or may not be membrane-bound. Our use of "protease" includes the meaning of an enzyme that catalyzes proteolysis (i.e., breaks down proteins into smaller polypeptides or individual amino acids). In one embodiment, the cleavage site is not the amino acid sequence DEVD.

[0221] It should be understood that a cleavable domain may or may not include a hinge region or a portion thereof. For example, a cleavable domain may include a cleavage site and a connection sequence that connects a therapeutic domain (such as OcoMYC) to a stable domain, wherein the stable domain is not an Fc region or a portion thereof (such as HSA).

[0222] In some embodiments, the cleavage site is located in the hinge region. The term "hinge region" encompasses the hydrophilic sequence of the antibody heavy chain. Typically, the hinge region is responsible for flexibly connecting the Fab and Fc regions. The hinge region can be divided into three parts: the upper hinge, the middle hinge, and the lower hinge. The middle hinge region is where the two heavy chains meet, and this is the part that binds the antibodies together. In some embodiments, the cleavable domain is located in the upper hinge, the middle hinge, or the lower hinge, preferably in the upper hinge or the lower hinge. Preferably, the cleavable domain is located in the upper hinge. The advantage of a cleavable domain in the hinge region includes providing the ability to completely separate the therapeutic and stabilizing domains after cleavage.

[0223] In some embodiments, the cleavable domain comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of:

[0224] The engineered trastuzumab design 1 includes a cleavable domain (SEQ ID NO:30).

[0225]

[0226] The engineered trastuzumab design 2 with a cleavable domain (SEQ ID NO:31)

[0227]

[0228] The engineered trastuzumab design includes a cleavable domain (SEQ ID NO:32).

[0229]

[0230] The engineered trastuzumab design 4 includes a cleavable domain (SEQ ID NO:33).

[0231]

[0232] The engineered trastuzumab design includes a cleavable domain (SEQ ID NO:34).

[0233]

[0234] Engineered trastuzumab design incorporates cleavable domains 6 and 7 (SEQ ID NO:35).

[0235]

[0236] Engineered trastuzumab design with cleavable domains (SEQ ID NO:36)

[0237]

[0238] Engineered design of cuttable structural domains 12, 13, 15, and 16 (SEQ ID NO:37)

[0239]

[0240] Engineered pembrolizumab design with cleavable domains of 14a (SEQ ID NO:38)

[0241]

[0242] Engineered pembrolizumab design with a cleavable 14b domain (SEQ ID NO:39)

[0243]

[0244] Engineered trastuzumab design for the cleavable domain of 17 (SEQ ID NO:40)

[0245]

[0246] Engineered trastuzumab design with cleavable domain 18 (SEQ ID NO:41)

[0247]

[0248] In one embodiment, the protease acting on the cleavage site is expressed by and / or accumulates in the vicinity of the unwanted cells or tumor. Preferably, the protease is a tumor-specific protease. Thus, when the unwanted cells are tumor cells, the cleavage site can be selectively cleaved by the protease present in the vicinity of the tumor cells.

[0249] Because the cleavage site is only cut in the region near the unwanted cells, specificity is increased. For example, tumor cells secrete proteases that are essential for tumor invasion of local tissues and metastasis; therefore, by including tumor-specific protease cleavage sites in the drug, the drug's specificity to the tumor can be increased.

[0250] The term "nearby region" or "tumor microenvironment" as used herein refers to the area on and / or near the cell surface, such as the environment closely surrounding the cell, including blood vessels, immune cells, fibroblasts, signaling molecules and / or extracellular matrix (ECM), blood, lymph, and other bodily fluids. In one embodiment, the near region begins at the tumor site and extends to a distance of at least 10 micrometers (μm), at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, at least 100 μm, at least 120 μm, at least 140 μm, at least 160 μm, at least 180 μm, or at least 200 μm from the tumor.

[0251] In some embodiments, the concentration of the enzyme may be low in tissues that the present invention cannot cut or penetrate deeper, such as in non-disease tissues. In some embodiments, the enzyme may be inactive or have significantly low activity in healthy (e.g., non-disease) tissues or tissues not used for therapy.

[0252] It should also be understood that because the cleavage sites in the drug give specificity to the release location of the therapeutic domain, the binding of the therapeutic domain to non-disease cells may also be tolerable in the vicinity of the cleavage site where it is not cleaved.

[0253] Proteases may include cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartic or aspartic proteases (including cathepsin D and E or novel aspartic protease A), and serine proteases (including cathepsin A and G, thrombin, plasmin, uPA, tissue plasminogen activator (tPA), proteolytic enzymes, MT-SP1, and fibroblast activating protein (FAP)).

[0254] Proteases can be metalloproteinases (also known as metallopeptidases or metalloproteinases) (MMP1-28), including both membrane-bound (MMP14-17 and MMP24-25) and secretory (MMP1-13, MMP18-23, and MMP26-28) types. Proteases can belong to the families of deintegrin A and metalloproteinases (ADAM), as well as the families of deintegrin A or metalloproteinases containing platelet-reactive protein motifs (ADAMTS). Other examples include CD10 (CALLA), prostate-specific antigen (PSA), and coagulation factors. It should be understood that proteases may or may not be membrane-bound. Proteases can include any one of the above or a combination thereof.

[0255] To treat a specific type of tumor, technicians typically select a protease cleavage site that is selectively cleaved by a protease known to be highly expressed in that tumor type. For example, for treating breast cancer, it is preferred to use a protease cleavage site that can be cleaved by any of uPA, novel aspartic protease A, tPA, podocyte lysin, proteolytic enzyme, proteolytic enzyme 2, cathepsin K, cathepsin O, MMP1, MMP2, MMP3, MMP11, MMP12, MMP17, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM28, or ADAMTS15.

[0256] In some embodiments, the cuttable domain includes one or more connector domains existing between the therapeutic domain and the stabilizing domain.

[0257] Such linker domains can improve enzyme accessibility and are selected from the group consisting of (G) m S n ) x Or (GGNGT) x (SEQ ID NO:42) or (YGNGT) x The amino acid sequence represented by (SEQ ID NO:43), wherein m and n are each independently selected from a group of integers from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and x is independently selected from a group of integers from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some specific embodiments, the adapter has (G4S) x The amino acid sequence represented by (SEQ ID NO:44), where x is independently selected from a group of integers from 1 to 6 (e.g., x is 4 or 5), such as (G4S)2 (SEQ ID NO:45). The linker may also be one having the same amino acid sequence as described above, consisting of (G4S)2. m S n ) x Or (GGNGT) x (SEQ ID NO:42) or (YGNGT) xAnother linker with similar flexibility and length to the amino acid sequence represented by (SEQ ID NO:43); the linker may also be selected from the group consisting of: GPGGS (SEQ ID NO:46), GSGPP (SEQ ID NO:47), AKTTPKLEEGEFSEAR (SEQ ID NO:48), AKTTPKLEEGEFSEARV (SEQ ID NO:49), AKTTPKLGG (SEQ ID NO:50), SAKTTPKLGG (SEQ ID NO:51), AKTTPKLEEGEFSEARV (SEQ ID NO:52), SAKTTP (SEQ ID NO:53), SAKTTPKLGG (SEQ ID NO:54), RADAAP (SEQ ID NO:55), RADAAPTVS (SEQ ID NO:56), RADAAAAGGPGS (SEQ ID NO:57), RADAAAA(G4S)4 (SEQ ID NO:58), SAKTTP (SEQ ID NO:59), SAKTTPKLGG (SEQ ID NO:60), SAKTTPKLEEGEFSEARV (SEQ ID NO:49), AKTTPKLGG (SEQ ID NO:60), AKTTPKLEEGEFSEARV (SEQ ID NO:49), AKTTPKLGG (SEQ ID NO:50), AKTTPKLEEGEFSEARV (SEQ ID NO:41), AKTTPKLEEGEFSEARV (SEQ ID NO:42), AKTTTP (SEQ ID NO:53), SAKTTPKLGG (SEQ ID NO:54), RADAAP (SEQ ID NO:55), RADAAPTVS (SEQ ID NO:56), RADAAAAGGPGS (SEQ ID NO:57), RADAAAA(G4S)4 (SEQ ID NO:58), SAKTTP (SEQ ID NO:59), SAKTTPKLGG (SEQ ID NO:60), AKTTPKLEEGEFSEARV (SEQ ID NO:49), AKTTPKLGG (SEQ ID NO:50), AKTTPKLEEGEFSEARV (SEQ ID NO:49), AKTTPKLGG (SEQ IDNO:61), ADAAP (SEQ ID NO:62), ADAAPTVSIFPP (SEQ ID NO:63), TVAAP (SEQ ID NO:64), TVAAPSVFIFPP (SEQ ID NO:65), QPKAAP (SEQ ID NO:66), QPKAAPSVTLFPP (SEQ ID NO:67), AKTTPP (SEQ ID NO:68), AKTTPPSVTPLAP (SEQ ID NO:69), AKTTAP (SEQ ID NO:70), AKTTAPSVYPLAP (SEQ ID NO:71), ASTKGP (SEQ ID NO:72), ASTKGPSVFPLAP (SEQ ID NO:73), GENKVEYAPALMALS (SEQ ID NO:74), GPAKELTPLKEAKVS (SEQ ID NO:75) and GHEAAAVMQVQYPAS (SEQ ID NO:76).

[0258] In some embodiments, the cleavable domain overlaps with the therapeutic domain and / or the stabilizing domain.

[0259] In one embodiment, the cleavable domain may be an additional sequence (which adds another amino acid to the biologic). In a further embodiment, the cleavable domain is generated by one or more mutations (e.g., addition, substitution, or deletion) of an endogenous sequence (which does not add any additional amino acids to the biologic). In yet another embodiment, it may be a combination of sequence addition and mutagenesis.

[0260] In some embodiments, the severable domain overlaps with the therapeutic domain and / or stable domain by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or at least 100%.

[0261] Small cleavage sites (including any adapters, if present) are generally advantageous because of the lower risk of immunogenicity (to foreign / non-host sequences).

[0262] In some embodiments, the length of at least one cleavage site is 4 to 20 amino acids. For example, the length of at least one cleavage site is 4 to 6 amino acids, 4 to 8 amino acids, 4 to 10 amino acids, 4 to 12 amino acids, 4 to 14 amino acids, 4 to 16 amino acids, 4 to 18 amino acids, 6 to 8 amino acids, 6 to 10 amino acids, 6 to 12 amino acids, 6 to 14 amino acids, 6 to 16 amino acids, 6 to 18 amino acids, 6 to 20 amino acids, 8 to 10 amino acids, 8 to 12 amino acids, 8 to 14 amino acids, or 8 to 16 amino acids. Amino acids, 8 to 18 amino acids, 8 to 20 amino acids, 10 to 12 amino acids, 10 to 14 amino acids, 10 to 16 amino acids, 10 to 18 amino acids, 10 to 20 amino acids, 12 to 14 amino acids, 12 to 16 amino acids, 12 to 18 amino acids, 12 to 20 amino acids, 14 to 16 amino acids, 14 to 18 amino acids, 14 to 20 amino acids, 16 to 18 amino acids, 16 to 20 amino acids, 18 to 20 amino acids.

[0263] In a further embodiment, the agent is a protein or peptide, the stable or therapeutic domain comprising an Fc region and a hinge region, and the cleavable domain is located: i. after the N-terminus of the hinge region and the therapeutic domain; or ii. before the C-terminus of the hinge region and the CH2 domain of the Fc region.

[0264] In some embodiments, the uncut agent has the following structural arrangement from the N-terminus to the C-terminus: i. therapeutic domain-cuttable domain-hinge region-Fc region; or ii. therapeutic domain-hinge region-cuttable domain-Fc region.

[0265] The cleavable domain may or may not appear before or after the hinge region. This positional difference will result in two separate portions, which can generate monovalent or multivalent (such as bivalent) binding proteins. The engineered design will depend on the preferred mode of action of the therapeutic agent.

[0266] In some embodiments, the cleavable domain is not cleaved by tumor-specific proteases selected from the list of the following: gelatinase A (MMP-2), matrix lysin 1 (MMP-3), matrix lysin (MMP-7), gelatinase B (MMP-9), macrophage metalloelastase (MMP-12), collagenase-3 (MMP-13), and cathepsin G9. In another embodiment, the cleavable domain is not cleaved by the protease Capsase-3 or PreScission protease. The term "PreScission protease" includes the meaning of a fusion of GST and human rhinovirus protease.

[0267] In a preferred embodiment, the therapeutic domain is Fab, the cleavable domain contains uPA cleavage sites, and the stable domain is the Fc region.

[0268] In a preferred embodiment, the therapeutic domain is Fab, the cleavable domain includes an ADAM10 cleavage site, and the stable domain is the Fc region.

[0269] In another preferred embodiment, the therapeutic domain is VHH, the cleavable domain contains a uPA cleavage site, and the stable domain is the Fc region.

[0270] In a preferred embodiment, the therapeutic domain is ScFv, the cleavable domain contains a uPA cleavage site, and the stable domain is the Fc region.

[0271] Therapeutic uses

[0272] This agent is suitable for use as a medicine, such as in therapies, such as in the treatment or prevention of tumors (optionally cancer).

[0273] Our use of the term "treatment" includes both therapeutic and preventative treatment for patients. The term "preventative" is used to cover the use of the pharmaceutical agents or formulations described herein for the prevention or reduction of the likelihood of cancer or the spread, transmission, or metastasis of localized cancer in a patient or subject. The term "preventative" also covers the use of the pharmaceutical agents or formulations described herein for the prevention of cancer recurrence in patients who have previously received cancer treatment.

[0274] As used herein, the term "treatment" or "treating" refers to a method used to obtain a beneficial or desired outcome (including, and preferably, a beneficial or desired clinical outcome). Such beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: reducing the proliferation of cancer cells or other diseased cells (or destroying cancer cells or other diseased cells); reducing the metastasis of cancer cells found in cancer; shrinking tumor size; alleviating symptoms caused by the disease; improving the quality of life of the patient; reducing the dosage of other drugs required to treat the disease; delaying disease progression; and / or prolonging the individual's survival.

[0275] Once bound, the drug can exert its therapeutic effect directly (e.g., by inducing cell death through antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or by carrying a radioactive isotope or other cytotoxic components). Alternatively, the bound drug can serve as a diagnostic (imaging) tool and can guide therapy selection or assist in the surgical removal of unwanted cells.

[0276] In a particularly preferred embodiment, the cleavage site is selectively cleaved at an unwanted extracellular location (on or near its surface), thereby releasing the therapeutic domain without requiring internalization.

[0277] Suitablely, unwanted cells or the area near the tumor contain high titers and / or high activity of tumor-specific proteases.

[0278] If the titer and / or activity of a protease in a region near unwanted cells or tumors is significantly higher than the titer and / or activity of the protease at other locations considered to be far from the region near unwanted cells or tumors, then the titer and / or activity of the protease will be considered "high". Those skilled in the art will be able to select appropriate assays to measure the titer and activity of the protease, such as ELISA, activity-based proteomic analysis (ABPP), mass spectrometry, or fluorescent probe imaging.

[0279] Suitablely, the subject of treatment or prevention is a mammal, preferably a human. In some embodiments, the subject is a non-human mammal, such as a rodent, a non-human primate, a companion animal (e.g., a cat, a dog, a horse), a farm animal, a draft animal, or a zoo animal.

[0280] Appropriately, a tumor is a solid tumor. In some embodiments, cancer is selected from the group consisting of: adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendiceal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, cholangiocarcinoma, bladder cancer, bone cancer, intestinal cancer, brain cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, chondrosarcoma, colon cancer, colorectal cancer, craniopharyngioma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal... Carcinoid tumors, gastrointestinal stromal tumors, common tumors, germ cell tumors, glioblastoma multiforme, glioma, head and neck cancer, hemangioendothelioma, Hodgkin's disease, hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, colorectal cancer, intrahepatic cholangiocarcinoma, invasive / invasive breast cancer, pancreatic islet cell carcinoma, jawbone cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal, mesothelioma, metastatic breast cancer, metastatic melanoma Pigmented cervical carcinoma, metastatic squamous cell carcinoma, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, nasal cavity carcinoma, nasopharyngeal carcinoma, cervical cancer, neuroblastoma, neuroendocrine tumor, non-small cell lung cancer, oat cell carcinoma, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian sex cord-stromal tumor, Paget's disease, pancreatic cancer, papillary carcinoma, paranasal sinus carcinoma, parathyroid carcinoma, pelvic cancer, penile cancer, peripheral nerve carcinoma, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pineal gland tumor Cell tumors, pituitary cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, tubular carcinoma, ureteral cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0281] In a further aspect, a method for treating or preventing tumors (optionally for treating or preventing cancer) is provided, wherein the method includes one or more of the following steps: a) obtaining a sample of a tumor and / or a region adjacent to the tumor; b) identifying one or more proteases expressed by the tumor and / or present in the region adjacent to the tumor; c) administering an agent to a subject, wherein the cleavable domain can be cleaved by one or more proteases identified as being expressed by the tumor and / or present in the region adjacent to the tumor.

[0282] The term "sample" includes any biological sample from an individual used for testing in the methods and uses of the present invention. It should be understood that a sample may include one or more tissues, cells, and / or biological fluids (e.g., blood; serum; plasma; serum plasma; urine; saliva; intestinal cells; biopsy; feces) collected from (e.g., isolated from) an individual.

[0283] Our statement that “identifying one or more proteases expressed by and / or present in the vicinity of a tumor” includes determining whether a sample contains one or more proteases expressed by and / or present in the vicinity of a tumor. Preferably, this includes exposing the sample to an agent and determining which proteases are expressed and / or present.

[0284] In a further aspect, the present invention provides a method for treating or preventing cancer, wherein the method comprises administering one or more agents according to the present invention.

[0285] Preferably, one or more agents are administered by injection or infusion. However, in practice, they can be administered by any suitable means. Those skilled in the art will be able to select an appropriate route of administration.

[0286] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutical agent according to the invention and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0287] The pharmaceutical compositions according to the invention can be administered together with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. By means of "pharmaceuticalally acceptable," we include formulations that are sterile and pyrogen-free. Suitable pharmaceutically acceptable carriers, excipients, or diluents are well known in the pharmaceutical field. A pharmaceutically acceptable carrier, excipient, or diluent must be "acceptable," meaning compatible with the pharmaceutical agents of the invention and harmless to its recipient. Typically, a pharmaceutically acceptable carrier, excipient, or diluent will be sterile and pyrogen-free water or saline; however, other pharmaceutically acceptable carriers, excipients, or diluents may also be used.

[0288] In another aspect, the present invention provides a medicament according to the invention for the prevention or treatment of a condition characterized by the presence of unwanted cells, optionally wherein the condition is cancer (i.e., the unwanted cells are tumor cells).

[0289] The term "condition characterized by the presence of unwanted cells" as used here includes any biological or medical condition or disease in which at least part of the condition is mediated by the presence of unwanted cells. The condition may be caused by the presence of unwanted cells, or the presence of unwanted cells may be a result of the condition. Examples of specific conditions include tumors (benign or malignant), autoimmune diseases, cardiovascular diseases, degenerative diseases, diabetes, allergic diseases (e.g., asthma), neurodegenerative diseases (such as Alzheimer's disease), transplant patients, and infectious diseases. It should be understood that the agent may also be used in regenerative medicine (e.g., laboratory-cultured organs or tissues). Particularly preferred is that the condition is a tumor (e.g., a malignant disease) and the unwanted cells are tumor cells or tumor-related tissue.

[0290] In another aspect, the present invention provides a method for improving the permeability of therapeutic domains into tissues or tumors, wherein the method includes engineering an agent comprising: i) a therapeutic domain; ii) a cleavable domain; and iii) a stabilizing domain.

[0291] Our use of "engineering a drug" includes the meaning of genetic modification. Such genetic modification can include: (i) mutations in the nucleotide bases of the drug (such as one or more additions, one or more deletions, one or more substitutions, or combinations thereof); and (ii) expression in cell culture systems (such as mammalian cells, bacteria, yeast, or insect cells) (such as recombinant expression).

[0292] In another aspect, the present invention provides the use of the pharmaceutical agent according to the invention in the manufacture of a medicament for treating or preventing tumors, optionally for treating or preventing cancer.

[0293] Subjects may receive treatment with a single or multiple effective doses of the pharmaceutical agent or pharmaceutical composition of the present invention. In the case of multiple administrations, these administrations may be performed, for example, once, twice, three times, four times, or more per day, week, or month, and may continue for a period of time until a therapeutic or preventative benefit is achieved. For example, treatment may last for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days or more, weeks, months, or years, or even for the remainder of the subject's life. The amount of the agent administered to an individual is the amount effective against the specific individual's condition.

[0294] In some embodiments, the agent is administered in combination with one or more additional agents, such as chemotherapeutic agents, immunotherapeutic agents, or radiotherapy agents. In some embodiments, the agent and additional agents of the present invention are formulated in a single composition. In alternative embodiments, the agent and additional agents of the present invention are administered in the form of two or more separate compositions. In yet another embodiment, the agent and additional agents of the present invention are administered simultaneously. In alternative embodiments, the agent and additional agents of the present invention are administered sequentially.

[0295] It may be appropriate to administer specific protease inhibitors to improve the target selectivity of the agents of the present invention. For example, if the therapeutic domain is known to bind to cells in both the heart and breast tissue, but only targets cells in the breast, it may be necessary to administer additional agents that selectively inhibit proteases in the heart but not in the breast.

[0296] In other words, additional agents are applied to inhibit proteases residing in the vicinity of desired cells but not in the vicinity of unwanted cells. This is particularly useful when the cleavable domain of the agent of the present invention can be cleaved by multiple enzymes, some of which reside in the vicinity of unwanted cells and some of which reside in the vicinity of desired cells. In this case, targeting specificity can be improved by applying a protease inhibitor that inhibits proteases residing in the vicinity of desired cells but still capable of cleaving the cleavable domain and thus releasing the therapeutic domain of the agent of the present invention. The role of applying the inhibitor is to ensure that the therapeutic domain is preferentially released in the vicinity of unwanted cells.

[0297] In another aspect, the present invention provides a method for improving the efficacy of a pharmaceutical agent, the method comprising engineering the agent to include: i) a therapeutic domain; ii) a cleavable domain; and iii) a stable domain, wherein the cleavable domain is positioned between the therapeutic domain and the stable domain. Any of the foregoing features may be used in this method and / or the pharmaceutical agent.

[0298] Smaller agents offer favorable pharmacokinetic properties (such as better penetration into solid tumors, higher tumor uptake levels, improved specificity and / or efficacy, and more predictable, linear elimination). However, smaller agents also have some significant drawbacks, such as rapid elimination (e.g. via GFB) and short half-life.

[0299] To overcome the disadvantages of small-sized agents, the inventors have created an engineered agent comprising: i) a therapeutic domain; ii) a cleavable domain; and iii) a stabilizing domain. This leverages the advantages of large agents (slow elimination and long half-life) until the agent contacts tumor-specific proteases in the region near the solid tumor, where cleavage causes a reduction in agent size. In the TME, the smaller portions of the agent (the stabilizing and therapeutic domains) benefit from the favorable pharmacokinetic properties discussed above.

[0300] According to the methods described above, the improvement in drug efficacy is manifested in achieving the desired clinical outcome of the drug using the same drug administration regimen (such as reducing the proliferation of cancer cells or other diseased cells, destroying cancer cells or other diseased cells, reducing the metastasis of cancer cells or other diseased cells, shrinking tumor size, alleviating disease-related symptoms, improving the subject's quality of life, delaying disease progression, and / or prolonging the subject's survival). Those skilled in the art will be able to select appropriate assays to measure the desired clinical outcome of the drug. For example, measuring tumor size by surgically removing a solid tumor from a subject, or visualizing cell proliferation by fluorescence imaging of in vivo tumors in a cancer animal model.

[0301] In another aspect, the present invention provides a method for improving the specificity of a pharmaceutical agent, the method comprising engineering the pharmaceutical agent to include: i) a therapeutic domain; ii) a cleavable domain; and iii) a stable domain, wherein the cleavable domain is positioned between the therapeutic domain and the stable domain.

[0302] In the methods described above, the improvement in drug specificity can be manifested as a higher titer of the therapeutic domain in diseased tissue than in unaffected tissue. Those skilled in the art will be able to select an appropriate assay to measure the titer of the therapeutic domain in the tissue. For example, biopsies can be taken from both diseased and unaffected tissues. The titer of the therapeutic domain in each sample can then be assessed by immunostaining with antibodies (such as anti-CH1 or anti-CL1 antibodies) or by ELISA.

[0303] The method may further include: i. administering the agent of the present invention; ii. cleaving the cleavable domain at the tumor site; and iii. separating the therapeutic domain from the stable domain. The terms "tumor site" and "region near the tumor" are used interchangeably.

[0304] In another aspect, the present invention provides the use of an agent in improving the permeability of a therapeutic domain into a tumor, the agent comprising: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilizing domain.

[0305] In another aspect, the present invention provides the use of an agent in reducing the agent size at a tumor site, the agent comprising: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilizing domain. It should be understood that the size of the agent is reduced through specific cleavage of the cleavable domain. Cleavage of the agent at the tumor site results in the formation of multiple smaller portions of the agent, which may contain both the stabilizing and therapeutic domains.

[0306] In another aspect, the present invention provides the use of an agent in improving the efficacy of the agent, the agent comprising: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilizing domain, wherein the cleavable domain is positioned between the therapeutic domain and the stabilizing domain.

[0307] In another aspect, the present invention provides the use of an agent in improving the specificity of the agent, the agent comprising: i. a therapeutic domain; ii. a cleavable domain; and iii. a stable domain, wherein the cleavable domain is located between the therapeutic domain and the stable domain.

[0308] In another aspect, the present invention provides the use of a cleavable domain in improving the permeability of a therapeutic domain into a tumor, the use including engineering a pharmaceutical agent to include: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilizing domain, wherein the cleavable domain is positioned between the therapeutic domain and the stabilizing domain.

[0309] In another aspect, the present invention provides the use of cleavable domains in reducing agent size at tumor sites, the use including engineering agents to include: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilizing domain, wherein the cleavable domain is positioned between the therapeutic domain and the stabilizing domain.

[0310] In another aspect, the present invention provides the use of cleavable domains in improving the efficacy of pharmaceutical agents, the use of which includes engineering pharmaceutical agents to include: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilizing domain, wherein the cleavable domain is positioned between the therapeutic domain and the stabilizing domain.

[0311] In another aspect, the present invention provides the use of cleavable domains in improving the specificity of pharmaceutical agents, the use of which includes engineering pharmaceutical agents to include: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilizing domain, wherein the cleavable domain is positioned between the therapeutic domain and the stabilizing domain.

[0312] In the embodiments, the improved drug specificity is manifested as a higher titer of the therapeutic domain in diseased tissue than in non-diseased tissue. Preferably, the drug is an agent according to the first aspect of the invention, wherein the cleavable domain is located between the therapeutic domain and the stable domain.

[0313] In another aspect, the present invention provides a pharmaceutical agent according to the first aspect of the invention for diagnosing diseases or ailments. Our use of "diagnosis" includes the detection of cancer cells in vivo (i.e., within a patient) or in vitro (i.e., within a tissue or cell sample taken from a patient). Those skilled in the art will be able to select an appropriate assay to detect cancer cells. As understood and discussed herein, the pharmaceutical agent, therapeutic domain, and stable domain can be detected by labeling (e.g., labeling the Fab region of an antibody with a fluorophore) and subsequently detecting the label (e.g., detecting the fluorophore using an antibody), by immunostaining using antibodies (e.g., anti-CH1 antibody, anti-λ antibody, or anti-κ antibody), or by ELISA.

[0314] In another aspect of the invention, the use includes: i. administering the agent to a subject; ii. cleaving the cleavable domain at the tumor site; and iii. separating the therapeutic domain from the stable domain; iv. increasing the accessibility of the therapeutic domain to cell binding compared to a situation where cleavage and separation according to steps (ii) and (iii) have not occurred. Our use of “accessibility to cell binding” includes the following meaning: cells that previously had limited ability to be contacted and bound by portions of the agent (such as the therapeutic domain or the stable domain) are now more easily contacted and more easily bound by portions of the agent. For example, prior to step (ii), portions of the agent would bind to a limited extent to portions of the tumor interior. After step (iii), portions of the agent would bind to a greater extent to portions of the tumor interior. Those skilled in the art will be able to select appropriate assays to assess the accessibility of the therapeutic domain to cell binding. For example, the same methods discussed above for measuring the ability of an agent to penetrate solid tumors can be used.

[0315] Particularly preferred is that selective cleavage of the cleavable domain enables the therapeutic domain to be released at or near the cell surface of unwanted cells. The terms "nearby region" and "near the tumor" are used interchangeably.

[0316] In the claims and / or specification, when used in conjunction with the term “comprising,” the use of the word “a / an” may mean “one,” but is also consistent with “one or more,” “at least one,” and “one or more than one.”

[0317] In this document, when “about” is used for a value or parameter, it includes (and describes) embodiments relating to that value or parameter itself. For example, a description referring to “about X” includes a description of “X”. Numerical ranges include the numbers that define the range. Although the numerical ranges and parameters that illustrate the broad scope of this disclosure are approximate, the numerical values ​​described in specific instances are reported as precisely as possible. However, any numerical value inherently contains some error necessarily caused by the standard deviation found in its corresponding test or measurement results. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges to which they fall.

[0318] These and other embodiments of the invention will be better understood and appreciated when considered in conjunction with the foregoing description and the accompanying drawings. However, it should be understood that while the foregoing description illustrates various embodiments of the invention and their numerous specific details, it is given by way of illustration rather than limitation. Many substitutions, modifications, additions, and / or rearrangements can be made within the scope of the invention without departing from its spirit, and the invention encompasses all such substitutions, modifications, additions, and / or rearrangements.

[0319] The enumeration or discussion of obviously previously disclosed documents in this specification is not necessarily an admission that such documents are part of the prior art or common general knowledge.

[0320] Example

[0321] Example 1: Antibody Design

[0322] In different regions of the exemplary antibody trastuzumab, cancer-specific protease cleavage sites (LSGRSDNH (SEQ ID NO:77), shown in bold below) for uPA were engineered to enable physical separation of the therapeutic domain and the stable domain at the tumor site. Figure 7 The following are seven different designs, involving the addition or replacement of different regions of the antibody at the protease cleavage site:

[0323] In the design of engineered trastuzumab antibodies, the cleavage site is... (SEQ ID NO:77) was added after the cysteine ​​residue at the end of the CH1 domain and before the hinge region (italicized) of DKTHT (SEQ ID NO:78). GS was added as an additional 2 amino acid linker for greater accessibility.

[0324] Engineered trastuzumab antibody design 1 (SEQ ID NO:1)

[0325]

[0326]

[0327] In the design of engineered trastuzumab antibodies, cleavage site (SEQ ID NO:77) was added after the cysteine ​​residue at the end of the CH1 domain and before the hinge region (italicized) of DKTHT (SEQ ID NO:78). No linker amino acid was added.

[0328] Engineered trastuzumab antibody design 2 (SEQ ID NO:2)

[0329]

[0330] In the design of engineered trastuzumab antibodies, cleavage site (SEQ ID NO:77) was added after the cysteine ​​residue at the end of the CH1 domain and before the hinge region (italicized) of DKTHT (SEQ ID NO:78). GS was added as two additional amino acid linkers to the C-terminus and N-terminus of the protease cleavage site for greater accessibility.

[0331] Engineered trastuzumab antibody design 3 (SEQ ID NO:3)

[0332]

[0333] In the engineered trastuzumab antibody design 4, the DKTHT (SEQ ID NO:78) hinge region is located after the cysteine ​​residue at the end of the CH1 domain and the hinge disulfide bond region ( (SEQ ID NO:79)) was previously (SEQ ID NO:77) Replacement. This is the minimum sequence modification required for insertion into the protease cleavage site.

[0334] Engineered trastuzumab antibody design 4 (SEQ ID NO:4)

[0335]

[0336] In the engineered trastuzumab antibody design 5, the DKTHT (SEQ ID NO:78) hinge region is located after the cysteine ​​residue at the end of the CH1 domain and the hinge disulfide bond region ( (SEQ ID NO:79)) was previously (SEQ ID NO:80) substitution. The additional threonine is thought to provide a similar proximity environment to the cysteine ​​in the hinge region, thereby enabling the region to fold correctly.

[0337] Engineered trastuzumab antibody design 5 (SEQ ID NO:5)

[0338]

[0339] In the design of engineered trastuzumab antibodies, 6, (SEQ ID NO:77) added to the hinge disulfide bond region ( (SEQ ID NO:79)) The F(ab')2 fragment is obtained by placing the three amino acids (PAP) into the CH2 domain of the protein to make the protease accessible.

[0340] Engineered trastuzumab antibody design 6 (SEQ ID NO:6)

[0341]

[0342] In the engineered trastuzumab antibody design 7, ELLGGPSV (SEQ ID NO:81) was replaced with the hinge disulfide bond region ( (SEQ ID NO:79) followed by three amino acids of (SEQ ID NO:77). This is the smallest mutation performed to obtain the F(ab')2 fragment.

[0343] Engineered trastuzumab antibody design 7 (SEQ ID NO:7)

[0344]

[0345] Example 2: Molecular Biology

[0346] Design of engineered trastuzumab gene blocks 1-3

[0347] Trastuzumab gene blocks were designed and synthesized using engineered trastuzumab gene blocks (IntegratedDNA Technologies), as shown below:

[0348] Engineered trastuzumab gene block design 1 (SEQ ID NO:82)

[0349] attaagctcttccctggccGAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGCGCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCTTGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATTCACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATACTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGCACCCTGGTCACTGTATCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGGCTCCAAGCTCTAAGAGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTGAACCTGTGACCGTCTCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTCAGTCAAGTGGCCTTTACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTTATATCTGCAACGTAAATCACAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTGGGTCTCTCTCCGGTCGATCCGACAACCACgctggaagagcgg

[0350] Engineered Trastuzumab Gene Block Design 2 (SEQ ID NO:83)

[0351] attaagctcttccctggccGAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGCGCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCTTGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATTCACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATACTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGCACCCTGGTCACTGTATCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGGCTCCAAGCTCTAAGAGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTGAACCTGTGACCGTCTCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTCAGTCAAGTGGCCTTTACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTTATATCTGCAACGTAAATCACAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTCTCTCCGGTCGATCCGACAACCACgctggaagagcgg

[0352] Engineered trastuzumab gene block design 3 (SEQ ID NO:84)

[0353] attaagctcttccctggccGAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGCGCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCTTGAGTGGGTTGCCCGTATTTATCCCACTAAC GGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATTCACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATACTGCAGTTTTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGCACCCTGGTCA CTGTATTCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGGCTCCAAGCTCTAAGAGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTGAACCTGTGACCGTCTCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTCA GTCAAGTGGCCTTTACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTTATATCTGCAACGTAAATCACAAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTGGGTCTCTCCGGTCGATCCGACAACCACGGGTCTgctggaagagcgg

[0354] All inserts contain the type II restriction enzyme site SapI to enable high-throughput cloning.

[0355] The target vector was modified with a SapI restriction site to enable high-throughput cloning of the VH+ / CH1 domain. The target vector, intended for mammalian antibody expression, contains a hinge region and an Fc region.

[0356] 100 ng of each gene block was mixed with 100 ng of pCREA high-throughput mammalian Fc vector, 5 units of SapI restriction enzyme (New England Biolabs), and 200 units of T4 DNA ligase (New England Biolabs). The sample was incubated at 37°C for 60 min, followed by enzyme inactivation at 65°C for 20 min. The DNA was transformed into NEB 5α cells (New England Biolabs) and plated on LB agar plates containing 100 μg / ml carbenicillin. The plates were incubated overnight at 37°C. The next day, five colonies were selected, and the insert was checked for correctness using mini-prep, followed by Sanger sequencing. For large-scale preparation of DNA for transfection, the correct construct was prepared using an endotoxin-free maximum preparation kit (Qiagen).

[0357] Engineered trastuzumab gene block design 4-7

[0358] Since designs 4-7 are closer to the hinge, these constructs are prepared by creating integrated DNA technologies that span CH1 and CH2, as shown below.

[0359] Engineered trastuzumab gene block (CH1 CH2) design 4 (SEQ ID NO:85)

[0360] atgccattgtaACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTCTCTCCGGTCGATCCGACAACCACTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTattcgcaatc

[0361] Engineered trastuzumab gene block (CH1 CH2) design 5 (SEQ ID NO:86)

[0362] atgccattgtaACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTCTCTCCGGTCGATCCGACAACCACacaTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTattcgcaatctc

[0363] Engineered trastuzumab gene block (CH1 CH2) design 6 (SEQ ID NO:87)

[0364] atgccattgtaACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACTCTCCGGTCGATCCGACAACCACCCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTattcgcaatc

[0365] Engineered trastuzumab gene block (CH1 CH2) Design 7 (SEQ ID NO:88)

[0366] atgccattgtaACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACTCTCCGGTCGATCCGACAACCACTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTattcgcaatc

[0367] The 5' restriction enzyme is AgeI, and the 3' restriction enzyme is BsrGI. The destination vector is trastuzumab with an engineered trastuzumab antibody design 1 (SEQ ID NO:1).

[0368] Gene blocks were digested using AgeI-HF (New England Biolabs) and BsrGI (New England Biolabs), as were the trastuzumab vector in the pCREA vector. Samples were digested overnight at 37°C. Gene blocks were purified using a PCR purification kit, but the vector was run on a 1% agarose gel in Tris-acetic acid-EDTA (TAE) buffer to separate the insert from the backbone. Larger backbone fragments were banded. Ligation was performed at a 3:1 molar ratio of insert to backbone in the gene block and the cut backbone. Ligation was performed using 400 units of T4 ligase (New England Biolabs) and incubated overnight at room temperature. Ligations were transformed into NEB 5α cells (New England Biolabs) and plated onto LB agar plates containing 100 μg / ml ampicillin. The presence of the correct insert was checked in 5 colonies using a mini prep, followed by Sanger sequencing. For large-scale preparation of DNA for transfection, the correct construct was prepared using an endotoxin-free maximum preparation kit (Qiagen).

[0369] Example 3: Antibody Expression and Purification

[0370] CHO-K1 cell line

[0371] Trastuzumab in the pCREA antibody mammalian expression vector and engineered trastuzumab antibody design 1 in the pCREA antibody mammalian expression vector were transfected into CHO-K1 cells in 30 ml shake flasks at a DNA:PEI ratio of 3:1 via polyethyleneimine (PEI) or expifectamine. Cells were grown for up to 7 days (96 hours post-transfection), during which cell health and titers were monitored from day 1 to day 4. Comparison of cell health on the first 4 days showed no difference between engineered trastuzumab antibody design 1 (D1) and the trastuzumab monoclonal antibody. Assessment of antibody concentration by Protein AOctet showed no difference between the trastuzumab monoclonal antibody and the design 1 equivalent (as shown in Table 1 and...). Figure 7 (As shown). At 96 hours post-transfection, expression levels ranged between 0.891 and 3.02 mg / L. SDS-PAGE was used to look for overexpression; however, due to the overall low expression levels, overexpression could not be detected (e.g., ...). Figure 8 (As shown).

[0372] Table 1 – Summary of Antibody Titers

[0373] Flask number Transfection method condition 96h Octet concentration (μg / mL) Flask 3 PEI Comparison with mAb 3.42 Flask 4 PEI Comparison with mAb 3.67 5 flasks PEI Crea-mAb 3.02 Flask 6 PEI Crea-mAb 3.01 Flask 7 Expifectamine Comparison with mAb 0.894 Flask 8 Expifectamine Comparison with mAb 0.891 Flask 9 Expifectamine Crea-mAb 0.924 Flask 10 Expifectamine Crea-mAb 0.964

[0374] Transfection conditions were optimized, and cells were grown for up to 7 days, during which cell health and titers were monitored from day 1 to day 4. Similarly, comparisons of cell health over the first 4 days showed no difference between engineered trastuzumab antibody design 1 and the trastuzumab monoclonal antibody. Assessment of antibody concentrations using Protein A Octet assays indicated no difference between the trastuzumab monoclonal antibody and the design 1 equivalent (as shown in Table 2 and...). Figure 9 (As shown). At 96 hours post-transfection, expression levels ranged between 1.83 and 5.46 mg / L. SDS-PAGE was used to identify overexpression, and overexpression of the antibody heavy chain was detected (see [link]). Figure 10 (The box in the middle).

[0375] Table 2 – Summary of antibody titers in the optimization experiment

[0376]

[0377] Expi-CHO cell line

[0378] After cloning, the trastuzumab in the pCREA antibody mammalian expression vector and the engineered trastuzumab antibody in the pCREA antibody mammalian expression vector were diluted to a concentration of 1 μg / μl (D1 to D7). These were then transfected into the Expi-CHO cell line (Invitrogen) in shake flasks using Optipro+ExpiFectamine and incubated overnight at 37°C. The culture was then fed, and the cells were allowed to continue growing for 9 days.

[0379] purification

[0380] On day 9, cells were collected by centrifugation at 3000x g for 30 min and filtered through a 0.2 μm filter to recover the overexpressed antibody from the cell supernatant. The supernatant was purified using an AKTA Go instrument (Cytiva) on a MabSelect PrismA 1 ml pre-packed column. MabSelect binding buffer (NaPO4) and Elution was performed in MabSelect elution buffer (glycine) at pH 2.7. Fractions were combined according to the chromatogram, and the buffer was exchanged in 1x DPBS at 3800x g for 20 minutes using a Vivaspin 20 concentrator. Figure 11 (See Table D1). Absorbance was measured using Nanodrop (ThermoScientific), and yield and concentration were calculated. Trastuzumab and engineered antibody _D1-7 had similar expression levels and yields (as shown in Table 3).

[0381] Table 3 – Summary of antibody expression levels and yields

[0382]

[0383] Purification control and SDS-page analysis of D1-D7

[0384] The purified antibody was heated at 85°C for 5 minutes, and then 10 μl was loaded onto a NuPAGE 4%–12% Bis-Tris gel inserted into a mini gel well containing 1X MES SDS running buffer, with protein markers as molecular weight standards. The gel was run at 200V for 25 minutes, and then... The protein dye was used for staining, and then decolorized with H2O.

[0385] Both reduced and non-reduced SDS-PAGE gels showed antibody profiles similar to mAbs (e.g. Figure 12 (As shown). All molecules appeared as expected, with D5 being the largest variant due to its higher molecular weight resulting from engineered glycosylation sites. These results are consistent with the size exclusion (SEC) high-performance liquid chromatography (HPLC) determinations and T... m and T agg The results observed in the determination were consistent.

[0386] SEC-HPLC determination of purified engineered antibodies

[0387] 10 μg of purified antibody sample was injected into a MAbPac SEC-1 (4x300 mm) column equipped with a MabPac SEC-1 (4x50 mm) pre-column, and SECHPLC was performed to determine the percentage of monomers and higher-order oligomers present.

[0388] SEC-HPLC assays showed good solution-state curves for D1-7 (as shown in Table 4), indicating that they both have acceptable purity levels compared to the control trastuzumab.

[0389] Table 4 – Thermal stability analysis of mAb comparison and D1-7

[0390]

[0391] Thermostability of Purified Engineered Antibodies

[0392] The sample was diluted to 1 mg / mL in DPBS (Duborough phosphate buffered saline), and then 9 μL of each sample was loaded into triplicate into a UNCLE instrument (Unchained Labs) to determine the melting temperature (T). m ) and aggregation temperature (T agg ).

[0393] All antibodies showed high T levels. M and T agg Values ​​(Table 4). T values ​​for all antibodies M All values ​​are above 65.0℃, thus meeting the typical requirements for CMC development, except for D7, which exhibits the lowest TM at 47.4℃ (as shown in Table 4). D7 is also the only one to show a second T. M The sample (76.1℃). T of D4 M1 The highest value was observed for D5 (70.8℃), while the lowest value was observed for D7 (47.4℃). The T value for D5 was also observed. agg The highest (74.5℃), and the T compared to trastuzumab. agg Lowest (67.1℃).

[0394] in conclusion

[0395] Considering the biophysical parameters analyzed, engineered D1-D7 has been shown to maintain the biophysical and biochemical properties required for trastuzumab to function as an antibody, and these properties have not been negatively affected by the addition of cleavable sites in the engineered form.

[0396] Example 4: Antibody Characterization

[0397] The binding of purified engineered antibody to HER2 (assessed by ELISA).

[0398] Maxisorp was prepared in the following manner. TM ELISA plate: Add 50 μl of phosphate-buffered saline (PBS), bovine serum albumin (BSA – 1 μg / ml), or hHER2 (1 μg / ml) to each well, and then refrigerate overnight. Wash the plate three times with PBS + 0.1% Tween 20, then wash three times with PBS, and block with 200 μl of PBS + 6% milk powder at room temperature for one hour, then wash again.

[0399] Purified trastuzumab mAb and _D1-7 antibody were diluted to 50 μl per well in PBS + 3% milk powder, added to the plate, and incubated at room temperature for 1 hour, followed by washing the plate again. Antibodies were added at the highest concentration of 150–50 nM and diluted 1:2 or 1:3 in an 8 or 12-point dilution series. 50 μl of anti-Vk antibody-HRP conjugate diluted 1:5000 in PBS + 3% milk powder was added to each well, and the plate was incubated at room temperature for 1 hour to bind to the sample. The plate was washed again, and 50 μl of 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate was added for color development. The signal was quenched with 50 μl of 1M HCl, and the absorbance of the plate was read at 450 nm.

[0400] ELISA showed that D1-D7 exhibited the same binding to hHER2 (as shown in Table 5 and...). Figure 13 (As shown).

[0401] Table 5 – IC50 of trastuzumab control and D1-D7 50 value

[0402] Antibody <![CDATA[IC 50 ]]> Trastuzumab mAb 0.1325 Trastuzumab D1 0.1282 Trastuzumab D2 0.1239 Trastuzumab D3 0.1395 Trastuzumab D4 0.1235 Trastuzumab D5 0.1423 Trastuzumab D6 0.1679 Trastuzumab D7 0.1548

[0403] The binding of purified engineered antibody to HER2 (assessed by BIAcore).

[0404] use Under the conditions of HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v / v surfactant P20) + running buffer and regeneration with 0.1M glycine pH 2.0, 12,500 RU units of anti-human IgG were coated onto a CM5 chip. hHER2 protein did not show binding to a blank CM5 chip.

[0405] Then, using amine coupling chemistry, the anti-human IgG Fc antibody was immobilized onto 10,215.8 RU units on the flow cell (Fc)2 of the CM5 chip using HBS-EP+ running buffer. It was subsequently confirmed that the HER2 protein did not bind to the immobilized anti-human IgG Fc antibody.

[0406] Trastuzumab (0.1, 0.5, and 1 μg / ml) was optimized for capture using hHER2 (1, 10, and 100 nM). Trastuzumab exhibited stable capture levels and concentrations, and its capture baseline did not vary significantly between cycles, indicating that efficient regeneration was possible using 0.1 M glycine HCl at pH 2.0.

[0407] Trastuzumab and D1-D7 were subjected to 12-point dilution kinetics measurements using HBS-EP+ run buffer, where 0.1M glycine regeneration resulted in similar KD binding to hHER2-HIS, and samples were assayed at room temperature. By BIAcore, all D1-D7 samples showed identical binding to HER2 (as shown in Table 6 and...). Figure 14 (As shown). This indicates that the engineered cleavable domain did not reduce the antibody's ability to bind to HER2 or otherwise negatively affect that ability.

[0408] Table 6–D1-7 and the combination of controls with HER2

[0409]

[0410] Protease assay of purified antibody

[0411] The conditions for protease assays were optimized, and purified D1-7 antibody and control trastuzumab were diluted with protease buffer (50 nM Tris-HCl and 0.01% Tween-20 pH 8.0). Each sample was digested with uPA enzyme at 0 nM, 23.5 nM, and 235 nM and incubated at 37 °C for 24 h. 5 μl of NuPAGE containing 10 nM dithiothreitol (DDT) was then used. TM LDS sample buffer (Thermo Fisher) Add the sample dye to each sample and boil at 95°C for 10 minutes, then cool to 10°C in a PCR machine. Load all contents onto a 4-12% SDS-PAGE gel and run at 200°C for 30 minutes. Allow the gel to cool to room temperature. The dye is applied for several hours, and then the color is removed in water for several days.

[0412] Studies have found that uPA protease releases Fab or F(ab')2 fragments (such as...) from D1-D4 and D6-D7. Figure 15 (As shown). uPA enzyme did not digest the trastuzumab control. This experiment shows that engineered D1-D4 and D6-D7 with protease cleavage sites were successfully cleaved, while control D5 and trastuzumab were not cleaved because they do not have protease cleavage sites.

[0413] Binding of uPA-treated, cleaved engineered antibody to HER2 (assessed by ELISA)

[0414] Four ELISA plates were prepared with 1 μg / ml hHER2-HIS (50 μl) in PBS, and two ELISA plates were prepared with PBS (50 μl) to test the binding of the cleaved therapeutic domain to its target. The plates were sealed and incubated overnight at 4°C.

[0415] Remove the plates from the refrigerator and wash them (as described above), then perform serial dilutions at a 1:4 ratio. Perform assays in triplicate for each antibody: one set for PBS plates, one set for hHER2-HIS plates (Vk detection), and one set for hHER2-HIS plates (Fc detection). Incubate the antibodies at room temperature for 1 hour. Wash the plates again, then add the antibodies to the plates. Dilute the anti-Vk antibody as described above and add it to one set of hHER2-HIS plates and one set of PBS plates. Add 50 μl to each well. Incubate the plates at room temperature for 1 hour, then wash them again. Develop the colorimetric assay, then quench the signal and read the absorbance at 450 nm.

[0416] Studies have found that uPA treatment does not affect the binding of the antibody to hHER2 (as shown in Table 7 and...). Figure 16 (As shown).

[0417] Table 7 – Estimated uPA processingTrastuzumab control and IC50 on D1-D7 50 value

[0418] Antibody <![CDATA[IC 50 ]]> Trastuzumab mAb 0.2843 Trastuzumab D1 0.2 Trastuzumab D2 0.1794 Trastuzumab D3 0.1848 Trastuzumab D4 0.3604 Trastuzumab D5 0.3466 Trastuzumab D6 0.2688 Trastuzumab D7 0.2623

[0419] Binding of uPA-treated, cleaved engineered antibodies to the Fcγ receptor (assessed by ELISA)

[0420] ELISA plates were prepared to detect the binding of the stable domain to Fcγ receptors (I, IIa, IIb, IIIa). 50 μl of 1 μl / ml FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a V176), and 50 μl of PBS were added to each well. The plates were incubated overnight at 4°C. The plates were washed, incubated at room temperature for 1 hour for blocking, and then washed again.

[0421] The antibody was tested in triplicate with an 8-point dilution (1:3). 50 μl of PBS + 3% milk powder was added to each well. The sample was incubated at room temperature for 1 hour to allow binding, followed by washing the plate. Anti-κIgG was diluted 1:5000 in PBS + 3% milk powder. The sample was incubated at room temperature for 1 hour, followed by washing the plate again. The plate was developed, the signal was quenched, and the absorbance was read at 450 nm.

[0422] The study found that engineered antibodies did not affect binding to Fcγ receptors Ia and IIa (as shown in Figures 17A and 17B). All D1-D7 antibodies strongly bound to Fcγ receptor Ia. Binding of D7 to Fcγ receptor Ia was affected by the protease site, which is expected to be due to the crystal structure of the Fc region interacting with the Fcγ receptor. No binding to Fcγ receptor IIb was observed (as shown in Figure 17C). Binding to Fcγ receptor IIIa was assessed using the 176V variant; both D6 and D7 showed slightly decreased binding, while D5 showed increased binding (Figure 17D), which is attributed to glycosylation. All other engineered trastuzumab variants (D1-D4) showed similar binding to Fcγ receptor IIIa as the trastuzumab mAb control.

[0423] in conclusion

[0424] The effect of engineering cleavable domains into antibody-derived fragments on the ability of stable domains to bind to Fcγ receptors is negligible.

[0425] Example 5: Manufacturability of Engineered Pharmaceuticals

[0426] trastuzumab and D3 transfection

[0427] Trastuzumab and D3 in the pCREA antibody mammalian expression vector were diluted to a concentration of 1 μg / μl and then transfected into the Expi-CHO cell line (Invitrogen) in shake flasks using Optipro+ExpiFectamine and incubated overnight at 37°C. The culture was then fed and the cells were allowed to continue growing for 10 days.

[0428] On day 10, the culture was aliquoted into 500 ml conical tubes and centrifuged at 3900 x g for 30 min. The supernatant was filtered through a 20 μm filter. 30 ml was transferred to a 50 ml Falcon tube and filtered, and the remaining 250 ml was purified to obtain the large-scale purified formulation.

[0429] Purification of trastuzumab and D3

[0430] The harvested trastuzumab and D3 were processed using the AKTA Go instrument (Cytiva) on the MabSelect PrismA. TM Purification was performed on a 1 mL column. Fractions were combined according to the chromatogram, and the buffer was exchanged in 1xDPBS at 3800 x g for 20 minutes using a Vivaspin 20 concentrator. Absorbance was measured using a Nanodrop (Thermo Scientific), and concentrations were determined (as shown in Table 8).

[0431] Table 8 – Concentrations of trastuzumab and D3 samples after Protein A purification

[0432] sample Concentration (mg / ml) Approximate total yield (mg) Expression level (mg / L) Trastuzumab mAb 6.6 48 240 Trastuzumab D3 5.1 36 180

[0433] Quality Control (QC) Analysis of Trastuzumab and D3

[0434] Using a Dionex UltiMate 3000U HPLC system (Thermo Scientific), samples of 1 mg / ml were analyzed at an isocratic rate of 0.35 ml / min for 15 minutes. SEC-HPLC analysis of trastuzumab showed a monomer purity of 98.5%, and SEC-HPLC analysis of D3 showed a monomer purity of 94.3% (e.g., ...). Figure 18 (As shown).

[0435] Each 1.5 μg sample was analyzed on a 4%–12% NuPAGE SDS gel, and the results showed that D3 had good purity (e.g., ...). Figure 19 (As shown in Table 9). Filter the sample and dispense it into 2ml test tubes (as shown in Table 9).

[0436] Table 9 – Final concentrations of trastuzumab and D3 samples after filtration

[0437] sample Concentration (mg / ml) Purity (% monomer) Endotoxin (EU / mg / ml) Trastuzumab mAb 1.05 98.5 <0.5 Trastuzumab D3 1.04 94.3 <0.5

[0438] Large-scale production of trastuzumab and the binding of D3 to Her2 (assessed by ELISA).

[0439] An ELISA plate was prepared using 1 μg / ml hHER2-HIS diluted in PBS, a second ELISA plate was prepared using 1 μg / ml BSA, and a third ELISA plate was prepared using PBS alone. The plates were placed in the refrigerator overnight. The plates were then washed, incubated at room temperature for 1 hour for blocking, and then washed again as described in Example 4.

[0440] 150 μl of the large-scale purified formulation (trastuzumab and D3 antibody) was transferred to 150 μl of PBS + 3% milk powder at a 12-spot dilution. 50 μl was added to each well and incubated at room temperature for 1 hour. The plate was washed, developed, and the signal quenched as described above. The absorbance was read at 450 nm.

[0441] Trastuzumab and D3 from large-scale expression formulations retained their ability to bind to HER2. Both trastuzumab and D3 were demonstrated to be active. The IC50 of trastuzumab binding to hHER2 was [not specified]. 50 The IC with D3 and hHER2 has a capacitance of 0.6095 nM. 50 It is 0.1035 nM (as shown in Table 10 and Figure 20 (As shown).

[0442] Table 10 – IC50 of trastuzumab and D3 from ELISA 50 value

[0443] Trastuzumab mAb Trastuzumab D3 <![CDATA[IC 50 (nM)]]> 0.6095 0.1035

[0444] Purification of trastuzumab and D1

[0445] Trastuzumab control and D1 were expressed in transfected CHO-K1 cells as described in Example 3. Samples were centrifuged at 3900 x g for 20 min, filtered through a 0.22 μm filter, loaded onto a 1 ml pre-packed MabSelect PrismA column, and purified using an AKTA Go instrument (Cytiva). Relevant fractions were pooled, and the buffer was exchanged in DPBS with Vivaspin 20 (30 kDa MWCO) and centrifuged at 3800 x g for 20 min in a benchtop centrifuge. Absorbance was measured using Nanodrop (ThermoScientific), and yields and concentrations were calculated. The study found that the expression levels of both trastuzumab and D1 were low, likely due to a lack of plasmid / cell line optimization (as shown in Table 11). However, the expression levels were comparable, indicating that engineered formulations maintained the purity of trastuzumab.

[0446] Table 11 – Expression levels of CHO-K1 trastuzumab and D1

[0447] sample Expression level (mg / L) Trastuzumab (CHO-K1) 6.5 D1 5.5

[0448] SDS-PAGE analysis of purified trastuzumab mAb and D1

[0449] The purified trastuzumab and D1 sample were boiled at 85°C for 5 minutes and then separated by SDS-PAGE. The gel showed that both trastuzumab and D1 antibodies remained intact (e.g., ...). Figure 21 (As shown).

[0450] Trastuzumab and _D1 expressed in Expi-CHO and CHO-K1 cells bind to HER2 (as assessed by ELISA). estimate)

[0451] Three Maxisorb ELISA plates were prepared as follows: 50 μl PBS, 1 μg / ml BSA, or 1 μg / ml hHER2 were added to each well and incubated overnight. The plates were washed and blocked at room temperature for 1 hour, then washed again. The antibody samples were purified according to Examples 3 and 6 above, diluted 1:3 to a final volume of 50 μl per well, and each antibody was measured in triplicate with 8-point dilutions. The samples were diluted in PBS + 3% milk powder and incubated at room temperature for 1 hour. The plates were prepared with anti-Vk antibody as described above. The samples were bound at room temperature for 1 hour. The plates were washed as described above. Color development was performed, the signal was quenched, and the absorbance of the plate was read at 450 nm.

[0452] ELISA analysis showed that trastuzumab expressed by Expi-CHO or CHO-K1 cells was identical to that expressed by D1, and the insertion of an exogenous cleavable domain did not affect the potency of trastuzumab (e.g., Figure 22 (as shown in Table 12).

[0453] Table 12 – IC50 values ​​of trastuzumab and D1 expression from CHO-K1 and Expi-CHO 50 value

[0454] Antibody <![CDATA[IC 50 ]]> Trastuzumab mAb (CHO-K1) 0.1262 Trastuzumab D1 (CHO-K1) 0.1171 Trastuzumab mAb (Expi-CHO) 0.1251 Trastuzumab D1 (Expi-CHO) 0.1270

[0455] Example 6: Different protease cleavage sites are suitable for engineered drug design

[0456] To demonstrate that this technology can be used for protease cleavage sites specific to different types of cancer, various agents containing the ADAM10 site (D9) were designed. This protease was chosen because it belongs to a different protease family than uPA (a serine protease). ADAM10 is a novel aspartic protease-A metalloproteinase. These designs are based on engineered trastuzumab antibody designs.

[0457] clone

[0458] Constructs incorporating different protease cleavage sites (ADAM10 site: PRAEALKGG (SEQ ID NO: 89), underlined) were designed and expressed. These constructs were based on engineered trastuzumab antibodies.3

[0459] Engineered antibodies containing the ADAM10 site

[0460] In engineered trastuzumab antibody design 9 (D9), GS PRAEALKGG GSAS (SEQ ID NO:90) was added after the cysteine ​​residue at the end of the CH1 domain and before the hinge region (italicized) of DKTHT (SEQ ID NO:78). This added two extra amino acids at the C and N ends of the protease cleavage site and four extra amino acids at the N end for greater accessibility.

[0461] Engineered trastuzumab antibody design 9, D9 (SEQ ID NO:8):

[0462]

[0463] The gene block for synthesizing D9 (SEQ ID NO:91) (Integrated DNA Technologies) is shown below:

[0464] attaagctcttccctggccGAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGCGCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCTTGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATTCACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATACTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGCACCCTGGTCACTGTATCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGGCTCCAAGCTCTAAGAGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTGAACCTGTGACCGTCTCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTCAGTCAAGTGGCCTTTACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTTATATCTGCAACGTAAATCACAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTGGGTCTCCACGAGGCTGGGATGCTCATACTGGGTCTgctggaagagcgg

[0465] 100 ng of each gene block was mixed with 100 ng of pCREA high-throughput mammalian Fc vector, 5 units of SapI restriction enzyme (New England Biolabs), and 200 units of T4 DNA ligase (New England Biolabs). The ligation was transformed into NEB 5α cells (New England Biolabs) and plated on LB agar plates containing 100 μg / ml ampicillin. The plates were incubated overnight at 37°C. The presence of the correct insert was checked in three colonies using a mini prep assay, followed by Sanger sequencing. For large-scale preparation of DNA for transfection, the correct construct was prepared using an endotoxin-free maximum preparation kit (Qiagen).

[0466] Purification of D9

[0467] Prior to purification, D9 was generated by transfecting Expi-CHO cells, with expression occurring at a 25 mL scale for 7–10 days. The purification buffer was filtered through a 20 μm filter. The supernatant was loaded onto MabSelect PrismA resin, and the samples were run on an AKTA Go instrument. Relevant fractions were pooled, and the buffer was exchanged in 1x PBS pH 7.2 with Vivaspin 20 (30 kDa MWCO), followed by centrifugation at 3800 x g for 20 min in a benchtop centrifuge. Absorbance was measured using Nanodrop (ThermoScientific), and yields and concentrations were calculated (see Table 14 below).

[0468] Table 14 – Yield and concentration of D9

[0469] sample A280 Ext.Coeff. Concentration (mg / ml) Volume (ml) Yield (mg) D9 4.97 1.441 3.4 0.7 2.4

[0470] SDS-PAGE analysis of D9

[0471] Boil the samples at 85°C for 5 minutes, then take 10 μl of each sample and load it onto a pre-prepared NuPAGE 4%-12% Bis-Tris gel inserted into a mini gel well containing 1X MES SDS running buffer, and add the protein markers. Run the gel at 200V for 25 minutes. Then use the gel... The protein dye was used for staining, and then decolorized with H2O.

[0472] All molecules met expectations on SDS-PAGE, and the band size and purity of D9 were comparable to other samples (e.g., Figure 23 (As shown).

[0473] These results indicate that replacing the uPA cleavage site with another cleavage site does not affect the expression of the trastuzumab engineered antibody at the D3 position.

[0474] Protease digestion

[0475] The protease ADAM10 was diluted 1:10, and digestion conditions were set so that 5 μg of D9 was digested with a single concentration of protease at a 1:1 antibody-to-protease molar ratio. The sample was prepared in 1.5 ml Eppendorf tubes and incubated at 37°C for 24 hours.

[0476] The engineered antibodies were cut and boiled at 85°C for 5 minutes, and then separated by SDS-PAGE.

[0477] D9 can be effectively cut by ADAM10 (see...) Figure 23 ), and therefore all designs are applicable to being cleaved by their respective proteases.

[0478] The combination of D9 and hHER2 (assessed via ELISA)

[0479] The plate setup was as follows: one hHER2-HIS plate diluted to 1 μg / ml, one BSA plate diluted to 1 μg / ml, and one PBS blank plate for D3 (control) and D9. 50 μl of each antigen was added to each well and diluted in PBS. The plates were washed and then incubated at room temperature for 1 hour for blocking, followed by another wash.

[0480] 150 μl of trastuzumab, D3, and D9 were transferred to 150 μl of PBS + 3% milk powder to prepare a 1:2 dilution. 50 μl of each solution was added to each well, and the protein was incubated at room temperature for 1 hour. Plates were prepared with anti-Vk antibody and samples as described above. Color development was performed, the signal was quenched, and the absorbance of the plate was read at 450 nm.

[0481] The results showed that D9 retained its potency and binding to HER2 (see [link to study]). Figure 24 (and Table 20).

[0482] Table 20 – IC50 of trastuzumab control, D3, and D9 50 value

[0483] Antibody <![CDATA[IC 50 ]]> Trastuzumab mAb 0.3363 Trastuzumab D9 0.3389

[0484] This example demonstrates that cancer-specific protease sites from different protease families can be used in this technology.

[0485] Example 7: Different antibody-derived fragments are suitable for engineered drug design

[0486] To demonstrate that this technology can be used with different types of antibody-derived fragments, various agents with fragments derived from pembrolizumab, cetuximab, bevacizumab, M5A, and ipilimumab were designed. These specific antibody-derived fragments were chosen because they each bind to different targets and are used to treat different types of cancer. These designs are based on engineered trastuzumab antibody designs.3

[0487] clone

[0488] Other clinical oncology-related antibodies were also evaluated for their suitability for hinge engineering. Antibodies selected included ipilimumab (as described in US20150283234), cetuximab, pembrolizumab, bevacizumab, and an anti-CEA antibody (M5A, as described in US7273608B2; Yazaki et al. 2004. Humanization of the anti-CEA T84.66 antibody based on crystal structure data. Protein Engineering, Design and Selection. 17(5):481–489). These designs, based on engineered trastuzumab antibody D3, added GS-[protein site]-GS after cysteine ​​in CH1 (upper hinge). Cleavable domains are shown in bold. uPA cleavage sites are shown underlined.

[0489] VRC01 antibody was used as a negative control because VRC01 targets a non-mammalian antigen, namely human immunodeficiency virus (HIV).

[0490] VRC01 comparative design (SEQ ID NO:92)

[0491]

[0492]

[0493] Cetuximab D12 (SEQ ID NO:11)

[0494]

[0495] Bevacizumab D13 (SEQ ID NO:12)

[0496]

[0497] Pembrolizumab D14a (SEQ ID NO:13)

[0498]

[0499] Pembrolizumab D14b (SEQ ID NO:14)

[0500]

[0501]

[0502] In engineered trastuzumab antibody designs 14 (D14a and D14b), three additional GPP residues (in italics) were added to the C-terminus of the cleavage site within the cleavable domain (D14a), or to the N-terminus of the cleavage site within the cleavable domain. This facilitates the expression and purification of the engineered antibody.

[0503] M5A D15 (SEQ ID NO:15)

[0504]

[0505] Ipilimumab D16 (SEQ ID NO:16)

[0506]

[0507] Transfection

[0508] All plasmids (vector: pCrea) used for transfection were diluted to a concentration of 1 μg / μl and then transfected into the Expi-CHO cell line (Invitrogen) in shake flasks using Optipro+ExpiFectamine, and incubated overnight at 37°C. The culture was fed and the cells were allowed to grow for 11 days. Afterward, the culture was centrifuged at 3900 x g for 30 min, and the supernatant was filtered through a 20 μm filter and stored at 4°C.

[0509] Purification and buffer exchange

[0510] Prepare purification buffer and then filter through a 20 μm filter. Purify the cell culture supernatant using MabSelect PrismA (Cytiva) and prepare it in 1X PBS pH 7.2 as described above. Combine the relevant fractions and exchange the buffer in DPBS using Vivaspin 20 (30 kDa MWCO), centrifuge at 3800 x g for approximately 60 min in a benchtop centrifuge as described above. Filter the buffer-exchanged molecules aseptically through a 20 μm filter.

[0511] Table 15 – Expression levels of different antibodies containing and without cleavable domains

[0512] Construct Volume (ml) Concentration (mg / ml) Yield (mg) Expression level (mg / L) bevacizumab 2.5 4.55 11.39 379.1 D13 2.5 4.94 12.35 418.7 Pembrolizumab 2.5 0.7 1.8 72.9 D14a 2.5 0.73 1.8 72.9 D14b 3.0 0.28 0.8 33.4 cetuximab 3.5 2.61 9.13 337.7 D12 2.3 3.93 9.05 341.0 M5A 3.0 2.57 7.70 310.6 D15 2.5 2.81 7.02 286.6 Ipilimumab 3.5 1.2 4.1 165.8 D16 3.0 3.6 10.7 427.2

[0513] SDS-PAGE analysis

[0514] Prepare 1 μg aliquots according to Table 16.

[0515] Table 16 – Preparation of purified antibodies for SDS-PAGE analysis

[0516] μl mAb R mAb Non-R Engineering Ab R Engineered Ab Non-R sample 2.3 2.3 2.8 2.8 <![CDATA[H2O]]> 11.7 12.7 11.2 12.2 SDS loading buffer (4X) 5 5 5 5 1M DTT (20X) 1 0 1 0 Total volume 20 20 20 20

[0517] The purified antibodies were boiled at 85°C for 5 minutes. 10 μl of each antibody was then loaded onto a NuPAGE 4%–12% Bis-Tris gel inserted into a mini gel well containing 1X MES SDS running buffer, and protein markers were added. The gel was run at 200V for 25 minutes, and then... The protein dye was used for staining, and then decolorized with H2O, as described above.

[0518] Both reduced and non-reduced SDS-PAGE showed the expected expression and purity curves for all loaded antibodies. Figure 25 ).

[0519] Protease digestion of antibody variants

[0520] Use the above protocols (see Examples 4 and 6) to test whether other clinical oncology-related antibody variants prepared as described above are easily digestible.

[0521] The study found that uPA cleaves the antibody variant and generates the F(ab) fragment in a manner similar to that described for trastuzumab D3 (as shown in Figures 27A, 27B, 27C, and 27D).

[0522] Binding of D12-D16 to their corresponding targets (assessed by ELISA)

[0523] Configure the ELISA board according to Table 17.

[0524] Table 17 – D12-D16 ELISA Plate Settings

[0525] Number of boards reagents Dilution 1 hEGFR-HIS 1μg / ml 1 hVEGF-HIS 1μg / ml 1 hPD1-HIS 1μg / ml 1 hCEACAM-5 1μg / ml 1 hCTLA-4 1μg / ml 2 BSA 1μg / ml 2 PBS blank

[0526] Add 50 μl to each well. Dilute all antigens in PBS. Prepare the plate, anti-Vk antibody, and samples as described above. Develop the colorimetric sample, sequence the signal, and read the absorbance of the plate at 450 nm.

[0527] Compared to non-engineered antibodies, all antibodies from D12 to D16 maintained similar binding characteristics to their respective targets (hEGFR, hVEGF, hPD-1, hCEACAM-5, and hCTLA-4) (e.g., Figure 26A, 26B, 28A, 28B and 28C and Table 18 are shown.

[0528] Table 18 – IC50 values ​​of unengineered and engineered antibodies D13-D16 50 value

[0529]

[0530]

[0531] D12-D16 is easily cleaved and has been found to retain potency and binding to its respective targets.

[0532] Examples show that different antibody-derived fragments that bind to multiple targets (and whose mechanisms of action in cancer may differ) can be used for this technology.

[0533] Example 9: Different types of therapeutic domains are suitable for engineered drug design

[0534] To demonstrate that this technology can be used for different types of therapeutic domains, different agents were designed. In the anti-EGFR VHH design 17 (D17), the anti-EGFR VHH (Llama 7D12) domain was used as the therapeutic domain. In the trastuzumab designs 18 (D18) and 19 (D19), the ScFv domain was used as the therapeutic domain. D19 differs from D18 in that the stabilizing domain contains a longer linker. These designs are based on engineered trastuzumab antibody design 3.

[0535] clone

[0536] VHH as a therapeutic domain

[0537] The llama 7D12 VHH used for the anti-EGFR antibody sequence is derived from the crystal structure of EGFR containing VHH nanobodies. This crystal structure is derived from Schmitz KR, Bagchi A, Roovers RC, van Bergen en Henegouwen PM, Ferguson KM. Structural evaluation of EGFR inhibition mechanisms for nanobodies / VHH domains. Structure. July 2, 2013; 21(7):1214-24. doi:10.1016 / j.str.2013.05.008. Electronic publication on June 20, 2013. PMID:23791944; PMCID:PMC3733345.

[0538] Gene block of anti-EGFR VHH design 17(D17) (SEQ ID NO:93)

[0539] attaagctcttccctggccCAAGTCCAACTGCAGGAGAGCGGGGGTGGCCTGGTACAGCCAGGAGGTAGCCTTCGGCTGTCATGTGCAGCCAGCGGACGCACATTTTCCAGTTACGCAATGGGTTGGTTTAGGCAAGCCCCTGGTAAACAGAGAGAATTCGTCGCTGCCATTCGGTGGAGTGGAGGTTATACATACTACACAGATAGTGTTAAGGGTCGTTTTACTA TTTCTCGGGACAATGCTAAAACCACAGTTTATCTCCAGATGAATAGTTTGAAGCCCGAGGACACAGCCGTATATTATTGTGCCGCCACTTATCTTTCTAGCGACTACTCCCGTTACGCCTTGCCACAACGTCCTCTTGACTATGACTATTGGGGACAAGGTACTCAAGTAACCGTTTCCTCATCTCTCTCCGGTCGATCCGACAACCACGGGTCTgctggaagagcgg

[0540] Using restriction sites NdeI and PacI (New England Biolabs), ScFv and VHH were cloned into pET49b+-based vectors for expression in *E. coli*. They also contain either a PelB or DSB leader sequence, allowing ScFv and VHH to be exported to the cell supernatant.

[0541] This design is based on engineered trastuzumab antibody D3. Cleavable domains are shown in bold, and cleavage sites are shown underlined.

[0542] Anti-EGFR VHH D17 (SEQ ID NO:9)

[0543]

[0544] ScFv as a therapeutic domain

[0545] Trastuzumab ScFv was prepared. Two types of trastuzumab ScFv were designed, as shown below.

[0546] Engineered trastuzumab ScFv 1(LH) (SEQ ID NO:94)

[0547]

[0548] Engineered trastuzumab ScFv(HL) (SEQ ID NO:95)

[0549]

[0550] This design is based on engineered trastuzumab antibody D3. Cleavable domains are shown in bold, and cleavage sites are shown underlined.

[0551] Trastuzumab D18 (SEQ ID NO:10)

[0552]

[0553]

[0554] A control ScFv for trastuzumab was also created. This design incorporates a longer linker rather than a cleavable domain. Different light chains were designed, as shown below:

[0555] Engineered trastuzumab ScFv control (LH) (SEQ ID NO:96)

[0556] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGG GLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGSGGGSGGGSHHHHHHHH

[0557] It was combined with heavy chain engineered trastuzumab ScFv (HL) to create an engineered trastuzumab ScFv control (long link).

[0558] Transformation

[0559] ScFv was expressed in the BL21(DE3) *E. coli* cell line. ScFv was induced in this cell line with isopropyl β-D-1-thiogalactoside (IPTG) when the cells were in logarithmic growth phase. Cells were grown at 30°C for 16 hours to express the protein. The bacteria were then harvested and the cells discarded. ScFv was purified via HIS tagging using nickel affinity chromatography and eluted with phosphate-buffered saline (PBS) containing a high concentration of imidazole. ScFv was dialyzed against PBS to remove trace amounts of imidazole, and purity was assessed by SDS-PAGE. The ScFv-FC construct was transformed into the EXP-CHO cell line as described above.

[0560] Purification, buffer exchange and SDS-PAGE analysis

[0561] All ScFv samples were purified on a nickel column (Cytiva) and prepared in 1X PBS pH 7.2. The PelB leader sequence yielded higher ScFv yields, and the LH or HL orientation of the ScFv was irrelevant. Engineered antibodies containing VHH and both ScFvs achieved good expression levels. The purification of ScFv-Fc samples was performed in the same manner as for monoclonal antibodies, as described above.

[0562] Sample quality control was performed by SDS-PAGE, as described above (see Examples 4 and 6-8). SDS-PAGE shows the expected expression and purity curves for all loaded reagents (Figure 30A).

[0563] Protease digestion of antibody variants

[0564] The above scheme (see Examples 4 and 6-8) was used to test whether the variants prepared as described above were easily digestible.

[0565] The study found that uPA cleaves antibody variants and generates F(ab) fragments in a manner similar to that described for other designs (Figure 30B).

[0566] The binding of D17 and D18 to their corresponding targets (assessed via ELISA)

[0567] Use an ELISA assay with Her2 to determine whether ScFv retains its binding properties. Use an ELISA assay with hEGFR to determine whether VHH retains its binding properties.

[0568] ELISA experiments were performed as described in Examples 4 and 6-8 above, but the antigen used for coating the plates was hHer2-Fc, and detection was performed using anti-HIS-HRP antibody instead of anti-Vk antibody after the addition of ScFv. Similarly, the LH or HL orientation in ScFv was irrelevant, and the introduction of protease sites did not affect expression. D18 and D19 were readily cleaved and were found to retain potency and binding to hEGFR and hHer2, respectively (as shown in Figures 30C, 30D, and Table 19).

[0569] Table 19 – IC50 of control and engineered reagents D7 and D18 50 value

[0570]

[0571] This example demonstrates that different therapeutic domains can be applied to this technique.

[0572] Example 9: In vivo validation

[0573] To verify in vivo that the protease-specific cleavage site does indeed lead to improved antibody permeability, trastuzumab and trastuzumab D3 were expressed at scale in an endotoxin-free manner and quality-controlled to ensure that they still bind to hHer2 at similar levels, but trastuzumab D3 was cleaved in the presence of uPA.

[0574] The human SVOK-3 ovarian cancer cell line was induced to grow and then transplanted into the right upper quadrant of Balb / C nude immunodeficient mice. 1×10 7 Cells were transplanted into PBS containing 1:1 matrix gel. When the tumor size reached 300 mm... 3 Dosing began at a specific time, with 5 mice in each group (10 mice in total) receiving either 5 mg / kg trastuzumab (Group 2 or G2) or trastuzumab D3 (Group 3 or G3). The study was terminated 24 hours later with a single dose administered. Tumor and serum samples were harvested and rapidly frozen for analysis.

[0575] Using ELISA, the binding of tumor samples to human Her2 was observed.

[0576] The plate setup was as follows: three hHer2-HIS plates diluted to 1 μg / ml, three BSA plates diluted to 1 μg / ml, and three PBS blank plates. 50 μl of each antigen was added to each well and diluted in PBS. The plates were coated overnight at 4°C. The plates were washed and then incubated at room temperature for 1 hour for blocking, followed by another wash.

[0577] Ten tumor samples were thawed on ice, and 150 μl of each sample was dissolved in 150 μl of PBS + 3% milk powder to prepare a 1:1 dilution. This was then serially diluted 1:2 in eight plates. 50 μl of each sample was added to each well, and the protein was incubated at room temperature for 1 hour. Plates were prepared with anti-Vk antibody and samples as described above. Color development was performed, the signal was quenched, and the absorbance of the plate was read at 450 nm.

[0578] Results and Conclusions

[0579] Combined analysis showed that tumors in group 3 mice had increased drug accumulation compared to tumors in group 2 mice (see Figure 31A). Data were pooled (see Figure 31b). The engineered drug concentration in tumors was more than twice that of non-engineered drugs, and this difference remained significant even 24 hours after a single 5 mg / kg dose.

[0580] This example demonstrates that engineered drugs can improve tumor penetration. It also shows that the level of endogenous cancer-specific proteases is sufficient to achieve effective cleavage of engineered drugs.

[0581] Example Embodiments

[0582] 1. An engineered pharmaceutical agent comprising:

[0583] i. Treatment of structural domains;

[0584] ii. Cuttable structural domains; and

[0585] iii. Stable structural domains

[0586] The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

[0587] 2. The pharmaceutical preparation according to Example 1, wherein the pharmaceutical preparation comprises amino acids.

[0588] 3. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the pharmaceutical agent is a protein, peptide, bicyclic peptide, tricyclic peptide or polypeptide.

[0589] 4. The pharmaceutical preparation according to any one of the foregoing embodiments, wherein the pharmaceutical preparation comprises a non-natural isomer or an amino acid.

[0590] 5. The agent according to any one of the foregoing embodiments, wherein the agent has a size that prevents it from penetrating the glomerular filtration barrier.

[0591] 6. The agent according to any one of the foregoing embodiments, wherein the agent has a size of at least 6 nanometers (nm).

[0592] 7. The agent according to any one of the foregoing embodiments, wherein the agent has a molecular weight of at least 40 kilodaltons (kDa), at least 50 kDa, at least 70 kDa, or at least 100 kDa.

[0593] 8. The agent according to any one of the foregoing embodiments, wherein the agent has a half-life of at least 10 hours in the biological system.

[0594] 9. The agent according to any one of the foregoing embodiments, wherein the half-life of the agent in the biological system is at least 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or at least 15 times longer than the half-life of the therapeutic domain in the biological system when it is present in isolation from a) the cleavable domain and the stable domain; or b) the stable domain.

[0595] 10. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain is a domain having a half-life of less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, or less than 5 hours when present in a biological system separately from the cleavable domain and the stable domain.

[0596] 11. The agent according to any one of the foregoing embodiments, wherein the ability of the agent to penetrate a solid tumor is reduced relative to the ability of the therapeutic domain to exist separately from a) the cleavable domain and the stable domain; or b) the stable domain.

[0597] 12. The agent according to any one of the foregoing embodiments, wherein the therapeutic domain has the ability to penetrate solid tumors when it exists separately from a) the cleavable domain and the stable domain; or b) the stable domain.

[0598] The ability relative to the stated agent has increased.

[0599] 13. The pharmaceutical preparation according to Example 12, wherein the increase is at least 5 times, at least 10 times, at least 15 times, or at least 20 times.

[0600] 14. The pharmaceutical preparation according to any one of the foregoing embodiments, wherein the tumor uptake level of the therapeutic domain when it is present separately from a) the cleavable domain and the stable domain; or b) the stable domain is at least 10% ID / g.

[0601] 15. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain is selected from any one or more of the following:

[0602] i. Antigen-binding domain;

[0603] ii. Fab area;

[0604] iii. F(ab')2 zone;

[0605] iv.scFv area;

[0606] v. Connect the scFv region in series;

[0607] vi. Domain-specific antibodies, preferably single-domain antibodies (sdAb);

[0608] vii. Nanobody;

[0609] viii. Monoclonal antibody;

[0610] ix. Polyclonal antibodies;

[0611] x. Double-chain antibody;

[0612] xi. Triple-chain antibody;

[0613] xii. Quadruplex antibody;

[0614] xiii. Five-chain antibodies;

[0615] xiv. Hexa-chain antibody;

[0616] xv. Antibody-drug conjugate;

[0617] xvi. Bispecific peptides, such as bispecific antibodies;

[0618] xvii. Multispecific peptides, such as multispecific antibodies;

[0619] xviii. Bicyclic peptides; and / or

[0620] xix. Tricyclic peptide;

[0621] xx. T cell receptor (TCR) domain;

[0622] xxi. Receptor domain;

[0623] xxi. Receptor mimicry domain;

[0624] xxiii. Cytokines;

[0625] xxiv. Hormones;

[0626] xxv. growth factors;

[0627] xxvi. peptides; and / or

[0628] xxvii. peptide derivatives

[0629] The preferred component is i. the antigen-binding domain;

[0630] ii. Fab area;

[0631] iii. F(ab')2 zone;

[0632] iv.scFv area;

[0633] v. Connect the scFv region in series;

[0634] vi.sdAb.

[0635] 16. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the pharmaceutical agent is:

[0636] i. Monoclonal antibodies;

[0637] ii. Polyclonal antibodies;

[0638] iii. Double-stranded antibody;

[0639] iv. Triple-chain antibodies;

[0640] v. Quadruple-chain antibody;

[0641] vi. Five-chain antibody;

[0642] vii. Six-chain antibody;

[0643] viii. Antibody-drug conjugates;

[0644] ix. Bispecific peptides, such as bispecific antibodies; and / or

[0645] x. Multispecific peptides, such as multispecific antibodies;

[0646] The preferred option is i. monoclonal antibody.

[0647] 17. The pharmaceutical preparation according to any one of the foregoing embodiments, wherein the therapeutic domain is monovalent, divalent, trivalent, or polyvalent.

[0648] 18. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain is a specific binding partner of an entity expressed by a target cell or target tissue.

[0649] 19. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain is a specific binding partner of an entity expressed by unwanted cells and / or associated with unwanted cells.

[0650] 20. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain is:

[0651] iT cell receptor (TCR) domain; or

[0652] ii. Receptor domain; or

[0653] iii. Receptor mimicry domain; or

[0654] iv. Cytokines; or

[0655] v. hormones; or

[0656] vi. growth factors; or

[0657] vii. peptide; or

[0658] viii. Peptide derivatives.

[0659] 21. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain binds to an antigen selected from the list comprising: Her2, VEGF, PD-1, PMSA, CEA, CTLA-4, or EGFR.

[0660] 22. The agent according to any one of the foregoing embodiments, wherein the cleavable structural domain does not interfere with the binding of the therapeutic structural domain to its binding partner.

[0661] 23. The agent according to any one of the foregoing embodiments, wherein the therapeutic domain binds to the binding partner when the cleavable domain has been cleaved or not.

[0662] 24. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain reduces or inhibits the proliferation of unwanted cells or the growth of tumors.

[0663] 25. The pharmaceutical preparation according to any one of the foregoing embodiments, wherein the therapeutic domain stimulates immune cells.

[0664] 26. The pharmaceutical preparation according to any one of the foregoing embodiments, wherein the therapeutic domain blocks immune cells.

[0665] 27. The pharmaceutical preparation according to Examples 24 to 26, wherein the tumor is a solid tumor.

[0666] 28. The pharmaceutical preparation according to Examples 24 to 27, wherein the tumor is an in vitro model.

[0667] 29. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the stable domain is based on a protein domain or a polymer domain.

[0668] 30. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the stable structural domain is:

[0669] i. Fc region, optionally wherein the Fc region is an IgG, IgE, IgM, IgD or IgA family Fc region or a bispecific Fc region; or

[0670] ii. Polyglycolic domain; or

[0671] iii. PAS-based structural domains; or

[0672] iv.XTENized structural domain; or

[0673] v.HES-encode the structural domain; or

[0674] vi. Lipid-modified domains; or

[0675] vii. Glycosylation domain; or

[0676] viii. Human serum albumin (HSA) protein or fragments thereof; or

[0677] ix.HSA binding protein; or

[0678] x. Proteins that bind to circulating blood cells; or

[0679] xi.Fab domain, wherein the Fab domain is directed to HSA or to an antigen that is the same as the antigen-binding domain;

[0680] Preferably, the Fc region is an i.Fc region, optionally wherein the Fc region is an IgG, IgE, IgM, IgD or IgA family Fc region or a bispecific Fc region.

[0681] 31. The pharmaceutical preparation according to any one of the foregoing embodiments, wherein the stable structural domain displays one or more N-glycosylation motifs.

[0682] 32. The pharmaceutical preparation according to any one of the foregoing embodiments, wherein the stable structural domain is selected from the sequences according to SEQ ID No: 27-29.

[0683] 33. The agent according to any one of the foregoing embodiments, wherein the selective cleavage of the cleavable domain enables the therapeutic domain to be released from the stable domain.

[0684] 34. The agent according to any one of the foregoing embodiments, wherein the cleavable domain comprises at least one cleavage site for an enzyme, optionally wherein the enzyme is a protease.

[0685] 35. The agent according to Example 34, wherein the protease is a protease expressed by unwanted cells or tumors and / or accumulated in the vicinity of unwanted cells or tumors.

[0686] 36. The pharmaceutical preparation according to Example 34 or 35, wherein the protease is a tumor-specific protease, such as urokinase plasminogen activator (uPA), novel aspartic protease A, ADAM10, FAP, proteolytic enzyme, podocyte protein, MT-SP1, cysteine ​​protease, serine protease, metalloproteinase, or a combination thereof.

[0687] 37. The agent according to any one of the foregoing embodiments, wherein the cleavable structural domain comprises one or more connector structural domains existing between the therapeutic structural domain and the stabilizing structural domain.

[0688] 38. The agent according to any one of the foregoing embodiments, wherein the cleavable domain overlaps with the therapeutic domain and / or the stable domain.

[0689] 39. The pharmaceutical preparation according to any one of the foregoing embodiments, wherein the length of the at least one cleavage site is 4 to 20 amino acids.

[0690] 40. The agent according to any one of the foregoing embodiments, wherein the cuttable structural domain is located in the hinge region, optionally wherein the cuttable structural domain is located in the upper hinge or the lower hinge.

[0691] 41. The agent according to any one of the foregoing embodiments, wherein the agent is a protein or polypeptide, the stable structural domain comprises an Fc region and a hinge region, and the cleavable structural domain is located at...

[0692] i. The N-terminus of the hinge region and after the therapeutic structural domain; or

[0693] ii. The C end of the hinge region and before the CH2 structural domain of the Fc region.

[0694] 42. The pharmaceutical preparation according to any one of Examples 1 to 41, which is used as a medicine, such as in a therapy.

[0695] 43. The pharmaceutical agent according to any one of Examples 1 to 41, which is used to treat or prevent tumors, and optionally to treat or prevent cancer.

[0696] 44. The agent according to Example 43, wherein the region near the unwanted cells or tumor contains high titers and / or high activity of tumor-specific proteases.

[0697] 45. The pharmaceutical preparation according to Example 43 or 44, wherein the tumor, optionally the cancer, is present in a subject, optionally a human subject.

[0698] 46. ​​The pharmaceutical agent according to any one of Examples 1 to 41, used for treating or preventing tumors, optionally used in a method of treating or preventing cancer, wherein the method comprises:

[0699] a) Obtain a sample of the tumor and / or the surrounding area of ​​the tumor;

[0700] b) Identify one or more proteases expressed by the tumor and / or present in the vicinity of the tumor;

[0701] c) Administering to the subject the agent according to any one of Examples 1-41, wherein the cleavable domain can be cleaved by one or more proteases determined to be expressed by the tumor and / or present in the vicinity of the tumor.

[0702] 47. A method for treating or preventing cancer, wherein the method comprises administering one or more agents according to any one of Examples 1-41.

[0703] 48. A pharmaceutical composition comprising a pharmaceutical agent according to any one of Examples 1-41 and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0704] 49. The medicament according to any one of Examples 1 to 41, for the prevention or treatment of a condition characterized by the presence of unwanted cells, optionally said condition being cancer.

[0705] 50. A method for improving the permeability of a therapeutic domain into a tissue or tumor, wherein the method includes engineering an agent comprising:

[0706] i. Treatment of structural domains;

[0707] ii. Cuttable structural domains; and

[0708] iii. Stable structural domains.

[0709] 51. Use of the pharmaceutical agent according to any one of Examples 1 to 41 in the manufacture of a medicament for treating or preventing tumors, optionally for treating or preventing cancer.

[0710] 52. A method for improving the efficacy of a pharmaceutical agent, the method comprising engineering the pharmaceutical agent to contain...

[0711] i. Treatment of structural domains;

[0712] ii. Cuttable structural domains; and

[0713] iii. Stable structural domains

[0714] The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

[0715] 53. A method for improving the specificity of a pharmaceutical agent, the method comprising engineering the pharmaceutical agent to contain...

[0716] i. Treatment of structural domains;

[0717] ii. Cuttable structural domains; and

[0718] iii. Stable structural domains

[0719] The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

[0720] 54. The method according to Example 53, wherein the improvement in drug specificity is manifested as a higher titer of the therapeutic domain in diseased tissue than in non-diseased tissue.

[0721] 55. The method according to any one of embodiments 50 to 54, wherein the method further comprises:

[0722] a) Apply the agent according to any one of Examples 1 to 41;

[0723] b) Cutting the cuttable domain at the tumor site; and

[0724] c) Separate the therapeutic domain from the stable domain.

[0725] 56. Use of an agent in improving the permeability of said therapeutic domain into a tumor, said agent comprising:

[0726] i. Treatment of structural domains;

[0727] ii. Cuttable structural domains; and

[0728] iii. Stable structural domains.

[0729] 57. Use of an agent in reducing the size of the agent at a tumor site, said agent comprising:

[0730] i. Treatment of structural domains;

[0731] ii. Cuttable structural domains; and

[0732] iii. Stable structural domains.

[0733] 58. Use of the pharmaceutical agent in improving the efficacy of the pharmaceutical agent, the pharmaceutical agent comprising:

[0734] i. Treatment of structural domains;

[0735] ii. Cuttable structural domains; and

[0736] iii. Stable structural domains

[0737] The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

[0738] 59. Use of a pharmaceutical agent in improving the specificity of the pharmaceutical agent, the pharmaceutical agent comprising:

[0739] i. Treatment of structural domains;

[0740] ii. Cuttable structural domains; and

[0741] iii. Stable structural domains

[0742] The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

[0743] 60. Use of cleavable domains in improving the permeability of therapeutic domains into tumors, said use including engineering agents to include:

[0744] i. Treatment of structural domains;

[0745] ii. Cuttable structural domains; and

[0746] iii. Stable structural domains

[0747] The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

[0748] 61. Use of cleavable domains in reducing agent size at tumor sites, said use including engineering agents to include:

[0749] i. Treatment of structural domains;

[0750] ii. Cuttable structural domains; and

[0751] iii. Stable structural domains

[0752] The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

[0753] 62. Use of cleavable structural domains in improving the efficacy of pharmaceutical agents, said use including engineering pharmaceutical agents to include:

[0754] i. Treatment of structural domains;

[0755] ii. Cuttable structural domains; and

[0756] iii. Stable structural domains

[0757] The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

[0758] 63. Use of cleavable domains in improving the specificity of pharmaceutical agents, said use including engineering pharmaceutical agents to include:

[0759] i. Treatment of structural domains;

[0760] ii. Cuttable structural domains; and

[0761] iii. Stable structural domains

[0762] The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

[0763] 64. The use according to Example 59 or 63, wherein the improvement in drug specificity is manifested as a higher titer of the therapeutic domain in diseased tissue than in non-diseased tissue.

[0764] 65. The use according to Examples 57 to 64, wherein the agent is the agent according to any one of Examples 1 to 41.

[0765] 66. The pharmaceutical preparation according to any one of Examples 1 to 41, which is used for diagnosing diseases or ailments.

[0766] 67. The pharmaceutical agent according to any one of Examples 1 to 41, for use as described in Example 66, wherein the use includes:

[0767] i. Administer the drug to the subject;

[0768] ii. Cutting the cuttable domain at the tumor site; and

[0769] iii. Separate the therapeutic domain from the stable domain;

[0770] iv. Increase the accessibility of the therapeutic domain to cell binding compared to the case where the cutting and separation according to steps (ii) and (iii) does not occur.

[0771] 68. The agent according to any one of the foregoing embodiments, wherein the selective cleavage of the cleavable domain enables the therapeutic domain to be released from the stable domain.

[0772] 69. The agent according to any one of the foregoing embodiments, wherein the selective cleavage of the cleavable domain enables the therapeutic domain to be released at or near the cell surface of the unwanted cell.

[0773] 70. The agent according to any one of the foregoing embodiments, wherein the cleaved therapeutic domain has a molecular weight of not more than 50 kDa, not more than 40 kDa, not more than 30 kDa, not more than 25 kDa, not more than 20 kDa, not more than 20 kDa, and not more than 15 kDa.

[0774] 71. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the pharmaceutical agent or the therapeutic domain is a trastuzumab-derived fragment, a bevacizumab-derived fragment, a pembrolizumab-derived fragment, a cetuximab-derived fragment, an M5A-derived fragment, or an ipilimumab-derived fragment.

[0775] 72. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain is at a half-maximal inhibitory concentration (IC50) of 0.2 to 1.4 nM, such as 0.22 to 0.36 nM. 50 Combined with its target.

[0776] 73. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain has a dissociation constant (K0) of 3 to 4.5 nanomolars (nM). D ).

[0777] 74. The agent according to any one of the foregoing embodiments, wherein the cleavable structural domain does not interfere with the thermal stability of the agent.

[0778] 75. The agent according to any one of the foregoing embodiments, wherein the agent has a melting temperature of 65°C to 75°C, such as 69°C to 71°C (T m ).

[0779] 76. The agent according to any one of the foregoing embodiments, wherein the agent has an aggregation temperature (Tg) of 65°C to 80°C, such as 66°C to 76°C. agg ).

[0780] 77. The agent according to any one of the foregoing embodiments, wherein the cleavable domain does not interfere with the binding of the stable domain to the Fcγ receptor, optionally wherein the Fcγ receptor is Fcγ receptor Ia and / or Fcγ receptor IIa.

Claims

1. An engineered pharmaceutical agent comprising: i. Treatment of structural domains; ii. Cuttable structural domains; and iii. Stable structural domains The slicable structural domain is located between the therapeutic structural domain and the stable structural domain; The engineered reagent is used as a drug.

2. The agent according to claim 1, wherein the agent has a size that prevents it from penetrating the glomerular filtration barrier.

3. The pharmaceutical agent according to claim 1 or 2, wherein the pharmaceutical agent comprises: i. A size of at least 6 nanometers (nm); and / or ii. Molecular weight of at least 40 kilodaltons (kDa), at least 50 kDa, at least 70 kDa, or at least 100 kDa.

4. The pharmaceutical agent according to any one of the preceding claims, wherein the half-life of the pharmaceutical agent in the biological system is: i. at least 10 hours; and / or ii. The half-life of the therapeutic domain in the biological system when it is present separately from a) the cleavable domain and the stable domain; or b) the stable domain is at least 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or at least 15 times longer.

5. The pharmaceutical agent according to any one of the preceding claims, wherein the therapeutic domain is a domain having a half-life of less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, or less than 5 hours when present in a biological system separately from the cleavable domain and the stable domain.

6. The pharmaceutical agent according to any one of the preceding claims, wherein: i. The ability of the agent to penetrate solid tumors is reduced relative to the ability of the therapeutic domain to exist separately from a) the cleavable domain and the stable domain; or b) the stable domain; and / or ii. The ability of the therapeutic domain to penetrate solid tumors relative to the ability of the agent is increased when it exists separately from a) the cleavable domain and the stable domain; or b) the stable domain, optionally wherein the increase is at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold.

7. The pharmaceutical agent according to any one of the preceding claims, wherein the tumor uptake level of the therapeutic domain, when present separately from a) the cleavable domain and the stable domain; or b) the stable domain, is at least 10% ID / g.

8. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the therapeutic domain is selected from any one or more of the following: i. Antigen-binding domain; ii. Fab area; iii. F(ab')2 zone; iv.scFv area; v. Connect the scFv region in series; vi. Domain-specific antibodies, preferably single-domain antibodies (sdAb); vii. Nanobody; viii. Double-chain antibody; ix. Monoclonal antibodies; x. Polyclonal antibodies; xi. Triple-chain antibody; xii. Quadruplex antibody; xiii. Five-chain antibodies; xiv. Hexa-chain antibody; xv. Antibody-drug conjugate; xvi. Bispecific peptides, such as bispecific antibodies; xvii. Multispecific peptides, such as multispecific antibodies; xviii. Bicyclic peptides; and / or xix. Tricyclic peptide; xx. T cell receptor (TCR) domain; xxi. Receptor domain; xxii. Receptor mimicry domain; xxiii. Cytokines; xxiv. Hormones; xxv. growth factors; xxvi. peptides; and / or xxvii. peptide derivatives, The preferred component is i. the antigen-binding domain; ii. Fab area; iii. F(ab')2 zone; iv.scFv area; v. Connect the scFv region in series; vi.sdAb.

9. The pharmaceutical agent according to any one of the preceding claims, wherein the cleaved therapeutic domain has a molecular weight of not more than 50 kDa, not more than 40 kDa, not more than 30 kDa, not more than 25 kDa, not more than 20 kDa, not more than 20 kDa, and not more than 15 kDa.

10. The pharmaceutical agent according to any one of the foregoing embodiments, wherein the pharmaceutical agent is: i. Monoclonal antibodies; ii. Polyclonal antibodies; iii. Double-stranded antibody; iv. Triple-chain antibodies; v. Quadruple-chain antibody; vi. Five-chain antibody; vii. Six-chain antibody; viii. Antibody-drug conjugates; ix. Bispecific peptides, such as bispecific antibodies; and / or x. Multispecific peptides, such as multispecific antibodies, The preferred option is i. monoclonal antibody.

11. The pharmaceutical agent according to any one of the preceding claims, wherein the therapeutic domain is a specific binding partner of an entity, the entity: i. Expressed by target cells or target tissues; and / or ii. Expressed by unwanted cells and / or associated with unwanted cells.

12. The pharmaceutical agent according to any one of the preceding claims, wherein the therapeutic domain binds to an antigen selected from the list comprising: Her2, VEGF, PD-1, PMSA, CEA, CTLA-4, or EGFR.

13. The pharmaceutical agent according to any one of the preceding claims, wherein the pharmaceutical agent or the therapeutic domain is a trastuzumab-derived fragment, a bevacizumab-derived fragment, a pembrolizumab-derived fragment, a cetuximab-derived fragment, an M5A-derived fragment, or an ipilimumab-derived fragment.

14. The agent according to any one of the preceding claims, wherein the cleavable domain does not interfere with the binding of the therapeutic domain to its binding partner.

15. The pharmaceutical agent according to any one of the preceding claims, wherein the therapeutic domain: i. When the cutable structural domain has been cut or not cut, it is coupled with the coupling partner; and / or ii. Reduce or inhibit the proliferation of unwanted cells or the growth of tumors.

16. The pharmaceutical agent according to any one of the preceding claims, wherein the therapeutic domain is at a concentration of 0.2 to 1.4 nM, such as 0.22 to 0.36 nM at an IC50 concentration of 0.2 to 1.4 nM. 50 Combined with its target.

17. The pharmaceutical agent according to any one of the preceding claims, wherein the therapeutic domain has a K+ of 3 to 4.5 nM. D .

18. The pharmaceutical agent according to any one of the preceding claims, wherein the therapeutic domain: i. Stimulating immune cells; and / or ii. Block immune cells.

19. The pharmaceutical agent according to any one of the preceding claims, wherein the stable structural domain is: i. Fc region, optionally wherein the Fc region is an IgG, IgE, IgM, IgD or IgA family Fc region or a bispecific Fc region; or ii. Polyglycolic domain; or iii. PAS-based structural domains; or iv.XTENized structural domain; or v.HES-encode the structural domain; or vi. Lipid-modified domains; or vii. Glycosylation domain; or viii. Human serum albumin (HSA) protein or fragments thereof; or ix.HSA binding protein; or x. Proteins that bind to circulating blood cells; or xi. Fab domain, optionally wherein the Fab domain is for HSA or for an antigen identical to the antigen-binding domain. Preferably, the Fc region is an i.Fc region, optionally wherein the Fc region is an IgG, IgE, IgM, IgD or IgA family Fc region or a bispecific Fc region.

20. The pharmaceutical agent according to any one of the preceding claims, wherein the cleavable domain comprises a cleavage site for an enzyme, optionally wherein the enzyme is a protease. Optionally, the protease is a protease expressed by unwanted cells or tumors and / or accumulated in the vicinity of unwanted cells or tumors, and further optionally, the protease is a tumor-specific protease, such as urokinase plasminogen activator (upA), novel aspartic protease A, ADAM10, fibroblast activating protein (FAP), proteolytic enzyme, podocyte protein, MT-SP1, cysteine ​​protease, serine protease, metalloproteinase, or a combination thereof.

21. The agent according to any one of the preceding claims, wherein the cleavable structural domain comprises one or more connector structural domains existing between the therapeutic structural domain and the stabilizing structural domain.

22. The pharmaceutical agent according to any one of the preceding claims, wherein the cleavable domain overlaps with the therapeutic domain and / or the stable domain.

23. The agent according to any one of the preceding claims, wherein the cuttable structural domain is located in the hinge region, optionally wherein the cuttable structural domain is located in the upper hinge or the lower hinge, preferably in the upper hinge.

24. The pharmaceutical agent according to any one of the preceding claims, wherein, in the case that the pharmaceutical agent is a protein or polypeptide, the stable structural domain comprises an Fc region and a hinge region, and the cleavable structural domain is located at: i. The N-terminus of the hinge region and after the therapeutic structural domain; or ii. The C end of the hinge region and before the CH2 structural domain of the Fc region.

25. The pharmaceutical agent according to any one of claims 1 to 24, for treating or preventing tumors, optionally for treating or preventing cancer, optionally wherein the region near the unwanted cells or tumor contains high titers and / or high activity of tumor-specific proteases, and further optionally wherein the tumor, optionally the cancer, is present in a subject, optionally a human subject.

26. The pharmaceutical agent according to any one of claims 1 to 24, used for treating or preventing tumors, optionally used for treating or preventing cancer, wherein the method comprises: a) Obtain a sample of the tumor and / or the surrounding area of ​​the tumor; b) Identify one or more proteases expressed by the tumor and / or present in the vicinity of the tumor; c) Administering the agent according to any one of claims 1 to 24 to the subject, wherein the cleavable domain can be cleaved by one or more proteases determined to be expressed by the tumor and / or present in the vicinity of the tumor.

27. A method for treating or preventing cancer, wherein the method comprises administering one or more agents according to any one of claims 1 to 24.

28. A pharmaceutical composition comprising a pharmaceutical agent according to any one of claims 1 to 24, and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

29. The medicament according to any one of claims 1 to 24, for the prevention or treatment of a condition characterized by the presence of unwanted cells, optionally said condition being cancer.

30. A method for improving the permeability of a therapeutic domain into a tissue or tumor, wherein the method includes engineering an agent comprising: i) Treating the structural domain; ii) Cuttable structural domains; and iii) Stable structural domains; The severable structural domain is located between the therapeutic structural domain and the stable structural domain.

31. Use of the pharmaceutical agent according to any one of claims 1 to 24 in the manufacture of a medicament for treating or preventing tumors, optionally for treating or preventing cancer.

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