Multiple specific protein prodrug constructs

Abstract

The present invention relates to a protein fusion construct comprising a protein prodrug construct, for example, a complement 3- and pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein (e.g., A2M) and one or more drugs, which functions as a protease-activatable prodrug. The present invention further relates to the release mechanism from the CPAMD protein and the development of multispecific drugs such as bispecificity.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and benefits of European Patent Application Publication No. 23 l 80912.0, filed on 22 June 2023, and the contents of the said patent document are thus incorporated in their entirety by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, thereby incorporating the entirety of it by reference. The XML file was created on June 20, 2024, named P52261WO-Sequence_listing.xml, and has a size of 487 bytes.

[0003] The present invention relates to a proteinaceous fusion construct comprising a proteinaceous prodrug construct, for example, a complement 3 and pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein (e.g., alpha-2-macroglobulin (A2M)), and one or more drugs, which functions as a protease-activatable prodrug. The present invention further relates to the release mechanism from CPAMD proteins and the development of multispecific drugs such as bispecificity. [Background technology]

[0004] When biopharmaceuticals are administered to patients in an active form from the outset, they can exert their biological effects in both diseased and healthy tissues. Drug effects in healthy tissues can be detrimental to the patient's health, quality of life, and / or therapeutic efficacy. One strategy to minimize these side effects is to induce the drug into a prodrug form, which is initially inactive and becomes active in the disease environment.

[0005] Proteases are enzymes that catalyze the hydrolysis of peptide bonds in other proteins. More than 600 human proteases are known, and the vast majority are tightly regulated under normal circumstances. In many diseases, specific proteases become dysregulated, and their activity is increased compared to a healthy state. Many protease-activated prodrug technologies have been developed for biopharmaceuticals, most of which are antibody-based. Some technologies use a masking moiety that blocks the antigen-binding region (paratope) of an antibody, preventing the antibody from binding to its cognitive epitope on a target antigen, and thus inactivating the antibody. The masking moiety is bound to the antibody by a linker that incorporates a protease-cleavable site. Cleavage of the linker by the protease separates the antibody from the masking moiety, releasing the paratope and restoring the antibody's activity. The masking portion may be designed to specifically bind to the antibody paratope (e.g., using a phage display-derived peptide, as in CytomX's Probody technology) or it may sterically surround the paratope to the extent that it sufficiently isolates it without specific interaction (e.g., using a long, bulky peptide, as in Amunix's XPAT technology). Another approach is to incorporate inactive VH and VL domains bound to the antibody by a protease-sensitive linker, thereby preventing the functional VH / VL domains from pairing in the prodrug (e.g., Maverick's COBRA technology). Cleavage of the linker removes the inactive domains, allowing for precise VH / VL pairing in the activated prodrug.

[0006] Each of these diverse prodrug technologies has its advantages and disadvantages. For example, CytomX's Probody minimizes the use of non-human and potentially immunogenic sequences, but it is not modular and requires identification of affine masking moieties for all antibodies incorporated into its platform. Amunix's XPAT platform does not require specific mask / antibody interactions and further exhibits differences in the hemodynamic half-lives before and after prodrug activation, which is achieved by using long non-human peptides. Therefore, a new prodrug technology that combines the key advantages of multiple technologies is needed and is expected to represent a significant advance in this field.

[0007] The concurrently pending application PCT / EP2023 / 052280 describes a novel technique for constructing prodrugs using CPAMD and antibodies or other biopharmaceuticals. Such CPAMD-based prodrugs would ultimately be large (>700kDa), monospecific, and tetravalent.

[0008] In cancer treatment, the penetration of therapeutic molecules into tumors can be limited by their size. Furthermore, some therapeutic approaches, such as altering the targeting of T cells (e.g., using the bispecific T cell engager akaBiTE), require the use of bispecific antibodies, which must also bind monovalently to T cells. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Therefore, there is a need for the development of prodrugs that can be released in smaller sizes, and / or for the released drug to be bispecific. [Means for solving the problem]

[0010] In particular, the protein prodrug constructs (e.g., protein fusion constructs) described herein enable specific drug delivery and controllable activity. This can be achieved by the protein prodrug construct undergoing a structural change that allows control of the activity of one or more drugs contained therein (e.g., a first drug such as an antibody). In the native (uncleaved) state of the protein prodrug construct, one or more drugs (e.g., the first drug / antibody) are not exposed and are therefore inactive. In the active (cleaved) state of the protein prodrug construct, the drugs (e.g., the first drug / antibody) are exposed and can interact with their targets. For example, the first drug is placed within the receptor-binding domain (RBD) of a CPAMD protein (e.g., A2M) contained in the protein prodrug construct of the present invention. The CPAMD protein (e.g., A2M) contains a bait region containing the first protease cleavage site. The structural change from the natural state to the active state is triggered by the cleavage of the first protease cleavage site in the bait region.

[0011] Typically, the protein prodrug constructs described herein include a second drug (e.g., a second antibody). The second drug can be fused to the C-terminus of the RBD. At the C-terminus, the second drug (e.g., a second antibody or other antigen-targeting moiety) is reachable in both its native and active states and can be used to direct the protein prodrug construct to a specific tissue, a specific cell type, and / or a specific receptor.

[0012] Specific drug delivery and controllable activity are provided by a release mechanism for the drug(s) (e.g., first and second antibodies) from a larger prodrug construct. For example, RBD contains a second protease cleavage site at its N-terminus. The release mechanism is enabled by cleavage of the second protease cleavage site. Typically, RBD remains bound to the CPAMD protein (e.g., A2M) via non-covalent interactions even after the second protease cleavage site is cleaved. Following proteolytic cleavage of the first protease cleavage site, the RBD containing the drug(s) (e.g., first and second antibodies) is released from the prodrug construct.

[0013] The cleavage site(s) contained within the protein prodrug construct can be modified to control where the drug is exposed. Depending on the cleavage site, the drug is exposed only at the location where a protease recognizing that cleavage site is present. When a specific protease is present and cleaves the first cleavage site, the structure of the protein prodrug construct changes from "native" to "active". Thus, the present invention provides protein prodrug constructs (e.g., protein fusion constructs) whose activity and specificity can be controlled and directed to specific regions (e.g., specific tissues) of an object requiring treatment with such constructs.

[0014] In this disclosure, the inventors describe a representative CPAMD-based prodrug in which a second protease cleavage site is incorporated into the sequence of the CPAMD protein (A2M), causing the portion responsible for the first drug after activation to be released from the rest of the CPAMD protein, resulting in both a smaller activated first drug and monovalent specificity (Examples 12 and 13). The inventors further describe creating a bispecific prodrug by simultaneously fusing the CPAMD protein (A2M) with a first drug (first antibody) and a second drug (second antibody), where the first drug is shielded by the CPAMD protein before release (Example 14). The inventors also demonstrate the release of a smaller activated multispecific (i.e., bispecific) RBD containing the first and second drugs, i.e., the first and second antibodies (Example 14).

[0015] Specifically, in one embodiment, the present invention relates to a proteinaceous prodrug construct comprising a complement 3 and pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein such as A2M, comprising a bait region comprising a first protease cleavage site and an RBD comprising a second protease cleavage site at the N-terminus of the receptor-binding domain, wherein the first drug is located inside the RBD and the second drug is fused to the C-terminus of the RBD, the CPAMD protein shields the first drug and the second drug is reachable, and the CPAMD protein can change its structure by proteolytic cleavage of the first protease cleavage site, releasing the RBD and thereby making the first drug reachable.

[0016] The inventors found that RBD remains bound to the CPAMD protein by non-covalent interactions even after the second protease cleavage site is cleaved. In other embodiments, the present invention relates to a protein prodrug construct comprising a complement 3 and pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein, such as A2M, comprising a receptor-binding domain (RBD) containing a bait region comprising a first protease cleavage site and a first drug located within the RBD and a second drug fused to the C-terminus of the RBD, wherein the CPAMD protein shields the first drug and makes the second drug reachable, the RBD is bound to the CPAMD protein by non-covalent interactions, and the CPAMD protein can change its structure by proteolytic cleavage of the first protease cleavage site, thereby releasing the RBD and making the first drug reachable.

[0017] Nucleic acids encoding the proteinaceous prodrugs described herein, as well as plasmid-like vectors containing nucleic acids, are also provided. These nucleic acids and vectors are therefore contained in host cells, and thus another aspect relates to host cells containing the above nucleic acids or vectors. Methods for the preparation, therapeutic methods and therapeutic uses of some of these aspects are also provided.

[0018] The following drawings illustrate the present invention using a protein prodrug construct containing alpha-2-macroglobulin (A2M) as the CPAMD protein. Those skilled in the art of protein prodrug design will recognize that other CPAMD proteins can be substituted for A2M. [Brief explanation of the drawing]

[0019] [Figure 1A]Figure 1A shows a schematic overview of a proteinaceous prodrug construct (1), for example, a CPAMD protein (2) fused to one or more drugs (e.g., one or more nanobodies / bio pharmaceutical moieties) (3) placed inside or in the vicinity of the RBD domain of the CPAMD protein, for example, A2M. One or more drugs (3) are inaccessible when the bait region of the CPAMD protein is not proteolytically cleaved (inactive or "native" structure I). One or more drugs (3) are accessible when the bait region is cleaved by a protease (4) (active structure II). When the protease (4) cleaves the "bait region", the protease (4) is trapped inside the proteinaceous prodrug construct (1). [Figure 1B] Figure 1B shows a schematic overview of different fusion strategies of a CPAMD protein (e.g., A2M) and a drug. [Figure 2A] Figure 2 shows native PAGE (A) and SDS - PAGE (B) analyses of wild - type A2M and fusion constructs of A2M with antibody scFvs derived from atezolizumab, ipilimumab, and nivolumab, as indicated. Prior to analysis, the samples were treated with methylamine (MA) or thermolysin as indicated. (C) is a schematic diagram of the domain organization of the A2M - antibody construct, showing the sizes of the products generated by thiolester autolysis and bait region cleavage. [Figure 2B] The same as above. [Figure 2C] The same as above. [Figure 3A]Figure 3 shows the structure-dependence of antigen binding by A2M-antibodies measured by the biolayer interferometry. (A) shows the interaction between A2M-atezolizumab (purified by one round of depletion using PD-L1 resin, see Example 4) and immobilized PD-L1-hFc. Control and methylalanine-treated A2M-atezolizumab show an approximately 149-fold difference in their effective concentration calculated from the fitting kobs value for their association. (B) shows the interaction between A2M-EgA1 (purified by two rounds of depletion using LRP1 resin, see Example 4) and immobilized EGFR-hFc. Control and methylalanine-treated or thermolysin-treated samples show an approximately 63-fold difference in their effective concentration. (C) shows the interaction between A2M-ipilimumab (purified by three rounds of depletion using LRP1 resin) and immobilized CTLA-4-hFc. (D) shows the interaction between A2M-nivolumab (purified by three rounds of depletion using LRP1 resin) and immobilized PD-1-hFc. (E) shows the interaction between A2M-KN035 (not concentrated for native A2M) and immobilized PD-L1-hFc. (F) shows the interaction between A2M-urelumab (purified by three rounds of depletion using LRP1 resin) and immobilized 4-1BB-hFc. (G) shows the interaction between A2M-foralumab (not concentrated for native A2M) and immobilized CD3γε-hFc. (H) shows the interaction between A2M-muromonab (not concentrated) and immobilized CD3γε-hFc. In panels G and H, the +thermolysin sensorgrams have signals from the biosensor associated with thermolysin that are only subtracted due to the low-intensity response. (I) shows the interaction between A2M-adalimumab (not concentrated) and immobilized TNFα. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above. [Figure 4A] Figure 4 shows the enrichment of native A2M-antibodies using affinity depletion. (A) A2M-atezolizumab was depleted using a resin coated with its cognitive antigen, PD-L1. One round of depletion was performed. Next, the antigen binding of untreated samples before (left) and after (right) depletion was compared using biolayer interferometry. (B-D) A2M-nivolumab, A2M-ipilimumab, and A2M-urerumab were depleted using an LRP1-coated resin. Three rounds of depletion were performed for each A2M-antibody, and then its antigen binding before and after depletion was compared using biolayer interferometry. (E) A2M-ipilimumab was depleted by three rounds using a protein L resin, and its antigen binding before and after was compared using biolayer interferometry. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 5] Figure 5 shows immune checkpoint blockade by A2M-atezolizumab in a cellular bioassay for PD-1 / PD-L1 blockade. PD-1+ Jarcut T cells possessing the NFAT-driven luciferase gene to report NFκB signaling were co-cultured with PD-L1+ CHO-K1 cells expressing a TCR agonist in the presence of a dilution series of A2M-atezolizumab in its native and methylamine-treated structures, or a dilution series of atezolizumab scFv fused to the human Fc region. Luminescence responses are shown after subtracting background from control cells and normalizing the subsequent response to the maximal response. EC50 curves were fitted using linear regression, and the maximal response and EC50 values ​​from the fitting are shown for each antibody. [Figure 6A]Figure 6 shows the structure and functionality of Tabula Rasa A2M, including a bait region that cannot be cleaved by proteases. (A) shows the wild-type, Tabula Rasa (TR), and TR K704 bait regions. Basic residues (i.e., cleavage sites for trypsin or LysC) are highlighted. (B) shows pore-restricted native PAGEs of A2M incorporating three given bait region sequences. All constructs initially showed slow electrophoretic mobility, characteristic of the native structure of A2M; upon methylamine aminolysis or bait region cleavage, A2M disintegrates and shows faster electrophoretic mobility. Both wild-type A2M and A2M TR K704 disintegrated with trypsin, and only A2M TR K704 disintegrated with LysC; A2M TR did not disintegrate with either protease. (C) shows reduced SDS-PAGEs of the same A2M samples as in panel B. The thiol ester-dependent thermal fragmentation bands (TE120 and TE60) disappeared upon methylamine treatment. Cleavage of the bait region of A2M yields approximately 85 and 95 N- and C-terminal fragment bands; the C-terminal fragment further forms a high MW multimer product through thiol ester-mediated conjugation. If the bait region is not cleavable by trypsin or LysC, A2M can be cleaved outside the bait region without any thiol ester activation. A2M TR K704 forms a strong approximately 250 kDa band upon proteolytic activation of the bait region lysine residue due to thiol ester-mediated conjugation. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 7A]Figure 7 shows the incorporation of MMP2 substrate sites into Tabula Rasa A2M. (A) shows the bait region sequences for wild-type A2M, TR A2M, and four TR bait regions, each incorporating a different MMP2 substrate sequence (A21A, B74, C9, and S1). The MMP2 recognition sequence is highlighted in each sequence; cleavage occurs at the N-terminus of the hydrophobic residue in bold. (B) A2M with these six bait regions was digested by MMP2, and cleavage was evaluated by nine other human proteases using SDS-PAGE. Proteases that cleave the bait region are indicated by + for complete cleavage and (+) for partial cleavage (compared to wild-type A2M). The TR bait region was not cleaved by any of the tested proteases, and each MMP2 substrate was cleaved by all tested MMPs. The TR S1 bait region was not cleaved by proteases other than MMPs, indicating increased inhibitory selectivity compared to the wild-type bait region. (C~D) shows pore-restricted native PAGE and reduced SDS-PAGE for six A2M structures with and without MMP2 cleavage. All constructs are similar bait regions cleaved by MMP2, resulting in structural breakdown and the appearance of high-MW multimer products in SDS-PAGE, with the exception of A2M TR. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 8A]Figure 8 shows the optimization of the production and inhibitory capacity of A2M TR S1. (A) Several modifications of the MMP2 substrate bait region, Tabula Rasa S1, were tested for their ability to improve the formation of native A2M and its inhibitory capacity with respect to MMP2. TR S1 QRT4 reintroduces a quarter of the wild-type bait region. Two different S1 positions (cleavage at position 710 or 703) were tested at TRΔ7, which shortens the TR bait region by 7 residues. (B) shows the pore-restricted native PAGE of A2M with the indicated bait region. A2M TR S1 is expressed in a considerable amount of non-native A2M. This non-native A2M could be removed by depletion using LRP1 conjugate resin. Instead, native content was improved at TRΔ7 and TR QRT4. (C) The ability of the indicated A2M to inhibit MMP2 digestion of DQ gelatin was determined. The fitted curve calculated from experimental data points by linear regression is shown as a dotted line. Error bars indicate standard; n=3. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A]Figure 9 shows the A2M antibody incorporating the manipulated bait region. (A) shows the bait region sequences for the wild-type A2M bait region, the shortened MMP2 substrate bait region "TRΔ7 S1 I703" described in Example 6, and the additional manipulated bait region "TRΔ7 S1 I703 P704". (B) Pore-restricted native PAGE and (C) Reduced SDS-PAGE are shown for wild-type A2M, A2M-atezolizumab with the wild-type bait region, and A2M-atezolizumab with the TRΔ7 S1 I703 bait region. A2M was analyzed untreated, methylalanine-treated, or treated with MMP2 to A2M in a 0.5:1 or 4:1 molar ratio as indicated. (D) Using biolayer interferometry, PD-L1 binding by A2M-atezolizumab was evaluated before and after MMP2 cleavage using three bait regions shown in Panel A (left corner above wild-type, right corner above TR△7 S1 I703, and below TR△7 S1 I703 P704). A biosensor related only to MMP2 without A2M-atezolizumab is included to account for this background binding. A2M-atezolizumab with wild-type bait regions was further cleaved using thermolysin for comparison. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10A]Figure 10 shows: (A) Reduced SDS-PAGE analysis of purified A2M-PD1. A2M-PD1 is expressed using the same protocol as wild-type A2M or A2M-antibodies and purified to high purity. Due to the formation of internal thiol esters in A2M-PD1, thermally induced fragmentation occurs at the thiol ester moiety under denaturing conditions, resulting in N-terminated and C-terminated product bands. (B) shows A2M-PD1 binding to immobilized PD-L1, evaluated by biolayer interferometry. PD-L1 binding by A2M-PD1 is shown without treatment that alters its structure, or after methylamine- or thermolysin treatment that disrupts its structure. A reference biosensor with thermolysin added but without A2M-PD1 was included to illustrate the nonspecific binding of thermolysin to the biosensor surface and was subtracted from the A2M-PD1 + thermolysin sensorgram. A2M-PD1 after LRP1 depletion was also included, both untreated and after methylamine treatment. [Figure 10B] Same as above. [Figure 11A] Figure 11 shows (A) the reduced SDS-PAGE analysis of purified A2M-IL2. A2M-IL2 is expressed using the same protocol as wild-type A2M or A2M-antibody and purified to high purity. Due to the formation of internal thiol esters in A2M-IL2, thermally induced fragmentation occurs at the thiol ester site under denaturing conditions, resulting in N-terminated and C-terminated product bands. (B) shows the binding of A2M-IL2 to immobilized IL-2Rα as evaluated by biolayer interferometry. This shows IL-2Rα binding by A2M-IL2 without treatment that alters its structure, or after methylamine- or thermolysin treatment that disrupts its structure. A2M-IL2 after three rounds of LRP1 depletion was evaluated similarly. [Figure 11B] Same as above. [Figure 12A-B]Figure 12 shows (A) the interaction between 5nM A2M-fusion-EgA1, measured using biolayer interferometry with immobilized human EGFR before and after methylalanine treatment, during 1 hour of association and 1 hour of dissociation. (B) shows the interaction between 5nM A2M-iRBD-EgA1, measured using biolayer interferometry with immobilized human EGFR before and after methylalanine or thermolysin treatment, during 1 hour of association and 1 hour of dissociation. (C-E) shows the interaction between 5nM A2M-miRBD-EgA1, A2M-miRBD-KN035, and A2M-miRBD-atezolizumab, measured using biolayer interferometry with immobilized EGFR or PD-L1 before and after methylalanine treatment, during 1 hour of association and 1 hour of dissociation (or 2 hours of association and 10 minutes of dissociation in the case of A2M-miRBD-atezolizumab). (F) shows the interaction between 10 nM A2M-tRBD-EgA1, measured during 1 hour of association and dissociation using immobilized EGFR before and after methylalanine treatment. [Figure 12C-D] Same as above. [Figure 12E-F] Same as above. [Figure 13]Figure 13 shows the RBD domain of A2M (residues 1335-1474 of SEQ ID NO: 1) and highlights four proposed sites that can be used for drug insertion to achieve structure-dependent binding. These sites, when indicated by the ciRBD, iRBD, miRBD, and tRBD fusion approaches, are residues 1392-1404 or 1391-1405 (loop 2), as well as residues 1368-1379 (loop 1), 1420-1426 (loop 3), and 1450-1457 (loop 4), all of which are flexible inter-beta linkers spatially adjacent to 1392-1404 and oriented in the same direction on the RBD domain. In contrast, residue 1468 dictates the position of the drug to be inserted in the A2M-fusion-EgA1 construct, and structure-dependent binding is not achieved, indicating that this opposite side of the RBD domain is unsuitable for achieving structure-dependent binding. The RBD domain structure (from PDB accession number 7VON) is shown as a schematic diagram, with the Cα atom of the indicated residue shown as a sphere. The RBD domain is shown from two different angles, as indicated. [Figure 14-1] Figure 14 shows an illustration of a further modified A2M protein. Schematic diagrams of the native structure and the disintegrated structure induced by proteolytic cleavage of the bait region are shown. Furthermore, an overview of the protein's domain structure is provided (note that all proteins are identical in the N-terminal region from MG1 to CUB2). The cleavage sites indicated by arrows on the domain structure differ from the bait region and can be cleaved without causing structural changes to A2M. The use of furin cleavage sites and TEV protease cleavage sites is shown in the examples. [Figure 14-2] Same as above. [Figure 15]Figure 15 shows SDS-PAGE(A) and native PAGE(B) analyses of wild-type A2M purified from plasma or recombinant A2M furin RBD, where the A2M furin RBD has a furin cleavage site between its CUB and MG8 domains. Both proteins were included in the analysis as untreated samples or after cleavage of the bait region using methylamine-induced aminolysis of the thiol ester or thermolysin. The A2M furin RBD was fully intracellularly processed by furin, produced using the intact thiol ester and native structures, and induced to undergo a structural change to a disintegrated structure by thiol ester aminolysis or bait region cleavage. [Figure 16A] Figure 16 shows (A) size exclusion chromatography (SEC) of A2M furin RBD following methylamine aminolysis of untreated or A2M-inducing thiol ester. On the right, a magnified chromogram image of elution of the MG8 domain is shown, as the MG8 domain has a very low extinction count and gives a low signal when absorbance is measured at 280 nm. (B) SDS-PAGE of A2M furin RBD samples before and after SEC separation is shown in panel A. The MG8 domain elutes in two fractions after methylamine treatment, but most of it elutes together with the remainder of the A2M protein before methylamine treatment. This indicates that the MG8 domain is released due to the structural change of A2M. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 17A-B]Figure 17 shows (A) SDS-PAGE of A2M tevRBD after overnight cleavage at room temperature using a titration series of TEV protease added in the indicated weight / weight ratio. TEV protease cleavage of A2M tevRBD is nearly complete at a 2.5:1 w / w ratio, resulting in an intact A2M band, a C-terminal autolysis band, and a truncated C-terminal bait region cleavage product. (B) SDS-PAGE and (C) native PAGE analysis of wild-type A2M purified from plasma or recombinant A2M tevRBD before or after TEV protease cleavage and removal of TEV protease by SEC. All proteins were included in the analysis as untreated samples or after cleavage of the bait region using methylamine-induced aminolysis of thiol esters or thermolysin. A2M tevRBD retains its thiol ester and native structure after TEV protease cleavage, and its structural transformation to a disintegrated structure was induced by thiol ester aminolysis or bait region cleavage using thermolysin. [Figure 17C] Same as above. [Figure 18A] Figure 18 shows (A) size exclusion chromatography (SEC) of A2M tevRBD after a preceding SEC purification step to remove its complete cleavage by TEV protease and the majority of the TEV protease. TEV-cleaved A2M tevRBD was treated either untreated or with methylamine for thiol ester aminolysis to induce structural changes in A2M. On the right, a magnified chromogram image of the eluate for elution of the MG8 domain is shown. (B) SDS-PAGE of A2M tevRBD samples before and after SEC separation is shown in panel A. The MG8 domain elutes in fractions 2 and 3 (depending on its glycosylation) after methylamine treatment, but elutes together with the remainder of the A2M protein before methylamine treatment. This indicates that the MG8 domain is released due to structural changes in A2M. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 19A-B]Figure 19 shows (A) SDS-PAGE and native PAGE (B) of plasma and wild-type A2M purified from recombinant A2M, containing scFv(tevRBD+2xAb) located at the TEV protease cleavable site (tevRBD), C-terminal nanobody, and ciRBD. Both A2M proteins are analyzed untreated, after methylamine aminolysis of the thiol ester, or after cleavage of the bait region with thermolysin. A2M tevRBD+2xAb is produced using intact thiol esters and is determined by the presence of autolytic fragments in SDS-PAGE. Thermolysin cleavage occurred mainly in the tevRBD+2xAb bait region, but also at the TEV protease site (ENLYFQS (SEQ ID NO: 226)), which contains several hydrophobic residues that are recognized and cleaved by thermolysin. This results in a ladder of five distinctly migrating bands seen for thermolysin-cleaved tevRBD+2xAb in native PAGE, following the removal of 0-4 MG8 domains and bound antibody fragments by thermolysin (shown in the image). - (C) SDS-PAGE and native PAGE (D) of wild-type A2M purified from plasma and recombinant A2M having scFv(tevRBD+2xAb_2) located at the modified TEV protease-cleavable site (tevRBD), C-terminal nanobody, and ciRBD. Recombinant A2M with two antibodies and the TEV site was initially native, containing a thiol ester and exhibiting slow mobility in native PAGE. Upon TEV protease cleavage, the MG8 domain remained non-covalently bound to A2M, and A2M remained native (having intact thiol ester and slow electrophoretic mobility). When TEV-processed A2M containing two antibodies is induced using methylamine, a structural change occurs, which releases the MG8 domain when the TEV site is cleaved. [Figure 19C-D] Same as above. [Figure 20]Figure 20 shows (A) a diagram illustrating the domain structure of a further modified bispecific A2M protein, i.e., a representative generalized domain structure of A2M-BiTE. Anti-CD3 = antibody targeting CD3. Anti-TAA = antibody targeting tumor-associated antigens. The first cleavage site located in the bait region and the second cleavage site located at the N-terminus of the MG8 domain are indicated by arrows. (B) Corresponding schematic diagrams of the "inactive" closed structure of A2M BiTE and the "active" open structure induced by proteolytic cleavage of the first cleavage site are shown. Anti-TAA is reachable in both the closed and open structures. Anti-CD3 is shielded until the first cleavage site is cleaved. Cleavage of the second cleavage site by a protease (shown in light gray) "stimulates" A2M-BiTE for the release of anti-CD3-MG8-anti-TAA BiTE. The release of BiTE depends on the cleavage of the first cleavage site shown, leaving behind activated A2M with a protease that cleaves the first cleavage site (shown in dark gray) trapped inside the molecule. [Figure 21] Figure 21 shows (A) a pore-restricted gel and (B) an SDS-PAGE of TEV-digested A2M and the A2M-BiTE construct BiTE2-TEV4 (SEQ ID NO: 258) samples. The pore-restricted gel in (A) shows that after digestion with TEV, the anti-CD3-MG8-anti-EGFR domain of A2M-BiTE is released by a structural change (induced by methylamine or trypsin), giving a "empty" A2M that moves as much as unmodified A2M. The release of the MG8 domain containing two antibodies required subjecting A2M-BiTE to TEV protease cleavage and a methylamine- or trypsin-induced structural change. [Figure 22]Figure 22 shows the levels of ERK1 / 2 phosphorylation in Jurcutt cells stimulated by A2M-BiTE under different structures: (A) BiTE2-TEV4 (SEQ ID NO: 258), (B) BiTE3-TEV5 (SEQ ID NO: 259) in the presence of EGFR-expressing human colon cancer cells (HCT116) as target cells, or (C) BiTE2-TEV4 (SEQ ID NO: 258) used with wild-type or EGFR-expressing CHO cells as target cells. Here, a trypsin-induced structural change of BiTE that does not occlude anti-CD3, and the presence of tumor-associated antigen-expressing target cells (EGFR) were required to induce ERK1 / 2 phosphorylation in Jurcutt cells. [Figure 23] Figure 23 shows antigen-specific target cell killing by primary T cells obtained from PBMCs after the A2M-BiTE construct has been structurally altered to allow anti-CD3 to reach the target cells. The structural alteration was induced by (A) thermolysin using CHO as the target cell, or (B) methylamine or trypsin using HCT116 as the target cell. The structural alteration was required to induce an effective cytotoxic T cell response. [Modes for carrying out the invention]

[0020] The present invention will be described in more detail below, to the minimum extent.

[0021] General To make the present invention more easily understood, certain terms are first defined below. Additional definitions of the following terms and other terms are given throughout this specification.

[0022] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context otherwise clearly indicates otherwise. For example, “biopharmaceutical portion” is understood to refer to one or more biopharmaceutical portions. Thus, the terms “a” (or “an”), “one or more,” and “at least one” are interchangeable herein.

[0023] Unless otherwise specifically stated or evident from the context, the term “or” as used herein is understood to be inclusive and encompass both “or” and “and.” Furthermore, where used herein, “and / or” should be interpreted as either together with or without the other, as a clear disclosure of each of two specific features or components. Thus, as used herein, the term “and / or” as used in phrases such as “A and / or B” is intended to encompass “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0024] Throughout this specification and the embodiments, variations of the words “have” and “comprise” or “has,” “having,” “comprises,” or “comprising” are understood to mean encompassing a stated element, function, or integer, or a group of elements, functions, or integers, but not excluding any other element, function, or integer, or a group of elements, functions, or integers. Wherever embodiments are described herein using the word “comprising” or its grammatical equivalent “having,” it is further understood that other similar embodiments are also provided, described using the terms “consisting of” and / or “consisting essentially of.”

[0025] As used herein, the term "about" refers to an interval of precision that a person skilled in the art would understand in order to still guarantee the technical effect of the function in question. This term indicates a deviation of ±10% from the indicated value. In some embodiments, the deviation is ±5% of the indicated value. In certain embodiments, the deviation is ±1% of the indicated value.

[0026] The terms “mutant” and “homologous” are used interchangeably and refer to proteins in which at least one function of the comparison protein is conserved (e.g., undergoing a structural change when cleaved by a protease). In some embodiments, the mutant or homologous is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to the wild-type version of the comparison protein (e.g., a CPAMD protein such as human A2M, containing or consisting of the amino acid sequence shown in SEQ ID NO: 1).

[0027] As used herein, the term “fragment” means a protein in which one or more amino acids are truncated (e.g., at the N-terminus and / or C-terminus) or which contains one or more deletions of amino acids while preserving at least one function of the comparison protein (e.g., in the case of an antibody or cytokine, specific binding to an antigen or receptor, or in the case of a CPAMD protein such as A2M, undergoing a structural change when cleaved by a protease).

[0028] As used herein, the terms “therapeutic” or “therapeutic active” mean any medicinal drug, medicine or composition that can be used to treat or prevent a disease, illness, condition or disorder or bodily function.

[0029] As used herein, the term “substantially” refers to a qualitative state that represents the entire or near-entire range of the characteristics or properties of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or progress to completeness or achieve or avoid absolute results. Therefore, the term “substantially” is used herein to express the potential lack of completeness inherent in many biological and chemical phenomena.

[0030] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in cell culture, in a test tube or reaction vessel, rather than within a multicellular organism.

[0031] As used herein, the term "in vivo" refers to events occurring within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events occurring within living cells (and, conversely, in vitro systems).

[0032] definition Before discussing the present invention in further detail, the following terms and conventions are first defined.

[0033] Alpha-2-macroglobulin (A2M) The term “A2M” should be understood to refer to an alpha-2-macroglobulin protein, or a variant or fragment thereof, comprising (1) a bait region having at least one protease cleavage site, and (2) a receptor-binding domain (RBD), and whose structure can be modified by proteolytic cleavage of at least one protease cleavage site. A2M is also known as C3 and PZP-like alpha-2-macroglobulin domain-containing protein 5 (CPAMD5). Preferably, A2M is the human A2M protein (NCBI#9606, Uniprot P01023). The amino acid sequence of human A2M is given in SEQ ID NO: 1, and it has naturally occurring polymorphs I1000V and N639D. Unless otherwise indicated, residue numbers provided herein for identifying specific amino acids or regions of A2M refer to the residues shown in SEQ ID NO: 1. It will be apparent to those skilled in the art that the numbering may differ in A2M variants containing one or more of the modifiers described herein.

[0034] CPAMD The term "CPAMD" should be understood to refer to members of the C3 and PZP-like alpha-2-macroglobulin domain-containing protein (CPAMD) protein family, to which A2M belongs. A descriptive list of CPAMD proteins is provided in Table 1. In some embodiments, the proteinaceous prodrug constructs of the present invention may include variants or fragments of naturally occurring CPAMD proteins. Such variants or fragments have the ability to shield one or more drugs and, when at least one protease cleavage site contained therein is proteolytically cleaved, alter their structure to make one or more drugs contained in the proteinaceous prodrug construct reachable. Typically, such proteins, and their variants or fragments, form multimers, specifically homodimers or homotetramers.

[0035] RBD domain The term "RBD" or "RBD domain" should be understood as the receptor-binding domain of a CPAMD protein (e.g., A2M). In the native human A2M protein, the RBD is located at its C-terminus and spans amino acids 1335–1474 of A2M. Y1452 and Y1453 are involved in the formation of a thiol ester group. The thiol ester group stabilizes the molecule in its "native" structure.

[0036] The amino acid sequence of the RBD domain of natural human A2M is given in Sequence ID No. 3. The RBD domain is also known as the macroglobulin 8 (MG8) domain (this is the term used in the examples). In the proteinaceous prodrug constructs described herein, one or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are placed inside or near the RBD such that the CPAMD protein (e.g., A2M) remains structurally modifiable when at least one protease cleavage site (e.g., a first protease cleavage site) contained in the bait region of the CPAMD protein is proteolytically cleaved.

[0037] Unreachable The term “unreachable” should be understood as meaning that a drug in a proteinaceous prodrug construct (e.g., a first drug or antibody) has a reduced ability to interact with its binding partner (e.g., a target antigen) if the construct is in a “closed” structure (i.e., the protease cleavage sites in the bait region are not proteolytically cleaved). In some embodiments, the ability of a drug to interact with its binding partner is reduced by 90% or more (e.g., 95% or 99% or more) in a closed structure compared to an unshielded (or “open”) structure. Thus, the drug is “unreachable” to its binding partner (e.g., if the drug is an antibody such as an scFv or nanobody).

[0038] Therefore, the term "unreachable" can also be understood as the drug being "inactive," "inactivated," or "shielded."

[0039] Therefore, in the embodiment, a. If the bait region in the CPAMD protein (e.g., A2M) is not proteolytically cleaved, one or more drugs will be unreachable; b. If the bait region in the CPAMD protein (e.g., A2M) is proteolytically cleaved, one or more drugs can reach it.

[0040] Reachable The term "reachable" should be understood as the ability of a protein-based prodrug construct (e.g., an antibody such as an scFv or nanobody) to interact with its binding partner (e.g., a target antigen). Therefore, the term "reachable" is understood as the drug being "unshielded."

[0041] For example, a drug fused to the C-terminus of an RBD can interact with its binding partner whether the construct is in a "closed" structure (i.e., the protease cleavage site in the bait region is not proteolytically cleaved) or an "open" structure, meaning its reachability is independent of protease cleavage. A drug placed inside an RBD becomes reachable in a protease-dependent manner; that is, a protease is required to cleave the protease cleavage site located in the bait region of the proteinaceous prodrug constructs described herein. The cleavage alters the structure of the proteinaceous prodrug, changing the CPAMD protein from a "closed" to an "open" structure.

[0042] Bait area The term "bait region" should be understood as a region of a CPAMD protein (e.g., A2M) that contains at least one protease cleavage site. In native human A2M protein, the bait region spans amino acids 690–728 of A2M. The sequence of the bait region of native human A2M is given in Sequence ID No. 4. The bait region of native human A2M is preferentially cleaved by most proteases, and bait region cleavage triggers structural changes in A2M. The bait region sequence can be modified to alter the selection of proteases that can cleave the bait region and trigger structural changes in the CPAMD protein.

[0043] Biopharmaceutical portion The term “biopharmaceutical moiety” should be understood as a protein or protein fragment (e.g., peptide or polypeptide) possessing therapeutic properties that can be incorporated into a proteinaceous prodrug construct together with a CPAMD protein (e.g., A2M) to create a protalytically activatable prodrug. This term is used herein interchangeably with the term “drug.” Examples of biopharmaceutical moieties include antibody fragments such as single-domain antibodies (e.g., nanobodies) or single-stranded variable fragments (scFvs), cytokines, or fragments of cell surface receptors or ligands. In a typical embodiment, the drug is an antigen-targeting moiety such as an antibody, e.g., a nanobody or a single-domain antibody such as an scFv. In some embodiments, the first and second antibodies are antigen-targeting moieties such as antibodies. In some embodiments, the first antibody is an scFv and the second antibody is a nanobody (or vice versa).

[0044] Sample sequences are provided for EGFR-binding nanobody EgA1 (SEQ ID NO: 27), scFv derived from PDL1-binding atezolizumab (SEQ ID NO: 28), PDL1-binding nanobody KN035 (SEQ ID NO: 29), scFv derived from PD1-binding nivolumab (SEQ ID NO: 30), scFv derived from CTLA-4-binding ipilimumab (SEQ ID NO: 31), scFv derived from CD3-binding foralumab (SEQ ID NO: 32), scFv derived from CD3-binding muromonab (SEQ ID NO: 33), scFv derived from 4-1BB-binding urelumab (SEQ ID NO: 34), scFv derived from TNF-alpha-binding nivolumab (SEQ ID NO: 35), IL2 cytokine (SEQ ID NO: 36), extracellular domain of PD1 receptor (SEQ ID NO: 39), or CD3-binding scFv derived from teventafuzp (SEQ ID NO: 252).

[0045] Terms such as “biopharmaceutical portion,” “drug,” “therapeutic peptide,” “therapeutic polypeptide,” or “therapeutic protein” and “activator” are used herein to refer to proteinaceous compounds that can be used to treat or prevent diseases, illnesses, conditions, or impairments of bodily functions.

[0046] Antigen targeting portion The term "antigen-targeting moiety" in this invention includes bispecific antibodies comprising single-stranded variable fragments, monoclonal, recombinant, chimeric, humanized, fully human, single-stranded, single-domain, and / or antibody fragments. Examples of such fragments include Fab, F(ab'), F(ab)', Fv, and sFv fragments. This term is used herein interchangeably with the term "antigen-binding moiety."

[0047] Antibodies are produced by enzymatic cleavage of full-length antibodies or by recombinant DNA techniques such as the expression of recombinant plasmids containing nucleic acid sequences encoding the antibody variable region. In a typical embodiment, the antigen-targeting portion in the proteinaceous prodrug construct of the present invention is a single-chain antibody such as an scFv or a single-domain antibody (e.g., a nanobody).

[0048] "Single-chain Fv," "sFv," or "scFv" antibodies contain a VH domain and a VL domain in a single polypeptide chain. The VH and VL domains are typically linked by a peptide linker. Any suitable linker can be used. In some embodiments, the linker is (GGGGS)n (SEQ ID NO: 223) or (GGS)n. In some embodiments, n = 1, 2, 3, 4, 5, or 6.

[0049] The term "single-domain antibody" refers to an antigen-targeting portion of an antibody in which one variable domain specifically binds to an antigen even without the presence of another variable domain. Single-domain antibodies contain nanobodies.

[0050] An antigen is a molecule or part of a molecule to which an antibody can bind, which can further induce an animal to produce an antibody that can bind to the epitope of that antigen. An antigen may have one or more epitopes. The specific reaction mentioned above is intended to indicate that the antigen reacts highly selectively with its corresponding antibody and does not react with a large number of other antibodies that can be induced by other antigens. Thus, the antigen-targeting moiety in the protein-prodrug construct of the present invention specifically binds to its respective target antigen.

[0051] The antigen-targeting moiety for use in the proteinaceous prodrug construct of the present invention can be obtained by various methods known to those skilled in the art of antibody production. For example, a monoclonal antibody (mAb) comprises a substantially homogeneous population of antibodies that are specific to an antigen, and this population comprises substantially similar epitope-binding sites. Such antibodies are any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and any of its subclasses. Hybridomas producing monoclonal antibodies for use in the present invention are cultured in vitro, in sights, or in vivo. High-titer production in vivo or in sights is a preferred production method.

[0052] Chimeric antibodies are molecules in which different parts originate from different animal species, such as molecules that have a variable region derived from a mouse monoclonal antibody and a constant region from a human immunoglobulin.

[0053] The term "chimeric antibody," as used herein, includes monovalent, divalent, or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer (HL) formed by a chimeric H chain linked to a chimeric L chain via a disulfide crosslink. A divalent chimeric antibody is a tetramer (H2L2) formed by two HL dimers linked via at least one disulfide crosslink. Multimeric chimeric antibodies can also be produced, for example, by using agglutinating CH regions (e.g., derived from IgM H chains or [micron] chains).

[0054] The mouse and chimeric antibodies, fragments, and regions of the present invention may individually comprise heavy (H) and / or light (L) immunoglobulin chains.

[0055] Selective binders, such as antibodies, fragments, or derivatives having chimeric H and L chains with the same or different variable region binding specificity, can also be prepared by the appropriate association of individual polypeptide chains.

[0056] In some embodiments, the term "antibody" as used herein refers to a single-chain or single-domain antibody.

[0057] Receptor-binding domain (RBD) This disclosure provides several methods for positioning a biopharmaceutical moiety (e.g., a first drug or antibody) inside the RBD in order to shield or make the biopharmaceutical moiety inaccessible until a protease cleavage site (e.g., a first protease cleavage site) located in the bait region of the CPAMD protein is cleaved.

[0058] ciRBD The term "ciRBD" should be understood as a protein fusion construct between a CPAMD protein (e.g., A2M) and a biopharmaceutical moiety (e.g., a therapeutic peptide, polypeptide, or protein), where the biopharmaceutical moiety is positioned within the RBD domain at a location corresponding to residues 1402 and 1403 of native human A2M, without removing any residues of the CPAMD protein. A linker sequence can be used to connect the N-terminus of the biopharmaceutical moiety to the carboxyl terminus of residue 1402 (SEQ ID NO: 78) or to connect the C-terminus of the biopharmaceutical moiety to the amino terminus of residue 1405 (SEQ ID NO: 79). An example of a ciRBD fusion construct incorporating an EgA1 nanobody (SEQ ID NO: 27) into A2M is given in SEQ ID NOs: 5-6.

[0059] iRBD The term "iRBD" should be understood as a protein fusion construct between a CPAMD protein (e.g., A2M) and a biopharmaceutical moiety (e.g., a therapeutic peptide, polypeptide, or protein), where the biopharmaceutical moiety replaces residues in the RBD domain corresponding to residues spanning from position 1392 (including position 1392) to position 1403 (including position 1403) in native human AM. The biopharmaceutical moiety is linked to residue 1391 by an N-terminal linker (SEQ ID NO: 80) and residue 1404 by a C-terminal linker (SEQ ID NO: 81). An example of an iRBD fusion construct incorporating an EgA1 nanobody (SEQ ID NO: 27) into A2M is given in SEQ ID NOs: 84-85.

[0060] miRBD The term "miRBD" should be understood as a protein fusion construct between a CPAMD protein (e.g., A2M) and a biopharmaceutical moiety (e.g., a therapeutic peptide, polypeptide, or protein), where the biopharmaceutical moiety replaces residues in the RBD domain corresponding to residues spanning from position 1393 (including position 1393) to position 1395 (including position 1395) of the native human AM. The biopharmaceutical moiety is linked to residue 1392 by an N-terminal linker (SEQ ID NO: 82) and residue 1396 by a C-terminal linker (SEQ ID NO: 83). An example of a miRBD fusion construct incorporating an EgA1 nanobody (SEQ ID NO: 27) into A2M is given in SEQ ID NOs: 86-87.

[0061] tRBD The term "tRBD" should be understood as a protein fusion construct between a CPAMD protein (e.g., A2M) and a biopharmaceutical moiety (e.g., a therapeutic peptide, polypeptide, or protein), where the biopharmaceutical moiety is incorporated into the RBD domain during its C-terminus. Furthermore, residues 1393 to 1402 of the RBD domain, or corresponding residues in the RBD domain of another CPAMD protein, are modified to allow the formation of an α-helix having a sequence complementary to the sequence of another α-helix located at the N-terminus of the biopharmaceutical moiety. The RBD domain α-helix and the N-terminus α-helix of the biopharmaceutical moiety are designed to interact with each other via coiled-coil interactions. These coiled-coil interactions transport the biopharmaceutical moiety to a position that facilitates shielding of the biopharmaceutical moiety by the CPAMD protein (e.g., A2M) relative to the RBD domain. The biopharmaceutical portion is linked at its N-terminus to the C-terminus of its adjacent α-helix by a 2-residue linker, and the α-helix itself is linked at its N-terminus to the C-terminus of the RBD domain by a 15-residue linker. An example of a tRBD fusion construct incorporating the EgA1 nanobody (SEQ ID NO: 27) into A2M is given in SEQ ID NOs: 92-93.

[0062] Epitope In this context, the term "epitope" refers to the portion of an antigen that is recognized by the immune system.

[0063] Eukaryotic expression vector In this context, a "eukaryotic expression vector" refers to a tool (e.g., nucleic acid) that contains an expression control sequence (e.g., a suitable promoter sequence) operably ligated to the nucleotide sequence to be expressed, and is used to introduce a specific coding polynucleotide sequence into target cells.

[0064] Sequence identity In this context, the term "sequence identity" is defined here as sequence identity at the nucleotide, base, or amino acid level between genes or proteins, respectively. Specifically, DNA and RNA sequences are considered identical if a transcript of the DNA sequence can be transcribed into the corresponding RNA sequence.

[0065] Therefore, in this context, "sequence identity" is a measure of identity between proteins at the amino acid level, and a measure of identity between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequences at a given position in each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequences at a given position in each sequence when the sequences are aligned.

[0066] To determine the percentage identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (for example, gaps may be introduced in the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). Next, amino acid residues or nucleotides are compared at their corresponding amino acid or nucleotide positions. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at its corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., duplicate positions) × 100). In one embodiment, the two sequences are of the same length.

[0067] In another embodiment, the two sequences are of different lengths and the gap is observed at different positions. The sequences may be manually aligned and the number of identical amino acids counted. Alternatively, the alignment of the two sequences for determining percentage identity may be accomplished using a mathematical algorithm. Such algorithms are incorporated into the BLASTN and BLASTX programs (Altschul et al., 1990). BLAST nucleotide search may be performed using the NBLAST program to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein search may be performed using the BLASTX program to obtain amino acid sequences homologous to the protein molecules of the present invention.

[0068] Gap BLAST can be used to obtain gap alignment for comparative purposes. Alternatively, PSI-Blast may be used to perform iterative searches to detect distant relationships between molecules. When using the BLASTN, BLASTX, and Gap BLAST programs, the default parameters of each program may be used. See http: / / www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned using the BLAST program in the EMBL database (www.ncbi.nlm.gov / cgi-bin / BLAST), for example. In general, default settings for "score matrix" and "gap penalty," for example, may be used for alignment. In the context of this invention, the BLASTN and PSI BLAST default settings may be advantageous.

[0069] The percentage identity between two sequences may be determined using a technique similar to the one described above, with or without gaps. When calculating the percentage identity, only exact matches are counted. Thus, embodiments of the present invention relate to sequences of the present invention having a certain degree of sequence diversity.

[0070] subject The term "subject" includes humans of all ages, other primates (e.g., crab-eating macaques, rhesus macaques); mammals in general, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, minks, ferrets, hamsters, cats, and dogs, as well as birds. The preferred subject is humans.

[0071] The term "target" includes healthy individuals within the population, particularly healthy individuals such as healthcare workers who are exposed to pathogens and require protection against infection.

[0072] It should be noted that embodiments and functions described in one context of the present invention also apply to other aspects of the present invention.

[0073] All patent and non-patent references cited in this application are thereby incorporated in their entirety by reference.

[0074] Detailed description of the invention In certain embodiments, the present disclosure relates to a fusion protein (e.g., a proteinaceous prodrug construct) comprising a complement 3- and pregnancy-related protein-like alpha-2-macroglobulin domain (CPAMD) protein such as A2M, (a) A bait region containing at least one first protease cleavage site; (b) Receptor-binding domain (RBD); (c) A second protease cleavage site introduced at the N-terminus of the RBD domain of the CPAMD protein, wherein when the second protease cleavage site is cleaved, the RBD domain remains bound to the CPAMD protein by non-covalent interactions; and (d) At least one biopharmaceutical moiety located from the C-terminus to a second protease cleavage site, wherein cleavage of the first protease cleavage site causes the release of the RBD domain from the CPAMD protein, thereby releasing at least one biopharmaceutical moiety. Regarding fusion proteins including...

[0075] In some embodiments, the fusion protein comprises a complement 3- and a pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein, (a) Bait region containing the first protease cleavage site; (b) A second protease cleavage site at the N-terminus of the RBD; and a fused drug at the C-terminus of the RBD. Receptor-binding domain (RBD) including Includes, (i) Even if the second protease cleavage site is cleaved, the RBD domain remains bound to the CPAMD protein due to non-covalent interactions; (ii) The CPAMD protein can change its structure and release RBD when the first protease cleavage site is proteolytically cleaved. A fusion protein is provided. In these embodiments, the drug is reachable.

[0076] While not wanting to be bound by any particular theory, the inventors believe that the release of RBDs containing drugs in a protease-activatable manner can improve tissue penetration. For example, a first cleavage site (and possibly a second protease cleavage) is made to be cleaved by one or more proteases present in the diseased tissue (e.g., tumor tissue or inflammatory site).

[0077] In some embodiments, the first and second protease cleavage sites are different. For example, it may be convenient to "pre-cleave" the second protease cleavage site during the production of the fusion protein or proteinaceous prodrug construct disclosed herein.

[0078] One suitable protease is furin. Furin is expressed in the Golgi apparatus of mammalian cells. When the furin cleavage site is used as a second protease cleavage site in the fusion protein or proteinaceous prodrug disclosed herein, cleavage of the second protease cleavage site occurs during recombinant expression in mammalian host cells.

[0079] Another suitable protease is tobacco etch virus (TEV) protease. TEV proteases are highly site-specific. TEV cleavage sites are generally found in recombinant proteins, enabling the removal of affinity tags, for example, used to purify recombinant proteins from host components.

[0080] As shown in Examples 12 and 13, specific prodrugs can be constructed in which the second protease cleavage site at the N-terminus of the RBD domain is a furin or TEV cleavage site. Thus, in certain embodiments, the present invention relates to a proteinaceous prodrug construct comprising a complement 3- and pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein such as A2M, (a) A bait region containing at least one first protease cleavage site; (b) Receptor-binding domain (RBD); (c) A furin protease cleavage site according to SEQ ID NO: 225 or a TEV protease cleavage site according to SEQ ID NO: 226, introduced at the N-terminus of the RBD domain of the CPAMD protein, such as one derived from residues 1334 to 1340 of A2M, wherein even if the furin or TEV protease cleavage site is cleaved, the RBD domain remains bound to the CPAMD protein due to non-covalent interactions; and (d) At least one biopharmaceutical moiety located from the C-terminus to a second protease cleavage site, wherein cleavage of the first protease cleavage site causes the release of the RBD domain from the CPAMD protein, thereby releasing at least one biopharmaceutical moiety. This relates to protein prodrug constructs containing [specific components].

[0081] Such fusion protein or protein prodrug constructs can be transformed into multispecific prodrugs, such as bispecificity, by introducing an additional biopharmaceutical moiety (e.g., an antibody). In some embodiments, the first biopharmaceutical moiety (e.g., a first drug or antibody) is located inside the RBD region, and the second biopharmaceutical moiety (e.g., a second drug or antibody) is located from the C-terminus to the CPAMD protein (e.g., fused to the C-terminus of the RBD). In such embodiments, the CPAMD protein shields the first biopharmaceutical moiety, and the CPAMD protein modulates its structure when the first protease cleavage site is proteolytically cleaved, releasing the RBD from the CPAMD protein, thereby releasing the first and second biopharmaceutical moieties, making the first biopharmaceutical moiety accessible.

[0082] Those skilled in the art will recognize that the functionality of a bispecific prodrug may not depend on the introduction of a second protease cleavage site. Therefore, in certain embodiments, the present invention relates to a protein prodrug construct (e.g., a poly- or bispecific protein prodrug construct) comprising a complement 3- and pregnancy-related protein-like alpha-2-macroglobulin domain (CPAMD) protein such as A2M, (a) A bait region containing at least one first protease cleavage site; (b) receptor-binding domain (RBD); and (c) At least two biopharmaceutical moieties (e.g., a first and a second antibody) located from the C-terminus to the RBD, wherein at least one first biopharmaceutical moiety (e.g., a first antibody) is located inside the RBD domain and at least one second biopharmaceutical moiety (e.g., a second antibody) is located at an unshielded position at the C-terminus relative to the RBD domain. Includes, The CPAMD protein shields the first biopharmaceutical moiety (e.g., the first antibody), and when the first protease cleavage site of the CPAMD protein or a fragment thereof is proteolytically cleaved, it can change its structure and deshim the first biopharmaceutical moiety. We provide protein-based prodrug constructs.

[0083] In some embodiments, the second protease cleavage site is introduced at the N-terminus of the RBD domain of the CPAMD protein, and the RBD domain remains bound to the CPAMD protein by non-covalent interactions even after the second protease cleavage site is cleaved, and when the first protease cleavage site is cleaved, it releases the RBD, thereby releasing the first and second biopharmaceutical moieties from the CPAMD protein.

[0084] Therefore, these embodiments may be combined, and thus, in another embodiment, the present invention is a proteinaceous prodrug construct comprising a complement 3- and pregnancy-related protein-like alpha-2-macroglobulin domain (CPAMD) protein such as A2M, (a) A bait region containing at least one first protease cleavage site; (b) Receptor-binding domain (RBD); (c) A second protease cleavage site introduced at the N-terminus of the RBD domain of the CPAMD protein, wherein even if the second protease cleavage site is cleaved, the RBD domain remains bound to the CPAMD protein due to non-covalent interactions; and (d) At least two biopharmaceutical moieties located from the C-terminus to a second protease cleavage site, wherein at least one first biopharmaceutical moiety is located inside the RBD domain and at least one second biopharmaceutical moiety is located in an unshielded position at the C-terminus relative to the RBD domain. Includes, The CPAMD protein or a fragment thereof shields the first biopharmaceutical moiety, and when the first protease cleavage site of the CPAMD protein or fragment thereof is proteolytically cleaved, it changes its structure to release RBD, thereby releasing the first and second biopharmaceutical moieties from the CPAMD protein, and deshielding the first biopharmaceutical moiety. Regarding protein-based prodrug constructs.

[0085] In certain embodiments, the proteinaceous prodrug construct comprises a complement 3- and a pregnancy-related protein-like alpha-2-macroglobulin domain (CPAMD) protein, (a) a bait region containing the first protease cleavage site; and (b) Second protease cleavage site at the N-terminus of the receptor-binding domain (RBD), A first biopharmaceutical component (e.g., a first drug or antibody) placed inside the RBD, and A second biopharmaceutical moiety (e.g., a second drug or antibody) fused to the C-terminus of the RBD. RBD including Includes, (i) The CPAMD protein shields the first biopharmaceutical moiety, leaving the second biopharmaceutical moiety accessible (i.e., unshielded); (ii) When the first protease cleavage site of the CPAMD protein is proteolytically cleaved, it changes its structure and releases RBD, thereby making the first biopharmaceutical moiety accessible. Protein prodrug constructs are provided. In these embodiments, RBD remains bound to CPAMD by non-covalent interactions even after the second protease cleavage site is cleaved.

[0086] In one particular embodiment, the first biopharmaceutical portion is an antigen-binding portion that specifically binds to CD3, and the second biopharmaceutical portion is an antigen-binding portion that specifically binds to tumor cell surface antigens.

[0087] In certain embodiments, the first biopharmaceutical portion is a harmful protein (e.g., a cytotoxic protein), and the second biopharmaceutical portion is an antigen-binding portion that specifically binds to tumor cell surface antigens.

[0088] Typically, the CPAMD protein is human. In some embodiments, the CPAMD protein is A2M. In some embodiments, the CPAMD protein is human A2M.

[0089] Targeted prodrugs A protein prodrug construct according to the present invention is manipulated to direct the prodrug to the site where it will perform its function. In some embodiments, the protein prodrug construct includes a biopharmaceutical moiety that can direct the protein prodrug construct to a specific tissue (e.g., cancerous or inflammatory tissue), a specific cell type (e.g., immune cells or tumor cells), and / or a specific receptor (e.g., T cell receptor or tumor cell surface antigen). In certain embodiments, such a biopharmaceutical moiety is fused to the C-terminus of the RBD.

[0090] The specific cells are immune cells, for example, when manipulating a bispecific protein prodrug construct (e.g., illustrated in Examples 14-17). Therefore, in some embodiments, the protein prodrug construct includes a biopharmaceutical moiety that can direct the protein prodrug construct to immune cells. In certain embodiments, such a biopharmaceutical moiety is fused to the C-terminus of the RBD. In some embodiments, the immune cells are NK cells, macrophages, T cells, or dendritic cells. In certain embodiments, the immune cells are T cells. In certain embodiments, the biopharmaceutical moiety is an antibody that specifically binds to a T cell receptor (e.g., CD3).

[0091] Since the prodrug can provide both shielded and unshielded portions, it is possible to choose whether the above targeting occurs before or after the structural change in the CPAMD protein. In some embodiments, at least one biopharmaceutical portion has specificity for a target and can bind to its target before cleavage at a first protease cleavage site. In some embodiments, at least one biopharmaceutical portion has specificity for a target and cannot bind to its target before cleavage at a second protease cleavage site. In some embodiments, at least one biopharmaceutical portion is a targeting portion.

[0092] As detailed below, a particularly preferred embodiment is the production of BiTF, and the essential part of BiTE is T cell specificity; however, without being bound by theory, prodrugs can be manufactured with T cell specificity even if they are not BiTF. In some embodiments, at least one biopharmaceutical moiety, e.g., a first biopharmaceutical moiety (i.e., a first antibody), is T cell specific, such as a T cell specificity moiety. In some embodiments, the T cell specificity moiety is specific to receptors expressed at increased levels on T cells, such as CD3, CD4 and / or CD8, preferably CD3. In some embodiments, the T cell specificity moiety is an anti-CD3 moiety.

[0093] When developing prodrugs with specificity to a particular T cell receptor, such as those using anti-CD3 BiTE, using multimeric CPAMD proteins, it is particularly important to develop proteinaceous prodrugs that include a second protease cleavage site that is processed before introduction into the target circulation, in order to avoid premature activation of T cells through crosslinking caused by receptor binding by multivalent antibodies.

[0094] In some embodiments, the biopharmaceutical portion is NK cell specific, such as an NK cell specificity portion. In some embodiments, the NK cell specificity portion is specific to a receptor expressed at increased levels on NK cells, such as CD16. In some embodiments, the NK cell specificity portion is an antibody against CD16.

[0095] In some embodiments, the biopharmaceutical portion is macrophage-specific, such as a macrophage-specific portion. In some embodiments, the macrophage-specific portion is specific to a receptor or molecule expressed at increased levels on macrophages, such as SIRPα. In some embodiments, the macrophage-specific portion is a SIRPα inhibitory antibody, such as a SIRPα antibody that blocks CD47.

[0096] In some embodiments, the biopharmaceutical moiety is specific to dendritic cells, such as a dendritic cell-specific moiety. In some embodiments, the biopharmaceutical moiety is specific to a receptor or molecule expressed at increased levels on dendritic cells, such as the DNGR1 receptor. In some embodiments, the dendritic cell-specific moiety is an antibody against the DNGR1 receptor.

[0097] Multiple specific drugs As presented herein, protein prodrugs are multispecific prodrugs, such as bispecific prodrugs. In some embodiments, RBD becomes a multispecific drug when released from the CPAMD protein. In some embodiments, RBD is a bispecific drug when released from the CPAMD protein. In certain embodiments, RBD comprises a first antibody and a second antibody, both of which become reachable when released from the CPAMD family proteins. In some embodiments, the CPAMD protein is a multispecific drug when its structure is altered. In some embodiments, the CPAMD protein is a bispecific drug when its structure is altered. In certain embodiments, the CPAMD protein comprises a first antibody and a second antibody, both of which become reachable when its structure is altered.

[0098] The at least two biopharmaceutical moieties selected to produce a multispecific prodrug, such as a bispecific prodrug, may be any combination of biopharmaceutical moieties, drugs, etc., as further described herein.

[0099] If a protein prodrug is designed to contain a first biopharmaceutical moiety in one of loops 1-4 of the RBD and a second biopharmaceutical moiety at the C-terminus of the RBD, the first biopharmaceutical moiety will be shielded until the bait region is cleaved, while the second biopharmaceutical moiety will not be shielded. This relies on the fact that the C-terminal biopharmaceutical moiety is not further adapted to be ligated within the RBD domain, as will be further described below.

[0100] In some embodiments, a bispecific drug comprises a protein prodrug construct that can be directed to immune cells as described in the previous section (e.g., a first antibody) and a drug as further described below (e.g., a second antibody that specifically binds to the surface of tumor cells).

[0101] In some embodiments, such proteins can be manipulated to target specific immune cells. In some embodiments, the bispecific drug (e.g., a bispecific antibody prodrug construct) is a bispecific T cell engager (BiTE), a bispecific NK cell engager (BiKE), a bispecific macrophage engager (BiME), or a bispecific dendritic cell engager (BiDE). In preferred embodiments, the bispecific drug is a bispecific T cell engager (BiTE). These acronyms are somewhat established terms in the art. The acronyms encompass bispecific molecules that include a portion targeting a specific cell type, e.g., an anti-CD3 antibody fragment, and a portion targeting a therapeutic target such as EGFR (e.g., a tumor cell surface antigen).

[0102] A bispecific drug (e.g., a bispecific antibody-prodrug construct) comprises at least two drugs (e.g., two antibodies) as further described below.

[0103] In some embodiments, the bispecific antibody prodrug construct is (a) Bait region containing the first protease cleavage site; (b) Second protease cleavage site at the N-terminus of RBD, The first antibody placed inside the RBD; and Second antibody fused to the C-terminus of RBD Receptor-binding domain (RBD) including Includes, (i) The CPAMD protein shields the first antibody, while the second antibody remains reachable; (ii) Even if the second protease cleavage site of RBD is cleaved, it remains bound to the CPAMD protein through non-covalent interactions; (iii) When the first protease cleavage site of the CPAMD protein is proteolytically cleaved, it changes its structure and releases RBD, thereby making the first antibody reachable. Contains CPAMD protein.

[0104] In some embodiments, the second protease cleavage site is cleaved (for example, during or after the production of the protein prodrug construct, e.g., during or after recombinant expression of the construct in host cells), (a) Bait region containing the first protease cleavage site; (b) A receptor-binding domain (RBD) containing a first antibody placed inside the RBD and a second antibody fused to the C-terminus of the RBD. Includes, (i) The CPAMD protein shields the first antibody, while the second antibody remains reachable; (ii) RBD is bound to the CPAMD protein by non-covalent interactions; (iii) When the first protease cleavage site of the CPAMD protein is proteolytically cleaved, it changes its structure and releases RBD, thereby making the first antibody reachable. It produces a bispecific antibody prodrug construct containing the CPAMD protein.

[0105] CPAMD protein The proteinaceous prodrug constructs described herein include complement 3 and pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) proteins. The CPAMD protein comprises a bait region having at least one protease cleavage site and a receptor-binding domain (RBD).

[0106] In some embodiments, one or more drugs are located inside or near one of loops 1-4 of the RBD (e.g., loop 1, loop 2, loop 3, or loop 4). In one particular embodiment, one or more drugs are located inside loop 2 (the most convenient site in the RBD for producing the prodrug construct disclosed herein, which shields one or more drugs until cleavage of the protease site within the bait region). In another particular embodiment, one or more drugs are located inside loop 3 (a suitable alternative to loop 2 as shown herein). In yet another particular embodiment, one or more drugs are located inside loop 4. In yet another particular embodiment, one or more drugs are located near loop 2 of the RBD.

[0107] In the protein-based prodrug construct of the present invention, the CPAMD protein shields the drug placed within the RBD. The CPAMD protein can change its structure and make the drug reachable when at least one protease cleavage site in the bait region is proteolytically cleaved.

[0108] While the present invention describes in more detail protein prodrug constructs in which the CPAMD protein is alpha-2-macroglobulin (A2M), or a variant or functional homolog thereof, those skilled in the art of protein prodrug design will recognize that other CPAMD proteins can substitute for A2M.

[0109] In some embodiments, the protein prodrug construct is a fusion protein. In one embodiment, the protein fusion construct comprises a member of the CPAMD family fused to one or more drugs; or a modified member of the CPAMD family fused to one or more drugs (2), wherein one or more drugs are located inside or near the RBD domain of A2M. The protein fusion construct and the protein prodrug are used interchangeably herein.

[0110] In some embodiments, one or more drugs are inserted into one of loops 1-4 of the RBD. In some embodiments, the loops are modified by the addition, substitution, or deletion of one or more amino acids to accommodate one or more drugs. In some embodiments, one or more drugs replace one or more amino acids in the loop. In certain embodiments, the loop is loop 2 of the RBD. In some embodiments, the loop is loop 3 of the RBD. In further embodiments, the loop is loop 4 of the RBD.

[0111] As discussed herein, placing one or more drugs near loop 2 of the RBD can be achieved by inserting them inside or within the five amino acid residues of loop 2 (e.g., by replacing one or more residues, or by direct insertion). Similarly, this can be achieved by inserting one or more drugs inside or within the five amino acid residues of loop 1, loop 3, or loop 4 (e.g., by replacing one or more residues, or by direct insertion). Loops 1, 3, and 4 are 27 Å, 21 Å, and 25 Å, respectively, from the center of the mass to loop 2. In the ciRBD fusion approach described herein, the shortest constraint between the drug and loop 2 is a 15-residue C-terminated linker. From the average length of 3.5 Å per amino acid residue, it can be calculated that one or more drugs can be located approximately 52 Å away from loop 2 (e.g., approximately 50 Å, 40 Å, 30 Å, or 20 Å), occupying positions whose reachability depends on the structure of the CPAMD protein (e.g., A2M).

[0112] As an alternative to direct fusion, the drug can be designed to be positioned within an equal distance to loop 2, with an orientation similar to that of the RBD domain, through other means, as achieved by the direct fusion approach. For example, the drug can be anchored to loop 2 using coiled-coil interactions or high-affinity interactions, as described herein (e.g., in the tRBD approach described herein).

[0113] Table 1 provides a descriptive list of CPAMD proteins that can be used to carry out the present invention, along with the location and sequence of each loop.

[0114] [Table 1-1] [Table 1-2]

[0115] In some embodiments, the CPAMD protein is selected from the group consisting of C3, C4A, C4B, C5, PZP, A2ML1, CD109, CPAMD8, ovostatin homolog 1, ovostatin homolog 2, and A2M. In some embodiments, the CPAMD protein is selected from A2M, PZP, ovostatin 1, and ovostatin 2, as well as their functional homologs. In some embodiments, the CPAMD protein is human A2M or its functional homolog, for example, mammalian A2M. In certain embodiments, the CPAMD protein is A2M.

[0116] In one embodiment, the CPAMD protein is a human CPAMD protein such as one of the proteins listed in Table 1 or its variants. In some embodiments, the human CPAMD protein is a modified variant as described herein, for example, the variant may include a modified bait region.

[0117] In some embodiments, the CPAMD protein has at least about 70% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In some embodiments, the CPAMD protein has at least about 75% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In one embodiment, the CPAMD protein has at least about 80% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In one embodiment, the CPAMD protein has at least about 85% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In one embodiment, the CPAMD protein has at least about 90% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1.

[0118] In one embodiment, the CPAMD protein has at least about 91% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In one embodiment, the CPAMD protein has at least about 92% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In one embodiment, the CPAMD protein has at least about 93% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In one embodiment, the CPAMD protein has at least about 94% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In one embodiment, the CPAMD protein has at least about 95% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In one embodiment, the CPAMD protein has at least about 96% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In one embodiment, the CPAMD protein has at least about 97% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In another embodiment, the CPAMD protein has at least about 98% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. In yet another embodiment, the CPAMD protein has at least about 99% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1.

[0119] In further embodiments, the CPAMD protein is a human CPAMD protein such as the proteins listed in Table 1. However, a. The bait area has been modified as described herein; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0120] In another embodiment, the CPAMD protein has at least approximately 70% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. However, a. The bait area has been modified as described herein; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0121] In one embodiment, the CPAMD protein has at least approximately 80% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. However, a. The bait area has been modified as described herein; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0122] In one embodiment, the CPAMD protein has at least approximately 85% sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. However, a. The bait area has been modified as described herein; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0123] In one embodiment, the CPAMD protein has at least about 90% (e.g., at least about 91%, at least about 92%, at least about 93%, or at least about 95%) sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. However, a. The bait area has been modified as described herein; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0124] In one embodiment, the CPAMD protein has at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity with at least one of the full-length CPAMD protein sequences listed in Table 1. However, a. The bait area has been modified as described herein; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0125] The RBD domains and bait regions of the CPAMD proteins listed in Table 1 are described in Table 2. The RBD domain (also called the "MG8 domain") and bait region (also called the "anaphylaxis domain" in some CPAMD proteins) were identified based on their functional equivalence to the corresponding domain / region in human A2M.

[0126] [Table 2-1] [Table 2-2]

[0127] When one or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are introduced into the RBD, they are sterically hindered from interacting with other proteins, such as their therapeutic targets. The RBD domain itself is a small domain (approximately 16 kDa). While we do not wish to be bound by any particular theory, the inventors believe that it is unlikely that the RBD domain alone could sterically hinder one or more drugs (e.g., therapeutic peptides, polypeptides, or proteins), especially considering that linkers are typically present between one or more drugs and the RBD domain. Again, while we do not wish to be bound by any particular theory, the inventors therefore believe that other parts or copies of the CPAMD protein contribute to surrounding and isolating one or more drugs. For example, naturally occurring CPAMD proteins (e.g., A2M) form homotetramers.

[0128] In some embodiments, the CPAMD protein (e.g., A2M) forms a multimer (e.g., a dimer or tetramer). In some embodiments, the multimer contains identical subunits (e.g., a homodimer or homotetramer). While we do not wish to be bound by any particular theory, the inventors believe that contributions from one or more adjacent subunits may contribute to the isolation of one or more drugs.

[0129] Two human CPAMD proteins are known to form dimers (typically stabilized by one or more disulfide crosslinks), namely A2M and pregnancy-related protein (PZP, aka CPAMD6). In A2M, the disulfide-crosslinked dimer forms a tetramer, primarily through its LNK domain, by engaging in additional non-covalent interactions with another disulfide-crosslinked dimer. This tetramerization is also seen in ovostatins, such as ovostatins, which are characteristic of ducks, chickens, and frogs. The two human ovostatins, ovostatin 1 and ovostatin 2, are also expected to be tetramers.

[0130] Therefore, in some embodiments, the proteinaceous prodrug construct according to the present invention can form a polymer, such as a dimer or a tetramer. In some embodiments, the polymer is a heteropolymer (e.g., a heterodimer or a heterotetramer). More typically, the polymer is a homodimer or a homotetramer.

[0131] In some embodiments, multimerization (e.g., dimerization or tetramerization) occurs via the LNK region of the CPAMD protein. In some embodiments, the tetramer is formed by two disulfide-bridged dimers (e.g., two homodimers). In preferred embodiments, the CPAMD protein is a multimer.

[0132] Cysteine, which forms the intersubunit disulfide bonds responsible for disulfide-bridged dimers, is found in two loops: one located on the MG3 domain of the CPAMD protein and the other on the MG4 domain of the CPAMD protein. These loops are defined in Table 3. The LNK region, which has been shown to be involved in the interaction between the two disulfide-bridged dimers in the tetramer-forming CPAMD protein, is also defined in Table 3.

[0133] [Table 3]

[0134] The iRBD, miRBD, ciRBD, and tRBD described herein create proteinaceous prodrug constructs by "locking" the position of a drug (e.g., a peptide, polypeptide, or protein) near loop 2 (residues 1392-1405) on the RBD of a CPAMD protein (e.g., A2M) by direct fusion in the iRBD / miRBD / ciRBD approach or by using coiled-coil interactions in the tRBD approach to anchor the drug to this position. Other approaches that can anchor a drug to this common position relative to the RBD domain will be apparent to those skilled in the art.

[0135] In some embodiments, the proteinaceous prodrug construct comprises a first interaction domain, and one or more drugs comprises a second interaction domain, and the first and second interaction domains form a complex that places one or more drugs near any one of loops 1-4 of the RBD (e.g., loop 1, loop 2, loop 3, or loop 4). In one particular embodiment, the first and second interaction domains form a complex that places one or more drugs near loop 2. In another particular embodiment, the first and second interaction domains form a complex that places one or more drugs near loop 4. In some embodiments, the first and second interaction domains form a coiled-coil structure.

[0136] While not wanting to be bound by any particular theory, the inventors hypothesize that if the first and second interaction domains are used to position one or more drugs near loop 2 of the RBD, the CPAMD protein can adopt its “native” structure, thereby isolating one or more drugs inside it (and thus shielding the CPAMD protein from interaction with one or more targets). Spatial proximity can be achieved, for example, by inserting the first interaction domain into loop 2 of the RBD, or into one of loops 1-4 of the RBD (e.g., loop 3).

[0137] One approach is to "dock" the drug into the CPAMD protein (e.g., A2M). This can be done using molecules with an inherent affinity for loop 2 of RBD, such as a functional fragment or antibody (e.g., a nanobody) of the LRP1 receptor that recognizes the loop 2 epitope.

[0138] Alternatively, the RBD of the CPAMD protein (e.g., A2M) could be modified to facilitate such docking. For example, a tag sequence could be introduced into the RBD (e.g., at the "ciRBD" position), and the drug could be fused to an antibody that recognizes the tag (e.g., a nanobody or similar small binding domain).

[0139] Therefore, in some embodiments, the first interaction domain is a tag or epitope sequence within loop 2 of the RBD, and the second interaction domain is a functional fragment of a receptor or antibody that can specifically bind to the tag or epitope sequence.

[0140] A2M Alpha-2-macroglobulin (A2M) is a protein found in human plasma at high concentrations (typically 1-5 g / L). A2M is a protease inhibitor with a clearly defined mechanism of action. Firstly, proteases cleave an exposed and fragile sequence of sequences called a bait region, which is tolerant of cleavage by most human proteases. Cleavage of the bait region triggers a structural change in A2M, causing it to disintegrate around the protease, trapping the protease within A2M and preventing it from accessing additional large protein substrates (Figure 1). If the cleavage is rapid and sequential, up to two proteases can be inhibited by a single A2M protein. In addition to the instigating confinement of the protease(s) that initiated the process, two additional consequences of the triggered structural changes are observed: (i) hidden binding sites on A2M to the LRP1 receptor are exposed, resulting in binding of the A2M-protease complex by cell surface LRP1 and rapid clearance of these complexes from circulation, for example, by LRP1-expressing hepatocytes; and (ii) the reactive thiol ester moiety is exposed on A2M, enabling the formation of covalent bonds to the confined protease.

[0141] This invention describes incorporating a biopharmaceutical moiety into A2M such that the binding ability of the biopharmaceutical moiety is regulated by the structure of A2M. Suitable biopharmaceutical moieties for use with this invention include therapeutic peptides, polypeptides, or proteins, such as antibodies (e.g., single-chain or single-domain antibodies such as scFv and nanobodies). In the native structure of A2M, the incorporated biopharmaceutical moiety occupies a shielded position where its ability to interact with its therapeutic target is reduced. After the structure of A2M is modified by proteolytic cleavage of the bait region (or, alternatively, by aminolysis of the thiol ester of A2M using a methylamine that triggers a similar structural change), the biopharmaceutical moiety shows an increased ability to interact with its target. By modifying the bait region sequence of A2M, a specific protease can be designated as one that can cleave the bait region and trigger this structural change. In summary, this allows for the creation of proteolytic fusion constructs of A2M and a biopharmaceutical moiety (e.g., therapeutic peptide, polypeptide, or protein) that function as a protease-activated prodrug version of the biopharmaceutical moiety.

[0142] In one embodiment, the present invention provides a protein prodrug construct comprising (a) an alpha-2-macroglobulin (A2M) protein and (b) one or more drugs, wherein (i) the A2M protein comprises (1) a bait region having at least one protease cleavage site and (2) a receptor-binding domain (RBD), (ii) one or more drugs are located inside or near the RBD, and (iii) the A2M protein shields one or more drugs and can change its structure when at least one protease cleavage site is proteolytically cleaved to make one or more drugs reachable.

[0143] In some embodiments, the present invention relates to a protein fusion construct comprising alpha-2-macroglobulin (A2M) fused to one or more drugs; or modified A2M fused to one or more drugs, wherein one or more drugs are located inside or near the RBD domain of A2M.

[0144] In some embodiments, one or more drugs are located inside or near one of loops 1-4 of the RBD. In some embodiments, the proteinaceous prodrug construct is a fusion protein. In some embodiments, one or more drugs are located inside one of loops 1-4 of the RBD. In some embodiments, the loop is loop 1. In some embodiments, the loop is loop 2. In some embodiments, the loop is loop 3. In some embodiments, the loop is loop 4. In some embodiments, the loop is modified compared to the wild-type loop sequence by the addition, substitution, or deletion of one or more amino acids to accommodate one or more drugs. In some embodiments, one or more drugs replace one or more amino acids in the loop.

[0145] In one embodiment, if the bait region in alpha-2-macroglobulin (A2M) is not proteolytically cleaved, one or more drugs are unreachable, while if the bait region in alpha-2-macroglobulin (A2M) is proteolytically cleaved, one or more drugs are reachable.

[0146] In one embodiment, cleavage of the bait region is achieved by serine-, cysteine-, aspartate-, and / or metallopropylinase.

[0147] The drug can be positioned at different locations within the sequence of the protein fusion construct.

[0148] Those skilled in the art can recognize the portion of the protein fusion construct derived from A2M. Therefore, in embodiments where a drug is inserted into the sequence of A2M, the resulting fusion construct can be considered as a first portion of A2M, the drug, and a second portion of A2M. In such cases, those skilled in the art can recognize the first and second portions of A2M as a complete molecule. Therefore, in certain embodiments, the sequence identity of A2M should be calculated from two separate portions based on the sequence derived from A2M, and thus without including one or more drugs.

[0149] In one embodiment, the A2M molecule is a mammalian A2M molecule, such as a human A2M molecule, or a variant thereof.

[0150] In one embodiment, the A2M molecule is a human A2M molecule such as the sequence according to Sequence ID No. 1, or a variant thereof. In some embodiments, the human A2M molecule is a variant that has been modified as described herein, for example, a variant that may include a modified bait region.

[0151] In some embodiments, the A2M molecule has at least about 70% sequence identity with the sequence according to Sequence ID No. 1. In some embodiments, the A2M molecule has at least about 75% sequence identity with the sequence according to Sequence ID No. 1. In one embodiment, the A2M molecule has at least about 80% sequence identity with the sequence according to Sequence ID No. 1. In one embodiment, the A2M molecule has at least about 85% sequence identity with the sequence according to Sequence ID No. 1. In one embodiment, the A2M molecule has at least about 90% sequence identity with the sequence according to Sequence ID No. 1.

[0152] In one embodiment, the A2M molecule has at least about 91% sequence identity with the sequence according to SEQ ID NO: 1. In one embodiment, the A2M molecule has at least about 92% sequence identity with the sequence according to SEQ ID NO: 1. In one embodiment, the A2M molecule has at least about 93% sequence identity with the sequence according to SEQ ID NO: 1. In one embodiment, the A2M molecule has at least about 94% sequence identity with the sequence according to SEQ ID NO: 1. In one embodiment, the A2M molecule has at least about 95% sequence identity with the sequence according to SEQ ID NO: 1. In one embodiment, the A2M molecule has at least about 96% sequence identity with the sequence according to SEQ ID NO: 1. In one embodiment, the A2M molecule has at least about 97% sequence identity with the sequence according to SEQ ID NO: 1. In one embodiment, the A2M molecule has at least about 98% sequence identity with the sequence according to SEQ ID NO: 1. In one embodiment, the A2M molecule has at least about 99% sequence identity with the sequence according to SEQ ID NO: 1.

[0153] In further embodiments, the A2M molecule is a human A2M molecule such that it follows the sequence of Sequence ID No. 1. However, a. The bait area has been modified as described above; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0154] In another embodiment, the A2M molecule has at least about 70% sequence identity with the sequence according to Sequence ID No. 1. However, a. The bait area has been modified as described above; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0155] In one embodiment, the A2M molecule has at least about 80% sequence identity with the sequence according to Sequence ID No. 1. However, a. The bait area has been modified as described above; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0156] In one embodiment, the A2M molecule has at least about 85% sequence identity with the sequence according to Sequence ID No. 1. However, a. The bait area has been modified as described above; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0157] In one embodiment, the A2M molecule has at least about 90% (e.g., at least 91%, at least 92%, at least 93%, or at least 95%) sequence identity with the sequence according to Sequence ID No. 1. However, a. The bait area has been modified as described above; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0158] In one embodiment, the A2M molecule has at least about 95% (e.g., at least 96%, at least 97%, at least 98%, or about 99%) sequence identity with the sequence according to Sequence ID No. 1. However, a. The bait area has been modified as described above; and / or b. One or more drugs (e.g., therapeutic peptides, polypeptides, or proteins) are inserted into the RBD region, for example, into loop 2, by removing one or more residues of loop 2 as described above.

[0159] In one embodiment, one or more drugs are located between 1391 and 1405 in SEQ ID NO: 1. In another embodiment, one or more drugs are located after position 1335 in SEQ ID NO: 1. In yet another embodiment, one or more drugs are located before position 1474 in SEQ ID NO: 1. In yet another embodiment, one or more drugs are located between 1391 and 1405 in SEQ ID NO: 1 or after position 1335 in A2M but before position 1474. In yet another embodiment, the A2M molecule includes one or more mutations K1393A, K1397A, T654C, and / or T661C.

[0160] K1393A and K1397A remove the A2M interaction with receptors LRP1 and Grp78, respectively. LRP1 mediates the clearance of cleaved A2M, and Grp78, upon binding, induces pro-mitotic signaling in cells. Both of these receptor interactions are potentially problematic for drugs, and therefore, removing these amino acids is beneficial.

[0161] The T654C and T661C mutations introduce disulfides that bridge the two disulfide dimers of A2M, so the entire A2M tetramer is stabilized by disulfide bonds. This prevents A2M from splitting into two halves. This can occur during physiological conditions such as inflammation (due to oxidative damage to A2M).

[0162] In aspects of the present invention, the present invention relates to a protein fusion construct comprising alpha-2-macroglobulin (A2M) having a bait region having at least one protease cleavage site, wherein the A2M is fused to a peptide drug located within residues 1392-1404, 1368-1379, or 1420-1426 of the receptor-binding domain (RBD) of the A2M. In particular, in such aspects, if the bait region in the A2M is not proteolytically cleaved, the peptide drug may be unreachable; if the bait region in the A2M is proteolytically cleaved, the peptide drug may be reachable.

[0163] While the preceding paragraphs describe placing one or more drugs within the RBD domain and introducing disulfide crosslinks in relation to A2M, those skilled in the art of proteinaceous prodrug design will recognize that other CPAMD proteins can substitute for A2M, and that corresponding residues can be identified in these CPAMD proteins for carrying out the present invention (for example, using the residue numbers provided in Tables 1 and 2 as a guide).

[0164] drugs A proteinaceous prodrug construct may contain one or more drug or biopharmaceutical components (e.g., therapeutic peptides, polypeptides, or proteins).

[0165] In some embodiments, a drug can increase or decrease the signal from a receptor when it binds to that receptor.

[0166] In one embodiment, the drug is selected from the group consisting of an antigen-targeting moiety (e.g., an antibody or antibody mimetic), a cytokine, an extracellular domain of a cell surface receptor, an extracellular domain of a cell surface ligand, and a receptor agonist.

[0167] In some embodiments, at least one biopharmaceutical portion is an antigen-binding portion (e.g., an antibody or an antigen-binding fragment of an antibody). In one embodiment, the antigen-targeting portion is selected from the group consisting of antibodies, nanobodies, diabodies, and single-stranded variable fragments. In some embodiments, the antigen-targeting portion is a single-stranded or single-domain antibody (e.g., a nanobody). In some embodiments, the antigen-targeting portion is a single-stranded variable fragment. In some embodiments, the antigen-targeting portion can direct the protein prodrug construct to a specific tissue, a specific cell type, and / or a specific receptor.

[0168] In another embodiment, the drug is selected from a group consisting of toxins, enzymes, and small molecule drug-protein conjugates similar to antibody-drug conjugates (ADCs). For example, the protein contains a site suitable for small molecule conjugation, such as a cysteine ​​residue. In some embodiments, the toxin is selected from bacterial toxins such as anthrax and diphtheria toxin. In certain embodiments, the drug, such as a toxin, enzyme, or protein conjugated to a small molecule drug analog, is placed inside the RBD and becomes inaccessible until the protease cleavage site in the bait region is cleaved.

[0169] In some embodiments, the proteinaceous prodrug construct comprises complement 3 and a pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein, (a) Bait region containing the first protease cleavage site; (b) Second protease cleavage site at the N-terminus of the receptor-binding domain (RBD); Toxins or enzymes placed inside the RBD; and The antigen-targeting portion (e.g., antibody) fused to the C-terminus of the RBD. RBD including Includes, (i) CPAMD proteins block toxins or enzymes; (ii) The antigen-targeting portion (e.g., an antibody) can direct the protein prodrug construct to a specific tissue, a cell type of a particular characteristic, and / or a specific receptor; (iii) Even after the second protease cleavage site of RBD is cleaved, RBD remains bound to the CPAMD protein due to non-covalent interactions. (iv) When the first protease cleavage site of the CPAMD protein is enzymatically cleaved, it changes its structure to release RBD, thereby making the toxin or enzyme reachable. A protein-based prodrug construct is provided.

[0170] In some embodiments, the proteinaceous prodrug construct comprises complement 3 and a pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein, (a) Bait region containing the first protease cleavage site; (b) An enzyme placed inside the receptor-binding domain (RBD); and an RBD containing an antigen-targeting moiety (e.g., an antibody) fused to the C-terminus of the RBD. Includes, There (i) CPAMD proteins block toxins or enzymes; (ii) The antigen-targeting portion (e.g., an antibody) can direct the protein prodrug construct to a specific tissue, a cell type of a particular characteristic, and / or a specific receptor; (iii) RBD remains bound to the CPAMD protein through non-covalent interactions. (iv) When the first protease cleavage site of the CPAMD protein is enzymatically cleaved, it changes its structure to release RBD, thereby making the toxin or enzyme reachable. A protein-based prodrug construct is provided.

[0171] In some embodiments, the antigen-targeting moiety specifically binds to the antigen as an antagonist (for example, the antigen-targeting moiety can inhibit the binding of a ligand to its receptor). In some embodiments, the antigen-targeting moiety specifically binds to the antigen as an agonist (for example, the antigen-targeting moiety can induce signal transduction by binding to a receptor).

[0172] In some embodiments, the antigen targeting portion includes BTLA, OX40, LAG3, NRP1, VEGF, HER2, CEA, CD19, CD20, amyloid beta, HER3, IGF-1R, MUC1, EpCAM, CD22, VEGFR-2, PSMA, GM-CSF, CXCR4, CD30, CD70, FGFR2, BCMA, CD44, ICAM-1, Notch1, MHC, CD28, IL-1R1, TCR, Notch3, F It specifically binds to antigens selected from the group consisting of GFR3, TGF-β, TGFBR1, TGFBR2, CD109, GITR, CD47, alpha-synuclein, CD26, LRP1, CD52, IL-4Rα, VAP-1, EPO receptor, integrin αv, TIM-3, Grp78, LIGHT, TLR2, TLR3, PAR-2, NRP2, GLP-1 receptor, Hedgehog, and Syndecan 1 (e.g., tumor cell surface antigens).

[0173] When developing multispecific prodrugs like BiTE (e.g., protein prodrug constructs containing at least two antigen-targeting moieties), the moieties that do not bind to immune cells, for example, the second drug typically binds to tumor cell surface antigens. In some embodiments, the antigen-targeting moieties include NRP1, HER2, CEA, CD19, CD20, DLL3, GPRC5D, gp100, HER3, IGF-1R, MUC1, EpCAM, CD22, VEGFR-2, PSMA, CD30, CD70, FGFR2, BCMA, CD44, ICAM-1, Notch1, MHC, IL-1R1, MCSP, CD66e, EphA2, Notch3, FGFR3, TGFBR1, T The antigen-targeting portion (e.g., antibody) specifically binds to an antigen selected from the group consisting of GFBR2, CD109, GITR, CD47, alpha-synuclein, CD26, LRP1, CD52, IL-4Rα, VAP-1, EPO receptor, integrin αv, TIM-3, Grp78, LIGHT, TLR2, TLR3, PAR-2, NRP2, GLP-1 receptor, Hedgehog, alpha-fetoprotein, CA-125, ETA, MAGE, and syndecan 1. The portion that binds to immune cells, e.g., the first drug, typically targets specific immune cells (e.g., T cells). The portion that binds to immune cells is typically placed inside the RBD to make it inaccessible until a protease cleavage site in the bait region is cleaved. The antigen-targeting portion (e.g., antibody) that specifically binds to the target antigen expressed on immune cells (e.g., T cells) is directed towards an immunostimulatory molecule, a co-stimulatory molecule, or an immunosuppressive molecule (such as a receptor or ligand). In some embodiments, the target antigen expressed on immune cells (e.g., T cells) is selected from the group consisting of CD3, CTLA-4, FcγRIIb, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, TIGIT, CD28, CD27, OX40, CD137, and ICOS. Therefore, in some embodiments, a protein prodrug construct comprising the CPAMD protein is used. (a) a bait region containing the first protease cleavage site; and (b) Second protease cleavage site at the N-terminus of the receptor-binding domain (RBD); The first antibody placed inside the RBD; and Second antibody fused to the C-terminus of RBD RBD including Includes, There (i) The first antibody (e.g., scFv or nanobody) specifically binds to a target antigen (e.g., an immunostimulatory receptor such as CD3) expressed on immune cells, and the second antibody (e.g., scFv or nanobody) specifically binds to tumor cell surface antigens; (ii) The CPAMD protein shields the first antibody, while the second antibody is not shielded, i.e., it is reachable. (iii) Even if the second protease cleavage site of RBD is cleaved, it remains bound to the CPAMD protein through non-covalent interactions. (iv) When the first protease cleavage site of the CPAMD protein is proteolytically cleaved, it changes its structure and releases RBD, thereby making the first antibody unblockable, i.e., reachable. A protein-based prodrug construct is provided.

[0174] In some embodiments, the first drug is an antigen-targeting moiety that specifically binds to CD3 (e.g., an anti-CD3 antibody), such as the CD3-targeting moiety contained in known BiTEs. Commercially available BiTEs include blinatumomab (CD19-targeted CD3 T cell activator), grofitamab (CD20-targeted CD3 T cell activator), mosnetuzumab (CD20-targeted CD3 T cell activator), solitomab (EpCAM-targeted CD3 T cell activator), talketamab (GPRC5D-targeted CD3 T cell activator), tarulatamab (DLL3-targeted CD3 T cell activator), and teventafusp (gp100-targeted CD3 T cell activator). While not wanting to be bound by any particular theory, the inventors hypothesize that reformatting existing BiTEs as proteinaceous prodrug constructs according to the methods disclosed herein will reduce common side effects such as cytokine release syndrome and / or toxicity such as immunoeffector cell-associated neurotoxicity syndrome (ICANS).

[0175] Other multispecific prodrug constructs other than BiTE that contain at least two antigen-targeting moieties are assumed herein.

[0176] In one embodiment, the antigen targeting moiety is selected from the group consisting of anti-PD1, anti-PD-L1, anti-EGFR, anti-CTLA4, anti-CD137, anti-CD3, and anti-TNFα.

[0177] In one embodiment, the antigen-targeting moiety is selected from the group consisting of atezolizumab, EgA1, ipilimumab, nivolumab, KN035, urerumab, foralumab, muromonab, and adalimumab, or one or more CDRs of any of the aforementioned antigen-targeting moieties, all three heavy chain CDRs, all three light chain CDRs, all three heavy chain CDRs and all three light chain CDRs, a heavy chain variable region, and / or a therapeutically active scFv, fragment, or variant thereof including the light chain variable region.

[0178] In some embodiments, the antigen-targeting moiety is selected from the group consisting of ANB032, rosnilimab, LY3361237, encelimab, covolimab, imsidolimab, dostarlimab, or one or more CDRs of any of the aforementioned antigen-targeting moieties, all three heavy chain CDRs, all three light chain CDRs, all three heavy chains and all three light chain CDRs, a heavy chain variable region, and / or a therapeutically active scFv, a fragment thereof, or a variant thereof.

[0179] As outlined above, cytokines may be used as drugs in the present invention. Cytokines are described as a category of small proteins that induce cell signaling.

[0180] In one embodiment, the one or more drugs are cytokines selected from the group consisting of chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.

[0181] In another embodiment, the one or more drugs are cytokines selected from the group consisting of IL1, IL1 alpha, IL1 beta, IL2, IL3, IL4, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL34, IL35, and IL36.

[0182] In further embodiments, the one or more drugs are cytokines selected from the group consisting of IL2, IFN-α, IL-15, IL-21, IL-10, IL-12, IL-17, GM-CSF, TGF-β, CSF-1, insulin, GLP-1, HGH, VEGF, PDGF, BMP, EPO, G-CSF, IL-11, IFN-γ, and IFN-β.

[0183] In a preferred embodiment, the one or more drugs are IL2, which is tested in Example 9.

[0184] In one embodiment, the antigen-targeting moiety is an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-43. In another embodiment, the antigen-targeting moiety has or includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-43. In a further embodiment, the antigen-targeting moiety has an amino acid sequence having at least about 80% sequence identity with the sequence selected from the group consisting of SEQ ID NOs: 27-43, for example, at least about 85%, 90%, or even up to about 95% sequence identity. If a variation is introduced in the antigen-targeting moiety, it is preferable that the CDR sequence is not modified.

[0185] In another embodiment, the nucleic acid sequence encoding the antigen-targeting portion is selected from the group consisting of fragments or variants thereof having at least about 90% sequence identity with any of SEQ ID NOs. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26: particularly about 95% identity with SEQ ID NOs. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26. In another embodiment, the amino acid sequence is encoded by a nucleic acid sequence selected from the group consisting of fragments or variants having at least about 90% sequence identity with any of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26: in particular about 95% identity with SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.

[0186] In one embodiment, one or more drugs (e.g., first and second antibodies) have a size of up to 100 kDa, such as up to 85 kDa, up to 75 kDa, up to 65 kDa, up to 55 kDa, up to 50 kDa, up to 40 kDa, up to 30 kDa, or at least 10 kDa.

[0187] In one embodiment, one or more drugs contain up to 900 amino acids, such as up to 770 amino acids, up to 680 amino acids, up to 590 amino acids, up to 500 amino acids, up to 450 amino acids, up to 360 amino acids, up to 270 amino acids, at least 90 amino acids, etc.

[0188] In further embodiments, the protein prodrug construct according to the present invention comprises 1 to 5, for example, 1 to 4, for example, 1 to 3, for example, 1 to 2 drugs. In a particular embodiment, the protein prodrug construct according to the present invention comprises 1 drug.

[0189] Bait area As previously described, the structural change of the protein prodrug construct (Figure 1) is initiated when the protease cleaves within the exposed and highly sensitive bait region.

[0190] In some embodiments, the proteinaceous prodrug construct according to the present invention comprises a CPAMD protein (e.g., A2M) having a modified bait region. In some embodiments, the bait region is modified to alter the selection of proteases that can cleave the bait region and trigger a structural change in the CPAMD protein (e.g., A2M). For example, the bait region may be modified to be cleaved by a specific protease or class of proteases (e.g., an MMP such as MMP2).

[0191] To manipulate specificity, it is sometimes convenient to create a bait region that cannot be cleaved by proteases through manipulation. This bait region that cannot be cleaved by proteases is referred to herein as the "tabula rasa bait region." For example, to prevent cleavage by proteases, the tabula rasa bait region may contain a manipulated amino acid sequence that is flexible and / or hydrophilic. In some embodiments, the manipulated amino acid sequence includes a sequence of glycine, serine, alanine, threonine, and / or proline residues. In some embodiments, the manipulated amino acid sequence replaces all or part of the wild-type bait region. In some embodiments, the manipulated amino acid sequence is approximately 15–51 amino acids, such as approximately 30–40, approximately 31–39, or approximately 32–35. In certain embodiments, the length of the manipulated amino acid sequence is approximately 32–33 amino acids. In some embodiments, the manipulated amino acid sequence replaces all of the wild-type bait region and has a length equal to that of the wild-type bait region.

[0192] For example, to prevent cleavage by proteases, the tabular survey region can consist of a series of amino acid repeats. This series of amino acid repeats may replace some or all of the native survey region. For example, the tabular survey region may contain a series of amino acid repeats. Examples of a series of 3-amino acid repeats are Gly-Gly-Ser, Gly-Gly-Gly, Gly-Ser-Gly, Gly-Ser-Ser, Ser-Gly-Gly, Ser-Gly-Ser, Ser-Ser-Gly, Ser-Ser-Ser. Each series of 3 amino acids can be repeated or combined with one another. For example, the tabular survey region may contain one or more amino acid repeats, the repeat being selected from the list consisting of Gly-Gly-Ser, Gly-Gly-Gly, Gly-Ser-Gly, Gly-Ser-Ser, Ser-Gly-Gly, Ser-Gly-Ser, Ser-Ser-Gly, and Ser-Ser-Ser. Alternatively, the tabular survey region may contain one or more amino acid repeats, the repeat being an amino acid triplet composed of Ser, Gly, and Ala residues. In some cases, the tabular survey region may consist of one or more amino acid repeats, the repeats selected from the list of Gly-Gly-Ser, Gly-Gly-Gly, Gly-Ser-Gly, Gly-Ser-Ser, Ser-Gly-Gly, Ser-Gly-Ser, Ser-Ser-Gly, Ser-Ser-Ser, and Ala.The tabular survey region can consist of one or more amino acid repeats, the repeats being Gly-Gly-Ser, Gly-Gly-Gly, Gly-Gly-Ala, Gly-Ser-Gly, Gly-Ser-Ser, Gly-Ser-Ala, Gly-Ala-Ser, Gly-Ala-Gly, Gly-Ala-Ala, Ser-Gly-Gly, Ser-Gly-Ser, Ser-Gly-Ala, Ser -Selected from a list consisting of Ser-Gly, Ser-Ser-Ser, Ser-Ser-Ala, Ser-Ala-Gly, Ser-Ala-Ser, Ser-Ala-Ala, Ala-Gly-Ser, Ala-Gly-Gly, Ala-Gly-Ala, Ala-Ser-Gly, Ala-Ser-Ser, Ala-Ser-Ala, Ala-Ala-Ser, Ala-Ala-Gly, and Ala-Ala-Ala. For example, a tabular survey region consisting of 13 Gly-Gly-Ser repeats can be seen in sequence number 124.

[0193] In one embodiment, the bait region includes 5, e.g., 7, e.g., 9, e.g., 11, e.g., 13, e.g., 15, e.g., 17 iterations. In one particular embodiment, the bait region includes 13 iterations. In another embodiment, the bait region includes about 5 to 17, e.g., about 7 to 15, e.g., about 9 to 13 iterations.

[0194] The total length of the bait region can vary from 15 to 51 amino acids. The length of the tabular bait region can be approximately 15 to 51, for example, approximately 30 to 40, for example, approximately 31 to 39, for example, approximately 32 to 35 amino acids. In certain embodiments, the length of the tabular bait region is approximately 32 to 33 amino acids.

[0195] At least one protease cleavage site can be introduced into the tabular survey domain. By using such a modified bait domain, it becomes possible to control which protease cleaves and thereby introduces a structural change into the protein prodrug construct.

[0196] Cleavage site In some embodiments, the present disclosure provides a prodrug comprising at least two protease cleavage sites. The first protease cleavage site is included in the bait region and causes the shielded biopharmaceutical moiety to be unshielded. The second protease cleavage site can be introduced at the N-terminus of the RBD domain of the CPAMD protein. The second protease cleavage site enables the release of the RBD from the CPAMD protein. If the first protease cleavage site is not cleaved, the RBD domain remains bound to the CPAMD protein by non-covalent interactions. Thus, the second protease cleavage site is optional in some embodiments of the present disclosure.

[0197] One skilled in the art can select protease cleavage sites to design prodrugs with different purposes. In some embodiments, the first protease cleavage site and the second protease cleavage site are specific to the same protease, i.e., the protease described herein as part of the bait region is introduced as the second protease cleavage site. For example, both the first protease cleavage site included in the bait region and the second protease cleavage site at the N-terminus of the RBD protein can be cleaved in vivo by the same protease(s) (e.g., one or more endogenous protease(s) present in diseased tissue such as tissue containing tumors or inflammatory sites).

[0198] In other embodiments, the second protease cleavage site is different from the first protease cleavage site such that the RBD domain, which necessarily includes the first and second biopharmaceutical moieties, is not released until the first protease cleavage site (e.g., in a tumor or at an inflammatory site) is cleaved. For example, when a BiTE is developed using the CPAMD proteins described herein, it is desirable to provide a proteinaceous prodrug that includes a second protease cleavage site that is processed before being introduced into the circulation of a subject. Premature cross-linking of T cells can be avoided because the first antibody placed within the RBD becomes accessible only when the first protease cleavage site is cleaved. Further, cross-linking can occur only when the RBD containing both the first and second antibodies is released from the CPAMD protein. Thus, in some embodiments, the first protease cleavage site can be cleaved by an endogenous protease (e.g., an endogenous protease present at the site of a disease such as cancer or an inflammatory disease), and the second protease can be cleaved by a protease that is not normally present extracellularly or in the circulation.

[0199] Specifically, preferred protease cleavage sites for the second protease cleavage site can be, for example, furin, TEV protease, enterokinase, or thrombin to facilitate in vitro cleavage of the proteinaceous prodrug construct before introduction into a living subject. In some embodiments, the second protease cleavage site is specific for furin, TEV protease, enterokinase, or thrombin. In a preferred embodiment, the second protease cleavage site is specific for furin. In another preferred embodiment, the second protease cleavage site is specific for TEV protease. Specificity for furin is achieved by incorporation of an amino acid sequence according to SEQ ID NO: 225. Specificity for TEV protease can be achieved by incorporation of an amino acid sequence according to SEQ ID NO: 226. As further provided in the section below, the second protease cleavage site is further surrounded by a linker.

[0200] In some embodiments, the first protease cleavage site is specific to aspartic acid-, cysteine-, glutamic acid-, aspartic acid-, serine-, threonine-, or metalloprotease.

[0201] The second protease cleavage site is inserted upstream of the RBD domain (i.e., at the N-terminus). In exchange, the first portion of the RBD domain is replaced by the second protease cleavage site. Once the second protease cleavage site is cleaved, the RBD domain and at least one biopharmaceutical moiety remain noncovalently bound to the CPAMD protein until the first protease cleavage site is cleaved, a structural change occurs, and as a result, the RBD domain and at least one biopharmaceutical moiety are released.

[0202] In some embodiments of the present invention, the second protease cleavage site is inserted according to the start of the RBD domain, as shown in Table 2. Those skilled in the art will recognize that it is not necessary to strictly adhere to this position, and that an insertion of + / - 20 amino acids at the N-terminus of the RBD may be acceptable. Therefore, in some embodiments of the present invention, the second protease cleavage site is inserted at any position selected according to the positions shown in Table 4. For example, for CPAMD5 (aka A2M), the insertion site is selected as 1335 according to Table 2; 20 amino acids from the N-terminus to it, such as position 1315 according to Table 4; or 20 amino acids from the C-terminus to it, such as position 1355 according to Table 4. Those skilled in the art will know that + / - 20 amino acids may mean any position in the approximately 40 amino acid region, for example, the insertion site for A2M may be selected as 1325 or 1345. As can be seen from Examples 12 and 13, an 11-amino acid second protease cleavage site can be efficiently inserted directly into the sequence of A2M. In some embodiments of the present invention, the second protease cleavage site is replaced with an appropriate number of amino acids according to the positions shown in Table 4.

[0203] [Table 4]

[0204] In some embodiments, the second protease cleavage site introduced at the N-terminus of the RBD domain of the CPAMD protein is introduced from residues 1334 to 1340 of A2M.

[0205] Individual protease cleavage sites can be introduced into the tabular survey region to make the protein prodrug construct effective as a drug and to control its activity. Therefore, those skilled in the art can control which proteases can cleave and thereby introduce structural changes into the protein prodrug construct.

[0206] The present invention is not limited to introducing a single protease cleavage site into the bait region. In some embodiments, the bait region may have several cleavage sites, which are cleaved by different proteases. Thus, in one embodiment, the bait region includes one or more protease cleavage sites (e.g., two, three, or four protease cleavage sites). In another embodiment, the bait region includes only one protease cleavage site.

[0207] In some embodiments, the protease cleavage sites for use in proteinaceous prodrugs (and particularly in the bait region) are activated protein C, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM9, ADAMMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, BMP-1, caspase 1, caspase 10, caspase 14, caspase 2, caspase 3, caspase -se 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, chymase, cruzipain, DESC1, DPP-4, elastase, FAP, granzyme B, guanidinobenzoate e) Hepsin, HtrA1, Neutrophil elastase, KLK10, KLK11, KLK13, KLK14, KLK4, KLK5, KLK6, KLK7, KLK8, Regmine, Malapsin, Matryptase-2, Meprin, MMP1, MMP8, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP2, MMP20, MMP23, MMP24, MMP26, MMP2 The group is selected from 7, MMP3, MMP7, MMP8, MMP9, MT-SP1 / matryptase, neprilysin, NS3 / 4A, Otubain-2, PACE4, plasmin, PSA, PSMA, renin, thrombin, TMPRSS2, TMPRSS3, TMPRSS4, tPA, tryptase, uPA, ADAM8, FVIIa, FIXa, furin, Fxa, FXIa, FXIIa, and TAFI.

[0208] In some embodiments, the bait region includes a single cleavage site selected from the group of sequence numbers 96-123. In some embodiments, the bait region includes only one single protease cleavage site that can be cleaved by a matrix metalloproteinase (MMP). In certain embodiments, the bait region includes one single protease cleavage site that can be cleaved by a protease selected from the group consisting of MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, MMP8, MMP10, and MMP19.

[0209] In some embodiments, the bait region includes two cleavage sites. For example, the bait region may include exactly two cleavage sites, one of which is cleavable by a group of proteases consisting of MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, MMP8, MMP10, and MMP19, and the other is cleavable by activated protein C, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, AD AM9, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, BMP-1, Caspase 1, Caspase 10, Caspase 14, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Kymase, Cruzipain, DESC1, DPP-4, Elastase, FAP, Granzyme B, Guanidinobenzoatase, Hepsin, HtrA1, Neutrophil Elastase, KLK10, KLK11, KLK13, KLK14, KLK4, KLK5, KLK6, KLK7, KLK8, Lactoferrin, Regmine, Malapsin, Matryptase 2. The region is cleavable by a group of proteases consisting of meprin, MT-SP1 / matryptase, neprilysin, NS3 / 4A, otsubine-2, PACE4, plasmin, PSA, PSMA, renin, thrombin, TMPRSS2, TMPRSS3, TMPRSS4, tPA, tryptase, uPA, ADAM8, FVIIa, FIXa, furin, Fxa, FXIa, FXIIa, and TAFI. In another embodiment, the bait region includes two cleavable sites selected from the group of sequence numbers 96-123.

[0210] In further embodiments, the bait region has no protease cleavage sites recognized by human proteases except for MMPs. In some embodiments, the bait region contains one or more (e.g., at least two or three) protease cleavage sites that can be cleaved by one or more (e.g., at least two or three) MMPs. In yet another embodiment, the bait region has no protease cleavage sites recognized by human proteases except for a single cleavage site.

[0211] As can be seen from the examples, the bait region can be highly modified, and those skilled in the art can select any suitable cleavage site within the bait region depending on the required specificity. Accordingly, in certain embodiments, a proteinaceous prodrug construct according to the present invention comprises a CPAMD protein (e.g., A2M) containing a modified bait region that can be selectively cleaved by one or more proteases.

[0212] A protease site is "selectively cleavable" if cleavage occurs only in the presence of, or primarily in the presence of, one specific protease. A modified bait region may contain one or more (e.g., at least two or three) cleavage sites, each of which can be manipulated to be "selectively cleavable" by a different protease. For example, a modified bait region may contain one, two, or three unique recognition sites, each of which can be manipulated to be specific to a different protease.

[0213] Exemplary MMP cleavage sites include A21A, B74, C9, and S1. In certain embodiments, the bait region includes one or more (e.g., at least two or three) of the A21A, B74, C9, and / or S1 cleavage sites. Exemplary modified bait regions containing the cleavage sites can be seen in SEQ ID NOs: 126-133. In another particular embodiment, the bait region includes lysine, as in SEQ ID NO: 125.

[0214] In some embodiments, the modified bait region includes a manipulated amino acid sequence that is flexible and / or hydrophilic. In some embodiments, the manipulated amino acid sequence includes a sequence of glycine, serine, alanine, threonine, and / or proline residues. In some embodiments, the manipulated amino acid sequence includes a combination of glycine, serine, and / or alanine residues. In some embodiments, the manipulated amino acid sequence replaces the wild-type bait region and has a length equal to that of the wild-type bait region. In one embodiment, the wild-type bait region is replaced by a combination of glycine, serine, and / or alanine residues having a length equal to that of the wild-type bait region.

[0215] Exemplary sequences in which the cleavage site is inserted into a tabula rasa region can be seen in any of the sequences identified by SEQ ID NOs: 125–133. In another embodiment, only a portion of the wild-type bait region is replaced by a tabula rasa region described herein, such as SEQ ID NO: 130, in which the C-terminal quarter of the wild-type bait region is retained.

[0216] In another embodiment, one or more cleavage sites in the bait region are replaced by a combination of glycine, serine, and / or alanine residues. In one embodiment, the bait region comprises one or more repeats, for example, at least 5, for example, at least 6, for example, at least 7, for example, at least 8. In a particular embodiment, the length of the tabular bait region is at least about 10 repeats.

[0217] In one embodiment, the bait region has a size of approximately 8 kDa, for example, up to approximately 5 kDa, for example, up to approximately 4 kDa, for example, up to approximately 3 kDa, for example, up to approximately 2 kDa. In a particular embodiment, the bait region has a size of up to approximately 2.5 kDa.

[0218] In one embodiment, the length of the bait region is about 15 - 51 amino acids. In one embodiment, the length of the bait region is about 30 - 40 amino acids, such as about 31 - 39 amino acids or 32 - 35 amino acids. In certain embodiments, the full length of the bait region is about 32 - 33 amino acids.

[0219] In one embodiment, the bait region is an engineered amino acid sequence that is globally flexible and / or hydrophilic, such as a random sequence of glycine, serine, alanine, threonine, and / or proline residues, and optionally includes one or more protease cleavage sites (e.g., MMP cleavage sites) such that the full length of the bait region, which may include repeats and cleavage site(s), is about 15 - 51 amino acids, such as about 32 - 33 amino acids.

[0220] In one embodiment, when the protease cleaves the "bait region", the protease is trapped inside the proteinaceous prodrug construct.

[0221] RBD domain As described above, the prodrug is produced by contacting a drug or biopharmaceutical moiety (e.g., a therapeutic peptide, polypeptide or protein) that must be shielded such that the drug is inaccessible, with the RBD domain such that the folding of the RBD domain shields the drug. The therapeutic protein can be contacted with the RBD domain by inserting it into the RBD domain or by replacing part of the RBD domain with the therapeutic protein.

[0222] Thus, in a typical embodiment of the proteinaceous prodrug construct of the invention, a drug (e.g., a therapeutic peptide, polypeptide or protein) is disposed inside the RBD such that the CPAMD protein (e.g., A2M) can change its structure when the protease cleavage site contained within the bait region is proteolytically cleaved, thereby making the drug accessible.

[0223] Considering the size of the RBD domain, there are numerous sites suitable for insertion into the RBD domain. As visualized in Figure 13, the RBD domain is largely composed of beta sheets, and the loops in the middle of the individual beta chains are suitable for drug insertion, as shown in the examples of the present invention. For example, in the native human A2M protein, loop 1 is formed by amino acid residues 1368-1379, loop 2 by amino acid residues 1392-1404, loop 3 by amino acid residues 1420-1426, and loop 4 by amino acid residues 1450-1457.

[0224] In one embodiment, the drug is located within loop 2 in the RBD domain of A2M (between residues 1391 and 1405 of native human A2M, for example, between 1392 and 1404). In one embodiment, the drug is located in the RBD domain of A2M by replacing one or more amino acids corresponding to the region-forming residues 1391-1405 or residues 1392-1404 of the native human protein. In one embodiment, the drug is located in the RBD domain of A2M between amino acids corresponding to residues 1391-1405 (for example, residues 1392-1404) of the native human protein. In another embodiment, one or more amino acids corresponding to residues 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, and / or 1405 of the native human protein are replaced by the drug. In another embodiment, the drug is positioned after one or more amino acids corresponding to residues 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, or 1404 of the native human protein.

[0225] In one embodiment, the drug is located within loop 1 in the RBD domain of A2M (between residues 1368–1379 of native human A2M). In one embodiment, one or more amino acids corresponding to residues 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, and / or 1378 of native human A2M are replaced by the drug. In another embodiment, the drug is located after one or more amino acids corresponding to residues 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, or 1378 of native human A2M.

[0226] In one embodiment, the drug is located within loop 3 in the RBD domain of A2M (between residues 1420–1426 of native human A2M). In another embodiment, one or more amino acids corresponding to residues 1420, 1421, 1422, 1423, and / or 1424 of native human A2M are replaced by the drug. In yet another embodiment, the drug is located after one or more amino acids corresponding to residues 1420, 1421, 1422, 1423, or 1424 of native human A2M.

[0227] In another embodiment, the drug is located near the RBD domain of A2M. In one embodiment, the drug is tethered to the C-terminus of the RBD domain of A2M and brought to very close proximity to residues 1391–1405 of the RBD domain through a specific interaction, such as an alpha-helix coiled-coil interaction. In some embodiments, the drug tethered to the C-terminus of the RBD domain of A2M is tethered to the amino acid corresponding to residue 1474 of human A2M.

[0228] Placing one or more drugs within the RBD domain is as described in the preceding paragraph with respect to A2M, but those skilled in the art of proteinaceous prodrug constructs will recognize that other CPAMD proteins can be substituted for A2M in order to carry out the present invention (for example, using the residue numbers provided in Table 1 as a guide), and that the corresponding residues can be identified in these CPAMD proteins.

[0229] The inventors have found that protein prodrug constructs in which a drug (e.g., a therapeutic peptide, polypeptide, or protein) is positioned within loop 2 or 3 (or loop 4) of the RBD domain of the CPAMD protein (e.g., by replacing one or more residues, or by direct insertion) can be successfully expressed at high levels (see, for example, the protein fusion constructs referred to herein as "ciRBD" and "miRBD"). For example, insertion of the drug between amino acids corresponding to residues 1402 and 1403 of native human A2M was found to be particularly advantageous. Replacing amino acids corresponding to residues 1393-1395 of native human A2M with the drug may be similarly advantageous.

[0230] Linker A linker can be used to insert a drug into a protein prodrug construct. Any suitable linker can be used. In some embodiments, the linker used to insert a drug into a protein prodrug construct is a GS linker. In some embodiments, the linker is (GGGGS)n (SEQ ID NO: 223) or (GGS)n. In some embodiments, n = 1, 2, 3, 4, 5, or 6.

[0231] In some embodiments, the second protease cleavage site is located between one or more linkers, such as two linkers. The linkers can have any suitable length. The linkers include, for example, a selection of small nonpolar (e.g., glycine, alanine) or polar (e.g., serine or lysine) amino acids. In some embodiments, one or more linkers are GS linkers. In some embodiments, at least one of the two linkers is a GS linker. In some embodiments, both linkers are GS linkers.

[0232] Linkers may have a length of 1 to 5 amino acids, e.g., 2, 3, or 4 amino acids. Alternatively, linkers may have a length of 5 to 30 amino acids, e.g., 5 to 25 amino acids. For example, linkers may be GS linkers having a length of 5 to 30 amino acids or 5 to 25 amino acids.

[0233] The representative second protease cleavage sites used in Examples 12 and 13 are inserted, for example, using the linker combinations described above. For example, the furin cleavage site RRRR (SEQ ID NO: 225) was inserted by including the N-terminal linker KASGSS (SEQ ID NO: 248) and a single serine residue from the C-terminus to the furin cleavage site. Thus, one representative sequence of the second protease cleavage site may also be, or include, the amino acid sequence KASGSSRRRRS (SEQ ID NO: 249).

[0234] The TEV protease cleavage site was inserted by including a single serine residue from the N-terminus to the TEV protease cleavage site and a GS linker having the amino acid sequence SSGS (SEQ ID NO: 250) from the C-terminus to the TEV protease cleavage site. Therefore, one representative sequence of the second protease cleavage site may be or include the amino acid sequence SENLYFQSSGS (SEQ ID NO: 251). In some embodiments, the second protease cleavage site is or includes the amino acid sequence SENLYFQSSGS (SEQ ID NO: 253). In some embodiments, the second protease cleavage site is or includes the amino acid sequence SGGSENLYFQS (SEQ ID NO: 254). In some embodiments, the second protease cleavage site is or includes the amino acid sequence SGGGSENLYFQSSGS (SEQ ID NO: 255). In some embodiments, the second protease cleavage site is or includes the amino acid sequence SGGSGGSGGSGENLYFQSSGS (SEQ ID NO: 256). In some embodiments, the second protease cleavage site is or includes the amino acid sequence SGGSGGSGGSGENLYFQSSGGSGGS (SEQ ID NO: 257).

[0235] Those skilled in the art will recognize that the specificity derives from the arrangement of the protease cleavage sites, and therefore the composition of the linker surrounding the protease cleavage sites can be easily replaced.

[0236] Representative protein fusion or prodrug constructs As illustrated by the following sequence, the prodrugs of the present invention can take many forms. In some embodiments, the proteinaceous prodrug is encoded by a single nucleic acid as a continuous peptide chain. In some embodiments, comprising at least one biopharmaceutical moiety, the RBD released from the CPAMD protein consists of a single protein chain when the first protease cleavage site is proteolytically cleaved.

[0237] Certain embodiments may involve a portion of a protein shown in the examples, such as a bispecific prodrug containing two antibodies and a TEV protease site, as illustrated in either SEQ ID NO: 231-A2M_tevRBD+2xAb or SEQ ID NO: 235-A2M_tevRBD+2xAb_2. In another embodiment, the protein fusion construct has at least about 80% sequence identity, such as at least about 85% sequence identity, about 90% sequence identity, or even more than about 95% sequence identity, to a sequence selected from the group consisting of SEQ ID NO: 231 and SEQ ID NO: 235.

[0238] In other specific embodiments of the present invention, intermediate proteins in which only a second cleavage site is inserted are possible, such as either SEQ ID NO: 227-A2M_furin_RBD or SEQ ID NO: 229-A2M_tevRBD. As will be apparent to those skilled in the art, these intermediate products can be combined with any of the prodrugs shown in Examples 1 to 11.

[0239] In one embodiment, the protein fusion construct is composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 5, 7, 9, 11, 13, 15, and 17, 19, 21, 23, and 25.

[0240] In another embodiment, the protein fusion construct has at least about 80% sequence identity, such as at least about 85% sequence identity, about 90% sequence identity, or even up to about 95% sequence identity, with a sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, and 17, 19, 21, 23, and 25.

[0241] In one embodiment, the amino acid sequence is encoded by a nucleic acid sequence selected from the group consisting of fragments or variants of SEQ ID NOs. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26: or SEQ ID NOs. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 having at least about 90% sequence identity, and particularly about 95% identity to any one of SEQ ID NOs. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26. In one embodiment, the amino acid sequence is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26: or by a fragment or variant thereof having at least about 90% sequence identity with any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, and particularly about 95% identity with any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.

[0242] Exemplary nucleic acids In one embodiment, the present invention relates to nucleic acids encoding protein fusion constructs according to the present invention. Such specific examples could be nucleic acids encoding bispecific prodrugs comprising two antibodies and TEV protease sites, as presented, for example, in SEQ ID NO: 232-A2M_tevRBD+2xAb or SEQ ID NO: 236-A2M_tevRBD+2xAb_2.

[0243] In the context of the present invention, particular embodiments relate to nucleic acids and vectors containing components relevant as intermediates when developing prodrugs. Such intermediates are, for example, prodrugs or nucleic acids encoding prodrugs, which contain a second protease cleavage site without the insertion of a biopharmaceutical moiety. Thus, in another embodiment, the present invention relates to nucleic acids or plasmid-like vectors encoding tetrameric multimeric CPAMD proteins or fragments or variants, such as A2M protein, CPAMD protein or fragments or variants thereof, which contain a second protease cleavage site at the N-terminus of the RBD domain of the CPAMD protein.

[0244] Such intermediates may be described elsewhere herein, and therefore, in some embodiments, the CPAMD protein or a fragment or variant thereof comprises a bait region, the bait region comprising at least one first protease cleavage site. In some embodiments, the CPAMD protein or a fragment thereof comprises a receptor-binding domain (RBD).

[0245] Specific examples of possible intermediate products are provided, for example, in either SEQ ID NO: 228 or SEQ ID NO: 230.

[0246] In another embodiment, the nucleic acid sequence is selected from the group consisting of fragments or variants thereof having at least about 90% sequence identity with any one of SEQ ID NOs: 232, 236, 228, and 230, and in particular about 95% identity with any one of SEQ ID NOs: 232, 236, 228, and 230. In another embodiment, the nucleic acid sequence is selected from the group consisting of fragments or variants having at least about 90% sequence identity to any one of SEQ ID NOs: 6, SEQ ID NOs: 8, SEQ ID NOs: 10, SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16, SEQ ID NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, SEQ ID NOs: 24, and SEQ ID NOs: 26, or at least about 90% sequence identity to any one of SEQ ID NOs: 6, SEQ ID NOs: 8, SEQ ID NOs: 10, SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16, SEQ ID NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, SEQ ID NOs: 24, and SEQ ID NOs: 26, particularly about 95% identity to any one of SEQ ID NOs: 6, SEQ ID NOs: 8, SEQ ID NOs: 10, SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16, SEQ ID NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, SEQ ID NOs: 24, and SEQ ID NOs: 26. In another embodiment, the nucleic acid is a denatured sequence of any of the nucleic acid sequences, where the nucleic acid sequence exhibits higher variability than at the protein level.

[0247] In one embodiment, the nucleic acid according to the present invention encodes a protein fusion construct according to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and any one of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25.

[0248] In another embodiment, the nucleic acid sequence is selected from the group consisting of fragments or variants thereof having at least about 90% sequence identity to any one of sequence numbers 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, and in particular about 95% identity to any one of sequence numbers 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26. In another embodiment, the nucleic acid sequence is selected from the group consisting of fragments or variants having at least about 90% sequence identity to any one of SEQ ID NOs: 6, SEQ ID NOs: 8, SEQ ID NOs: 10, SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16, SEQ ID NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, SEQ ID NOs: 24, and SEQ ID NOs: 26, or at least about 90% sequence identity to any one of SEQ ID NOs: 6, SEQ ID NOs: 8, SEQ ID NOs: 10, SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16, SEQ ID NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, SEQ ID NOs: 24, and SEQ ID NOs: 26, particularly about 95% identity to any one of SEQ ID NOs: 6, SEQ ID NOs: 8, SEQ ID NOs: 10, SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16, SEQ ID NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, SEQ ID NOs: 24, and SEQ ID NOs: 26. In another embodiment, the nucleic acid is a denatured sequence of any of the nucleic acid sequences, where the nucleic acid sequence exhibits higher variability than at the protein level.

[0249] vector For a protein fusion construct to be expressed, the nucleic acid according to the present invention is inserted into or can be inserted into an expression vector, which is typically a plasmid or virus designed to regulate gene expression in cells. The vector is engineered to contain a regulatory sequence that acts as an enhancer or promoter for the efficient expression of the desired coding sequence contained within the vector. In non-limiting examples, the use of a naked circular plasmid having the essential functions necessary for expression, including a promoter, the coding sequence of interest, and a polyadenylation signal, is provided.

[0250] Furthermore, to facilitate production, which may be carried out using E. coli, the plasmid includes a selection marker. This allows for bacterial production with or without the use of conventional bacterial resistance selection.

[0251] In another embodiment, the present invention relates to a vector comprising nucleic acid according to the present invention.

[0252] In one embodiment, a nucleic acid encoding a protein fusion construct is operably linked to a promoter and, optionally, to a regulatory sequence that modulates the expression of the nucleic acid.

[0253] In one embodiment, the vector is a eukaryotic expression vector, particularly a mammalian expression vector, such as a human expression vector. In one embodiment, the vector is selected from the group consisting of plasmids, cosmids, phages, bacterial artificial chromosomes (BACs), phagemids, and P1-derived artificial chromosomes.

[0254] In one embodiment, the vector is a plasmid. In one embodiment, the plasmid is selected from the group consisting of TA cloning vectors, Gateway cloning vectors, restriction cloning vectors, Topo cloning vectors, pET vector systems, and pBAD vector systems.

[0255] host cell A vector according to the present invention can be inserted into a host cell for the expression of a protein fusion construct according to the present invention.

[0256] In one embodiment, the present invention relates to a host cell containing a vector according to the present invention.

[0257] The cells can be prokaryotic cells, such as bacteria, or eukaryotic cells.

[0258] In one embodiment, the host cell is selected from the group consisting of bacteria and eukaryotes; typically, the host cell is a eukaryote.

[0259] In another embodiment, the host cell is yeast.

[0260] In a typical embodiment, the host cell is a mammalian cell, such as a CHO (Chinese hamster) cell.

[0261] In one embodiment, the host cells are human.

[0262] In another embodiment, the host cells are or are derived from the HEK293 cell line.

[0263] composition Further aspects of this disclosure relate to compositions comprising proteinaceous prodrug constructs described herein. Compositions comprising nucleic acids, vectors, or host cells described herein are also provided.

[0264] In one embodiment, the composition comprises a pharmaceutically acceptable carrier. Such a composition may also be called a pharmaceutical composition.

[0265] therapeutic use Protein fusion constructs according to the present invention can be used in the treatment of diseases. In further embodiments, compositions, nucleic acids, vectors, or host cells described herein can be used in the treatment of diseases.

[0266] In one aspect, the present invention relates to a proteinaceous prodrug construct for use in therapy, for example, as a drug. In a further aspect, the present invention relates to compositions, nucleic acids, vectors, or host cells described herein for use in therapy, as a drug.

[0267] In some embodiments, the protein prodrug constructs according to the present invention are intended for use in treating diseases or disorders of the nervous system, eye, circulatory system, respiratory system, digestive system, or skin. In some embodiments, the disease or disorder is a neoplasm, hematological disorder, metabolic disorder, autoimmune disease, immunodeficiency, or infectious disease. In some embodiments, the neoplasm is a cancer selected from brain cancer, glioblastoma, lung cancer, colorectal cancer, skin cancer, malignant melanoma, pancreatic cancer, bladder cancer, liver cancer, breast cancer, eye cancer, and prostate cancer; the cancer is a hematological cancer selected from the group consisting of multiple myeloma, acute myeloblastic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia; or the cancer is malignant melanoma, breast cancer, non-small cell lung cancer, pancreatic cancer, head and neck cancer, liver cancer, sarcoma, and B-cell lymphoma. In some embodiments, the autoimmune disease is selected from arthritis (e.g., rheumatoid arthritis or psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus, and inflammatory bowel disease.

[0268] In one embodiment, a protein prodrug construct according to the present invention is intended for use in the treatment of cancer. Therefore, at least one or more protease cleavage sites are specific to proteases expressed by cancer. In another embodiment, a protein prodrug construct according to the present invention is intended for use in the treatment of arthritis. In a further embodiment, a composition, nucleic acid, vector, or host cell according to the present invention is intended for use in the treatment of cancer. In yet another embodiment, a composition, nucleic acid, vector, or host cell according to the present invention is intended for use in the treatment of arthritis.

[0269] The protein prodrug constructs, compositions, nucleic acids, vectors, or host cells described herein can also be used in treatment methods. In other embodiments, this disclosure relates to a treatment method comprising administering a therapeutic dose of the protein prodrug constructs, compositions, nucleic acids, vectors, or host cells described herein to a subject in need thereof. The subject in need thereof may be a subject suffering from cancer or arthritis.

[0270] In one embodiment, the cancer is a solid tumor. In some embodiments, the cancer is selected from a list consisting of brain cancer, glioblastoma, lung cancer, colorectal cancer, skin cancer, malignant melanoma, pancreatic cancer, bladder cancer, liver cancer, breast cancer, eye cancer, and prostate cancer.

[0271] In another embodiment, the cancer is a hematological cancer selected from the group consisting of multiple myeloma, acute myeloblastic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia.

[0272] In further embodiments, the cancer is malignant melanoma, breast cancer, non-small cell lung cancer, pancreatic cancer, head and neck cancer, liver cancer, sarcoma, or B-cell lymphoma.

[0273] In some embodiments, the proteinaceous prodrug construct according to the present invention comprises a CPAMD protein (e.g., A2M) having a modified bait region. In some embodiments, the bait region is modified to alter the selection of proteases that can cleave the bait region and trigger a structural change in the CPAMD protein (e.g., A2M). For example, the bait region is modified to be cleaved by a specific protease or class of proteases (e.g., an MMP such as MMP2). In other embodiments, the second protease cleavage site is selected to induce release specificity.

[0274] In one embodiment, cancer expresses one or more proteases that are specific to the cleavage site in the bait region of the CPAMD protein (e.g., A2M). In a particular embodiment, the proteinaceous prodrug construct according to the present invention comprises a CPAMD protein (e.g., A2M) containing a modified bait region that can be selectively cleaved by one or more proteases expressed by cancer.

[0275] In one embodiment, cancer is controlled by activated protein C, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM9, ADAMMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, BMP-1, caspase 1, caspase 10, caspase 14, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Kymase, Cruzipain, DESC1, DPP-4, Elastase, FAP, Granzyme B, Guanidinobenzoate, Hepsin, HtrA1, Neutrophil elastase, KLK10, KLK11, KLK13, KLK14 KLK4, KLK5, KLK6, KLK7, KLK8, lactoferrin, regmine, malapsin, matryptase-2, meprin, MMP1, MMP8, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP2, MMP20, MMP23, MMP24, MMP26, MMP27, MMP3, MMP7, MMP8, MMP9, MT-S It expresses one or more proteases selected from the list consisting of P1 / matryptase, neprilysin, NS3 / 4A, otubein-2, PACE4, plasmin, PSA, PSMA, renin, thrombin, TMPRSS2, TMPRSS3, TMPRSS4, tPA, tryptase, uPA, ADAM8, FVIIa, FIXa, furin, Fxa, FXIa, FXIIa, and TAFI.

[0276] Another aspect relates to a method of treatment, in which an appropriate biopharmaceutical portion and, optionally, a protease for a first and / or second protease cleavage site are selected, and then the final construct is designed, and therefore, as appropriate, an intermediate nucleic acid is selected as described above. Thus, in another aspect, the present invention is 1. To provide the above nucleic acids or vectors as intermediates; 2. Determining a first biopharmaceutical portion suitable for treating the target, or determining first and second biopharmaceutical portions, and, if applicable, determining appropriate first and / or second protease cleavage sites; 3. Inserting the first and / or second biopharmaceutical moieties, as well as any first and / or second protease cleavage sites of step 2, into the nucleic acid or vector such that the nucleic acid encodes a proteinaceous prodrug according to the present invention; 4. Introducing nucleic acids or vectors into host cells; 5. Growing host cells under conditions that enable the expression of protein fusion constructs from nucleic acids or vectors; 6. Purifying the protein prodrug and / or cleaving the second protease cleavage site, as applicable; and 7. Administering protein prodrugs to subjects who require them. This concerns methods for treating objects that require treatment, including those that need treatment.

[0277] Similar embodiments relate to nucleic acids administered as drugs, and therefore, in further embodiments, this disclosure 1. To provide the above nucleic acids or vectors as intermediates; 2. Determining a first biopharmaceutical portion suitable for treating the target, or determining first and second biopharmaceutical portions, and, if applicable, determining appropriate first and / or second protease cleavage sites; 3. Inserting the first and / or second biopharmaceutical moieties, as well as any first and / or second protease cleavage sites of step 2, into the nucleic acid or vector such that the nucleic acid encodes a proteinaceous prodrug according to the present invention; 4. Administering nucleic acids to those who need them. This concerns methods for treating objects that require treatment, including those that need treatment.

[0278] Target and Administration The “Subjects” as described herein include humans of all ages, other primates (e.g., crab-eating macaques, rhesus macaques); mammals in general, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, minks, ferrets, hamsters, cats, and dogs; and / or birds. In typical embodiments, the subject is human.

[0279] The term "target" includes healthy individuals within the population, particularly healthy individuals such as healthcare workers who are exposed to pathogens and require protection against infection.

[0280] Furthermore, pathogenic infections caused by respiratory viruses can be particularly serious in elderly and frail patients, as well as in patients with chronic or congenital respiratory disorders such as asthma, cystic fibrosis, or chronic obstructive pulmonary disease (COPD).

[0281] Accordingly, in embodiments of the present invention, the subjects are selected from the group consisting of humans of all ages, other primates (e.g., crab-eating macaques, rhesus macaques); mammals in general, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, minks, ferrets, hamsters, cats and dogs, and birds.

[0282] In certain embodiments, the subject is a human being.

[0283] Method for preparing protein fusion constructs Methods for producing the fusion protein and proteinaceous prodrug constructs of the present invention are also provided herein.

[0284] In some embodiments, a method for producing a protein prodrug construct of the present invention comprises providing a host cell containing a nucleic acid encoding the protein prodrug construct, and culturing the host cell under conditions that enable the expression of the protein prodrug construct from the nucleic acid. In some embodiments, the protein prodrug construct is contacted with a protease (e.g., a TEV protease) that specifically cleaves a protease cleavage site provided at the N-terminus of the RBD (e.g., a second protease cleavage site such as a TEV cleavage site).

[0285] In some embodiments, a method for producing a proteinaceous prodrug construct of the present invention comprises providing a proteinaceous prodrug construct and contacting it with a protease (e.g., a TEV protease) that specifically cleaves a protease cleavage site provided at the N-terminus of the RBD (e.g., a second protease cleavage site such as a TEV cleavage site).

[0286] In some embodiments, a method for producing a proteinaceous prodrug construct of the present invention includes one or more purification steps using chromatography, such as size exclusion chromatography.

[0287] In some embodiments, the present invention is 1. To provide nucleic acids encoding the prodrugs described herein, or vectors encoding the prodrugs described herein; 2. Introducing nucleic acids or vectors into host cells; 3. Growing host cells under conditions that enable the expression of protein fusion constructs from nucleic acids or vectors; 4. If necessary, purify the protein prodrug and / or cleave the second protease cleavage site. The present invention relates to a method for producing a protein fusion construct containing the present invention.

[0288] In some embodiments, the present invention is 1. To provide the above nucleic acids or vectors as intermediates; 2. Determining the first biopharmaceutical portion or the first and second biopharmaceutical portions, and, if applicable, determining the appropriate first and / or second protease cleavage sites; 3. Inserting the first and / or second biopharmaceutical moieties, as well as any first and / or second protease cleavage sites of step 2, into the nucleic acid or vector such that the nucleic acid encodes a proteinaceous prodrug according to the present invention; 4. Introducing nucleic acids or vectors into host cells; 5. Growing host cells under conditions that enable the expression of protein fusion constructs from nucleic acids or vectors; 6. If necessary, purify the protein prodrug and / or cleave the second protease cleavage site. The present invention relates to a method for producing a protein prodrug containing [a specific substance].

[0289] In some embodiments, it is particularly appropriate to cleave the second protease cleavage site as part of the production method, because, as previously described, the application of polyvalent molecules can be harmful.

[0290] therefore, (a) To provide a host cell containing nucleic acid encoding a protein fusion construct according to the present invention; (b) Culturing host cells under conditions that enable the expression of a protein prodrug construct encoded by nucleic acid; (c) Contacting the protein prodrug construct with a protease that specifically cleaves the second protease cleavage site. Methods for producing such proteinaceous prodrugs, including the above, are provided herein.

[0291] Numbered Embodiments The present invention is further described by reference to the following numbered embodiments. 1. A proteinaceous prodrug construct comprising a complement 3 and pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein, such as A2M, or a fragment thereof, wherein the CPAMD protein or fragment thereof is (a) A bait region containing at least one first protease cleavage site; (b) Receptor-binding domain (RBD); (c) A second protease cleavage site introduced at the N-terminus of the RBD domain of the CPAMD protein, wherein the RBD domain remains bound to the CPAMD protein by non-covalent interactions even after the second protease cleavage site is cleaved; and (d) At least one biopharmaceutical moiety located from the C-terminus to a second protease cleavage site, wherein cleavage of the first protease cleavage site releases the RBD domain and thereby the at least one biopharmaceutical moiety from the CPAMD protein. A protein prodrug construct containing the above. 2. The proteinaceous prodrug construct according to Embodiment 1, wherein at least one biopharmaceutical moiety is a first biopharmaceutical moiety located within the RBD region and a second biopharmaceutical moiety located from the C-terminus to the CPAMD protein, the CPAMD protein or a fragment thereof shields the first biopharmaceutical moiety, and the CPAMD protein or a fragment thereof changes its structure when the first protease cleavage site is proteolytically cleaved, thereby releasing the RBD from the CPAMD protein, and thereby the first and second biopharmaceutical moieties, making the first biopharmaceutical moiety reachable. 3. A protein prodrug construct according to any one of Embodiments 1 to 2, wherein at least one of the biopharmaceutical portions can direct the protein prodrug construct to a specific tissue, a specific cell type, and / or a specific receptor. 4. A protein prodrug construct according to any one of embodiments 1 to 3, wherein at least one of the biopharmaceutical parts is capable of directing the protein prodrug construct to immune cells. 5. The protein prodrug construct according to Embodiment 4, wherein the immune cells are NK cells, macrophages, T cells, or dendritic cells. 6. A protein prodrug construct according to any one of Embodiments 1 to 5, wherein the biopharmaceutical portion is T cell specific, such as a T cell specific portion. 7. The protein prodrug construct according to Embodiment 6, wherein the T cell-specific portion is specific to a receptor expressed at increased levels on T cells, such as CD3, CD4 and / or CD8, preferably CD3. 8. A protein prodrug construct according to any one of embodiments 6 to 7, wherein the T cell-specific portion is an anti-CD3 portion. 9. A protein prodrug construct according to any one of Embodiments 1 to 5, wherein the biopharmaceutical portion is NK cell specific, such as an NK cell specific portion. 10. The protein prodrug construct according to Embodiment 9, wherein the NK cell-specific portion is specific to a receptor such as CD16, which is expressed at increased levels on NK cells. 11. The biopharmaceutical portion is a macrophage-specific protein prodrug construct according to any one of Embodiments 1 to 5, such as a macrophage-specific portion. 12. The protein prodrug construct according to Embodiment 11, wherein the macrophage-specific portion is specific to a receptor or molecule expressed at increased levels on macrophages, such as a SIRPα antibody that blocks CD47 or a SIRP-suppressing antibody. 13. The biopharmaceutical portion is a protein prodrug construct according to any one of Embodiments 1 to 5, which is specific to dendritic cells, such as an antibody against the DNGR1 receptor, such as the DNGR1 receptor, such as a dendritic cell-specific portion that is specific to a receptor or molecule expressed at increased levels on macrophages, such as the DNGR1 receptor, such as an antibody against the DNGR1 receptor, such as a dendritic cell-specific portion. 14. A proteinaceous prodrug construct according to any one of embodiments 1 to 13, wherein at least one of the biopharmaceutical parts is a drug. 15. A proteinaceous prodrug construct according to any of Embodiments 9 to 10, wherein the drug, upon binding to a receptor, can increase or decrease the signal from that receptor. 16. A proteinaceous prodrug construct according to any of Embodiments 1 to 15, wherein RBD is a bispecific drug when released from the CPAMD protein. 17. The bispecific drug is a protein prodrug construct according to Embodiment 16, comprising one biopharmaceutical moiety capable of directing the protein prodrug construct to immune cells according to any one embodiment of Embodiments 3 to 13 and a drug according to any one embodiment of Embodiments 14 to 16. 18. A bispecific drug is a protein prodrug construct according to Embodiment 12, comprising at least two drugs described in any of Embodiments 14 to 16. 19. A protein prodrug construct according to any one of embodiments 16 to 17, wherein the bispecific drug is a bispecific T cell engager (BiTE), a bispecific NK cell engager (BiKE), a bispecific macrophage engager (BiME), or a bispecific dendritic cell engager (BiDE). 20. The bispecific drug is a bispecific T cell engager (BiTE), a protein prodrug construct according to any one of embodiments 16 to 17. 21. A proteinaceous prodrug construct according to any one of Embodiments 1 to 20, wherein at least one biopharmaceutical portion is a protein such as an ScFv or a single-domain antibody, such as an antigen-binding fragment of an antibody. 22. A protein prodrug construct according to any one of Embodiments 1 to 21, wherein the biopharmaceutical portion is an antigen-binding fragment of an antibody, such as a single-domain antibody, that specifically binds to an antigen selected from the group consisting of IL-2, EGFR, PDL-1, PD-1, CTLA-4, CD3γε, 4-1BB, IL-2Rα, and TNFα. 23. A protein prodrug construct according to any one of Embodiments 1 to 22, wherein the biopharmaceutical portion is selected from the group consisting of atezolizumab, EgA1, ipilimumab, nivolumab, KN035, urerumab, foralumab, muromonab, adalimumab, and their respective therapeutically active antigen-binding fragments or variants. 24. The biopharmaceutical portion is a cytokine selected from the group consisting of IL1, IL1α, IL1β, IL2, IL3, IL4, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36, GM-CSF, TGF-β, CSF-1, insulin, GLP-1, HGH, VEGF, PDGF, BMP, EPO, G-CSF, IL-11, IFN-α, IFN-β, and IFN-γ, or a therapeutically active fragment or variant thereof, as described in any of Embodiments 1 to 23. 25. The CPAMD protein is selected from the group consisting of CPAMD1(akaC3), CPAMD2(akaC4A), CPAMD3(akaC4B), CPAMD4(akaC5), CPAMD5(akaA2M), CPAMD6(akaPZP), CPAMD7(akaCD109), CPAMD8, CPAMD9(akaA2ML1), ovostatin 1, and ovostatin 2, and is a protein prodrug construct according to any one of Embodiments 1 to 24. 26. A protein prodrug construct according to any one of Embodiments 1 to 25, wherein the biopharmaceutical portion is inserted between residues 1402 to 1403 of A2M. 27. A protein prodrug construct according to any one of Embodiments 1 to 26, wherein the protein prodrug is encoded by a single nucleic acid as a continuous peptide chain, preferably at least one biopharmaceutical moiety, and the RBD consists of a single protein chain when the first protease cleavage site is proteolytically cleaved. 28. A protein prodrug construct according to any one of embodiments 1 to 27, wherein the second protease cleavage site is located between two linkers, such as a linker of 5 to 30 amino acids in length, a linker of 5 to 25 amino acids in length, a GS linker of 5 to 30 amino acids in length, and a GS linker of 5 to 25 amino acids in length. 29. A proteinaceous prodrug construct according to any one of Embodiments 1 to 28, wherein the first protease cleavage site and the second protease cleavage site are specific to the same protease. 30. A proteinaceous prodrug construct according to any one of Embodiments 1 to 29, wherein the second protease cleavage site is specific to furin, TEV, enterokinase, or thrombin. 31. A proteinaceous prodrug construct according to any one of Embodiments 1 to 30, wherein the second protease cleavage site introduced at the N-terminus of the RBD domain of the CPAMD protein is introduced from residue positions 1334-1340 of A2M. 32. A proteinaceous prodrug construct according to any one of Embodiments 1 to 31, wherein the first protease cleavage site is specific to serine-, cysteine-, aspartate-, and / or metalloproteinase. 33. A plasmid-like nucleic acid or vector encoding a CPAMD protein or fragment thereof, such as a tetramer-like multimeric CPAMD protein or a fragment thereof, wherein the CPAMD protein or fragment thereof contains a second protease cleavage site at the N-terminus of the RBD domain of the CPAMD protein. 34. The nucleic acid according to Embodiment 33, wherein the CPAMD protein or a fragment thereof comprises a bait region, the bait region comprising at least one first protease cleavage site. 35. The nucleic acid according to any one of embodiments 33 to 34, wherein the CPAMD protein or a fragment thereof comprises a receptor-binding domain (RBD). 36. A nucleic acid encoding a proteinaceous prodrug construct according to any one of Embodiments 1 to 32. 37. A plasmid-like vector comprising the nucleic acid described in Embodiment 36. 38. A host cell comprising the nucleic acid described in Embodiment 36 or the vector described in Embodiment 37. 39. The host cell according to Embodiment 38, wherein the host cell is a bacterium or a eukaryote, for example, a mammalian cell. 40. A proteinaceous prodrug construct according to any of Embodiments 1 to 32, a nucleic acid according to Embodiment 36, a vector according to Embodiment 37, or a host cell according to any of Embodiments 38 to 39, for use as a pharmaceutical agent. 41. A protein prodrug construct according to any of Embodiments 1 to 32, a nucleic acid according to Embodiment 36, a vector according to Embodiment 37, or a host cell according to any of Embodiments 38 to 39, for use in the treatment of diseases or disorders of the nervous system, eye, circulatory system, respiratory system, digestive system, or skin. 42. A protein prodrug construct according to any of Embodiments 1 to 32, a nucleic acid according to Embodiment 36, a vector according to Embodiment 37, or a host cell according to any of Embodiments 38 to 39, for use in the treatment of neoplasms, hematological disorders, metabolic disorders, autoimmune diseases, immunodeficiency, or infectious diseases. 43. The neoplasm is a cancer selected from brain cancer, glioblastoma, lung cancer, colorectal cancer, skin cancer, malignant melanoma, pancreatic cancer, bladder cancer, liver cancer, breast cancer, eye cancer, and prostate cancer, and the cancer is a hematological cancer selected from the group consisting of multiple myeloma, acute myeloblastic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia, or the cancer is a protein prodrug construct, nucleic acid, vector, or host cell for use as described in Embodiment 42, wherein the neoplasm is a cancer selected from brain cancer, glioblastoma, lung cancer, colorectal cancer, skin cancer, malignant melanoma, liver cancer, sarcoma, and B-cell lymphoma. 44. An autoimmune disease is selected from arthritis (e.g., rheumatoid arthritis or psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus, and inflammatory bowel disease, and is a protein prodrug construct, nucleic acid, vector, or host cell for use as described in Embodiment 42. 45.1. To provide nucleic acids or vectors according to any of embodiments 33 to 35; 2. Determining a first biopharmaceutical portion or a first and second biopharmaceutical portion suitable for treating the target, and, if applicable, determining appropriate first and / or second protease cleavage sites; 3. Insert the first and / or second biopharmaceutical moieties, as well as any of the first and / or second protease cleavage sites of step 2, into the nucleic acid or vector such that the nucleic acid encodes the proteinaceous prodrug described in any of Embodiments 1 to 32; 4. Introducing nucleic acids or vectors into host cells; 5. Growing host cells under conditions that enable the expression of protein fusion constructs from nucleic acids or vectors; 6. Purifying the protein prodrug and / or cleaving the second protease cleavage site, as applicable; and 7. Administering a protein prodrug to the subject who needs it [US treatment method] A method for treating objects that require treatment, including those that need treatment. 46.1. To provide nucleic acids or vectors according to any of embodiments 33 to 35; 2. Determining a first biopharmaceutical portion or a first and second biopharmaceutical portion suitable for treating the target, and, if applicable, determining appropriate first and / or second protease cleavage sites; 3. Insert the first and / or second biopharmaceutical moieties, as well as any of the first and / or second protease cleavage sites of step 2, into the nucleic acid or vector such that the nucleic acid encodes the proteinaceous prodrug described in any of Embodiments 1 to 32; 4. Administering nucleic acids to those who need them [US treatment method] A method for treating objects that require treatment, including those that need treatment. 47.1. To provide nucleic acids or vectors according to any of embodiments 33 to 35; 2. Determining the first biopharmaceutical component or determining the first and second biopharmaceutical components; 3. Inserting the first and / or second drug biopharmaceutical moieties into the nucleic acid or vector such that the nucleic acid encodes the proteinaceous prodrug described in any of Embodiments 1 to 32; 4. Introducing nucleic acids or vectors into host cells; 5. Growing host cells under conditions that enable the expression of protein fusion constructs from nucleic acids or vectors; 6. If necessary, purify the protein prodrug and / or cleave the second protease cleavage site. A method for producing a protein-based prodrug containing [a specific substance]. 48.1. To provide the nucleic acid described in Embodiment 36, or the vector described in Embodiment 37; 2. Introducing nucleic acids or vectors into host cells; 3. Growing host cells under conditions that enable the expression of protein fusion constructs from nucleic acids or vectors; 4. If necessary, purify the protein prodrug and / or cleave the second protease cleavage site. A method for producing a protein-based prodrug containing [a specific substance]. 49. A method for treating or preventing a disease or disorder in a subject that needs to be treated or prevented, comprising administering a therapeutically effective amount of a protein prodrug construct according to any of Embodiments 1 to 32, a nucleic acid according to Embodiment 36, a vector according to Embodiment 37, or a host cell according to any of Embodiments 38 to 39 to the subject.

[0292] Equal parts Methods and materials similar to or equivalent to those described herein may be used in the execution or testing of the present invention, but suitable methods and materials are listed below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. References cited herein are not considered prior art to the claimed invention. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0293] Examples The present invention is now described in more detail in the following non-limiting embodiments. [Examples]

[0294] Summary of the present invention This paper illustrates the overall design and mechanism of the present invention, which relates to a technique for producing protease-activated prodrug versions of biopharmaceuticals. In Figure 1A, a proteinoid prodrug construct (1) comprises a CPAMD protein, e.g., human alpha-2-macroglobulin (A2M)(2), fused to one or more drugs (3), such that drug accessibility depends on the structural state of the CPAMD protein (e.g., A2M)(2). The CPAMD protein (e.g., A2M)(2) is converted from its initial "native" structure to an "activated" structure by one or more proteases (4). The drug (3) can genetically fuse to the CPAMD protein (e.g., A2M)(2) at a location where the drug is inaccessible to its therapeutic target in the "native" structure (I) of the CPAMD protein (e.g., A2M)(2) but accessible in the "activated" structure (II) of the CPAMD protein (e.g., A2M)(2). In this way, the activity of one or more drugs (3) is spatially restricted to tissues that are proteolytically competent and that contain one or more proteases (4) capable of activating the CPAMD protein (e.g., A2M) (2). Since the CPAMD protein (e.g., A2M) (2) can be modified to be activated by one or more designated proteases (4), this technique enables drug targeting to tissues expressing disease-related proteases, e.g., diseased tissues, thereby potentially improving the efficacy of the drug while minimizing the side effects resulting from target binding to healthy tissue. [Examples]

[0295] Preparation of A2M and antibody protein fusion constructs the goal This data demonstrates the expression and purification of the A2M-antibody construct as a correctly folded tetrameric protein, indicating that A2M adopts a functional native structure containing thiol esters.

[0296] material and method Expression and purification of A2M antibody fusion constructs. The nucleotide sequences and corresponding amino acid sequences encoding the A2M-antibody fusion constructs are given (SEQ ID NOs. 5-22).

[0297] Protein fusion constructs were expressed in HEK293 FreeStyle cells using a standard transient transfection protocol. Briefly, 25 kDa linear polyethyleneimine (Polysciences) and plasmid DNA were incubated in antibiotic-free FreeStyle medium (Thermo Fisher Scientific) at a 4:1 w / w PEI to DNA ratio for 10 minutes, and then slowly added to cell cultures at a density of 1 million cells per mL to a final DNA concentration of 1 μg per mL. After 4 days, cells were centrifuged at 1500 × g, and the supernatant was collected by adding pH 7.4 HEPES to a final concentration of 50 mM.

[0298] The construct was purified using an established protocol for purifying A2M. The supernatant was first treated with Zn. 2+The eluent was run through a supported chelated HiTrap column (GE Healthcare) and eluted using 50 mM EDTA, 150 mM NaCl, 100 mM sodium acetate, and pH 7.4. The EDTA eluent was dialyzed against 20 mM HEPES at pH 7.4, then loaded onto a HiTrap Q column (GE Healthcare) and eluted with a gradient of 0 to 400 mM NaCl (with a constant 20 mM HEPES at pH 7.4). The fraction containing A2M was pooled, concentrated by ultrafiltration, and purified by size exclusion chromatography on a Sephacryl S-300 HR (GE Healthcare) using a running buffer (HEPES-buffered saline, HBS) with 20 mM HEPES, 150 mM NaCl, and pH 7.4.

[0299] SDS-PAGE and Pore-Restricted Native PAGE Native pore-restricted PAGE was performed as previously described (36) using a homemade gel in TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA) with an acrylamide gradient of 5–10% for A2M analysis and 10–15% for C3 analysis. The pore-restricted electrophoresis gel was run overnight in TBE buffer at 100V.

[0300] Modified SDS-PAGE was performed on homemade 5-15% acrylamide gradient gels using a discontinuous 2-amino-2-methyl-1,3-propanediol and glycine buffer system. Samples were reduced with 25 mM DTT at 95°C for 5 minutes.

[0301] Reaction of A2M with methylamine and protease To aminolyze the thiol ester of A2M, methylamine (pH 8) was added up to 250 mM and incubated at 37°C for 16 hours. To evaluate the cleavage of A2M by thermolysin, thermolysin was added to a protease-to-A2M ratio of 2.2:1 molar ratio and incubated at 37°C for 5 minutes. Digestion was then inhibited using EDTA (10 mM, 15 min, room temperature).

[0302] result Protein fusion constructs were prepared in yields of several mg / L by transient HEK293F transfection. After purification, the constructs migrated as native homotetramers on native PAGE (Figure 2A) and as approximately 190 kDa monomer subunits on reduced, denatured SDS-PAGE (Figure 2B). The presence of thiol esters in the constructs was confirmed by characteristic thermally induced fragmentation at the thiol ester sites, which produced visible Nt and Ct autodegradation fragments on SDS-PAGE (Figures 2B-C). These Nt and Ct autodegradation fragments disappeared when the thiol esters were aminolysed with methylamine prior to SDS-PAGE analysis. Proteolytic processing of the bait region was evaluated by treatment with thermolysin. The bait region of the constructs was preferentially cleaved by thermolysin, resulting in the formation of Nt and Ct cleavage fragments (Figures 2B-C). Bait region cleavage induced structural changes in the construct that increased its movement in native PAGE, but not to the same extent as wild-type A2M (Figure 2A); this is because the exposed antibody fragment increases the electrophoretic resistance experienced by the activated A2M-antibody construct.

[0303] conclusion The protein fusion construct of A2M and the antibody scFv is constructed as a homotetramer. In these proteins, the A2M component adopts its native structure, forms a thiol ester, and is preferentially cleaved by proteases in its bait region, thus remaining functionally normal. [Examples]

[0304] Structure dependence of binding in biolayer interferometry This embodiment demonstrates how structural changes in a protein fusion construct can control drug activity. In the native state, the drug is not exposed and is therefore inactive. In the active state, the drug is exposed and can interact with its target.

[0305] the purpose In binding experiments using purified antigens immobilized on a biosensor, the objective is to determine the antigen-binding ability of an A2M-antibody fusion construct, and to determine the extent to which this antigen-binding ability is influenced by the structure of A2M.

[0306] material and method Proteins used in binding studies Antigens for the antibodies under investigation were recombinantly expressed in HEK293F cells using a standard transient transfection protocol (see Example 1). The antigens were expressed together with the A2M leader peptide, an N-terminal Strep II tag, and a C-terminal Fc region derived from human IgG1 (uniprot ID P01857, residues 100-330, SEQ ID NO: 40). The residues included for each antigen, using the numbering before signal peptide removal, were as follows: • EGFR (uniprot ID P00533, residues 25-645, SEQ ID NO: 37) • PD-L1 (uniprot ID Q9NZQ7, residues 19-239, SEQ ID NO: 38) • PD-1 (uniprot ID Q15116, residues 26-150 with Cys93Ser mutation, SEQ ID NO: 39) CTLA-4 (uniprot ID P16410, residues 36-161, SEQ ID NO: 40) • CD3γε (uniprot ID P09693, γ chain residues 23-103 followed by 26 glycine-serine linker residues and uniprot ID P07766, ε chain residues 23-118, SEQ ID NO: 41) • 4-1BB (uniprot ID Q07011, residues 24-186, SEQ ID NO: 42)

[0307] The final sequences of these expressed and fused antigens are given as both amino acid and nucleotide sequences (SEQ ID NOs. 45-58).

[0308] An additional antigen, TNFα (uniprot P01375, residues 77-233), was expressed as a StrepII-tagged protein but lacked the C-terminal Fc region (SEQ ID NOs. 59, 60). This antigen was also purified by StrepTactin affinity chromatography and size exclusion chromatography on a Superdex 200 Increase to isolate the TNFα trimer.

[0309] The supernatant containing the expressed antigen was purified using StrepTactin affinity chromatography (Iba Life Sciences), followed by size exclusion chromatography on a Superdex 200 Increase (GE Healthcare).

[0310] The A2M-antibody fusion constructs were prepared as described in Example 1. Where noted, the native A2M-antibody was purified by affinity depletion of pre-activated A2M; see Example 5 for further details. The amino acid and nucleotide sequences of the A2M-antibodies are given (SEQ ID NOs. 5-22).

[0311] Reaction of A2M antibody with methylamine and protease When A2M-antibody was treated with methylamine, 200 mM methylamine (pH 8) was added to the A2M-antibody and incubated at 37°C for 16 hours. When A2M-antibody was treated with thermolysin, thermolysin derived from Geobacillus stearothermophilus (Sigma-Aldrich) was added to the A2M-antibody in a 2.2:1 molar ratio of protease to A2M and incubated at 37°C for 5 minutes. After that, thermolysin was inhibited by the addition of 25 mM EDTA.

[0312] Biolayer Interferometry HEPES-buffered saline (HBS; 20 mM HEPES, 150 mM NaCl, pH 7.4) was used as a buffer in all biolayer interferometry experiments. The antigen was immobilized on an anti-human Fc capture biosensor (AHC biosensor; Fortebio) at 30 nM in HBS for 20 minutes. Next, the A2M-antibody fusion constructs were incubated with the antigen-coated biosensor at various concentrations to measure association, followed by measurement of dissociation in HBS. Where stated, the A2M-antibody fusion constructs were activated by methylamine or protease treatment using the same method as in Example 1.

[0313] result The structure-dependent antigen binding by eight different A2M antibodies was evaluated using biolayer interferometry. Antigens were expressed as fusion proteins possessing a human IgG1 Fc region, and the antigens were immobilized on the biosensor surface using a standardized anti-human Fc capture biosensor. The A2M antibodies were then associated with the immobilized antigens either without any treatment of the A2M antibodies or by inducing structural changes in A2M using methylamine aminolysis and / or proteolysis with thermolysin. In some cases, the A2M antibodies were enriched for the native structure of A2M by affinity depletion using the antigen (PD-L1) or LRP1 resin, as described in the figure caption and detailed in Example 4.

[0314] In all antibodies examined, antigen binding was strongly dependent on the structure of A2M (Figure 3A-I), with thermolysin proteolysis of A2M consistently yielding the highest binding response. Methylamine treatment produced a variable binding response; in some cases, methylamine induced a binding response similar to proteolysis (Figure 3B, 3I), while in others, methylamine produced an intermediate response (Figure 3C, 3F-H) or a very slight response (Figure 3D). When the native structure of the A2M antibody was enriched by affinity depletion, there was little to no antigen binding detected in the native sample without methylamine / proteolysis (Figure 3A-D, 3F).

[0315] conclusion Antibodies incorporated into A2M fusion constructs retain their ability to bind to their cognitive antigens. This antigen binding is determined by the structure of A2M, with little to no antigen binding in the native structure of A2M. Activation of A2M by proteolytic cleavage significantly increased antigen binding, while activation by methylamine treatment varied depending on the A2M-antibody in question. [Examples]

[0316] Concentration of native A2M antibody constructs due to affinity depletion This embodiment demonstrates how modification of a protein fusion construct can be used to control where a drug is exposed. Depending on the cleavage site, the drug is exposed only at locations where a protease that recognizes that cleavage site is present. When a specific protease is present and cleaves the cleavage site, the structure of the protein fusion construct changes from "naive" to "active".

[0317] the purpose Recombinantly expressed A2M-antibody fusion constructs are not only made with A2M in its native structure, but also with trace amounts of components pre-activated. Here, we investigate whether these pre-activated components can be removed by affinity depletion using the antibody's cognitive antigen, activated A2M receptor LRP1, or kappa light chain binding protein L.

[0318] material and method The proteins used The A2M antibody was prepared as described in Example 1. Recombinant LRP1 (residues 20-974, SEQ ID NOs. 63-64) was prepared as a StrepII-tagged fusion protein containing the human IgG1 Fc region, as described for the antigen in Example 2. resin preparation The resin coated with LRP1 was prepared using amine-reactive chemistry. 200 mg of NHS-activated agarose (Pierce) and 600 μg of recombinant LRP1 were mixed in 0.15 M triethylammonium bicarbonate, 0.15 M HEPES, pH 8.3, on a rotating plate at room temperature for 2 hours. Following incubation, the resin was washed twice in HBS, and the reaction was quenched in 50 mM Tris-HCl, pH 8 for 20 minutes, followed by a final washing step using HBS. The resin coated with PD-L1 was prepared as described for LRP1. Protein L-coated agarose was purchased from Pierce (Thermo Scientific).

[0319] Affinity depletion To deplete the pre-activated A2M, A2M-antibody fusion constructs were incubated overnight with the resin at room temperature with shaking using a helicopter rotor, up to 2 mg / mL in HBS. For LRP1-based depletion, 10 mM CaCl2 was added to the HBS. After overnight incubation, the supernatant was collected, and the resin was regenerated using acidic elution with 25 mM EDTA in HBS for LRP1, or with pH 2.7 and 10 mM KH2PO4 buffer for PD-L1 and Protein L. The collected supernatant was tested using biolayer interferometry as described in Example 2.

[0320] result A2M-atezolizumab was incubated with a resin coated with its cognitive antigen, PD-L1. A single round of depletion was performed. Next, the binding of A2M-atezolizumab to PD-L1 before and after this depletion was evaluated using biolayer interferometry. A2M-atezolizumab from both pre- and post-depletion samples bound to PD-L1 similarly after methylamine treatment, but antigen binding from the untreated sample after depletion was significantly reduced compared to the untreated sample before depletion, indicating that PD-L1 depletion enriched the content of A2M-antibodies that had unreachable antibodies (Figure 4A).

[0321] A2M-ipilimumab, A2M-nivolumab, and A2M-urerumab were incubated with a resin coated with LRP1, a receptor that specifically binds to activated A2M but not to native A2M. Three rounds of depletion were performed for each A2M-antibody, and then their binding to CTLA-4, PD-1, or 4-1BB was evaluated using biolayer interference complement (Figures 4B-D). Antigen binding by untreated A2M-antibodies before LRP1 depletion was approximately 25% of the maximum binding defined by thermolysin activation for all three antibodies. After LRP1 depletion, there was no detectable antigen binding in the untreated A2M-ipilimumab and A2M-nivolumab samples, and only very little detectable antigen binding in the untreated A2M-urerumab sample. Equal binding was observed with thermolysin-activated A2M-antibodies before and after LRP1 depletion. These data indicate that LRP1 depletion was able to deplete A2M antibodies, which allow antibodies to bind to antigens faster than usual, further demonstrating a correlation between A2M structure and antibody reachability.

[0322] A2M-ipilimumab was also depleted using a protein L-coated resin that specifically binds to the κ light chain of the human antibody. Three rounds of depletion were performed. Biolayer interferometry demonstrated that protein L-based depletion could remove antigen binding in untreated A2M-ipilimumab samples (Figure 4E).

[0323] conclusion The native and activated A2M antibodies can be distinguished by affinity depletion based on their binding to the antigen, LRP1, or protein L. This binding allows for the removal of activated A2M antibodies and the preparation of native A2M antibodies to a higher purity. Binding experiments comparing antigen binding before and after depletion have shown that enrichment of native A2M antibodies minimizes or eliminates antigen binding by the native protein, demonstrating that antigen binding by untreated A2M antibodies is caused by contamination with non-native A2M antibodies. [Examples]

[0324] Investigating immune checkpoint blockade in cell assays the purpose To investigate whether the A2M antibody exhibits structure-dependent target binding in cellular conditions and retains the biological activity of its parent antibody, A2M-atezolizumab was examined in a PD-1 / PD-L1 blockade bioassay.

[0325] material and method The proteins used A2M-atezolizumab was expressed and purified as described for the A2M-antibody fusion construct in Example 2. Native A2M-atezolizumab was enriched using PD-L1-based affinity depletion as described in Example 4. Methylamine-treated A2M-atezolizumab was prepared by incubation with 200 mM methylamine at 37°C for 16 hours, followed by desalting and return to HBS on a PD-10 column. Atezolizumab scFv was also expressed fused to the human IgG1 Fc region with an N-terminal Strep II tag, and this atezolizumab-hFc was purified using the same protocol as for the antigen-hFc fusion construct described in Example 3, namely StrepTactin affinity chromatography followed by size exclusion chromatography.

[0326] Cell-based evaluation of immune checkpoint blockade The ability of A2M-atezolizumab and atezolizumab-hFc to block the PD-1 / PD-L1 pathway on human T cells was tested using a PD-1 / PD-L1 blockade bioassay developed by Promega. Jarcut cells were cultured in RPMI 1640 medium supplemented with penicillin / streptomycin and 10% fetal bovine serum, and CHO-K1 cells were cultured in DMEM medium supplemented with penicillin / streptomycin and 10% fetal bovine serum. The day before performing the assay, 40 cells per well were used. * 10 3CHO-K1 cells were seeded on a 96-well plate. On the day of the assay, the culture medium was removed from the wells and 40 μL of antibody solution diluted in assay buffer (RPMI 1640 medium with 1% fetal bovine serum) and 50 μL of assay buffer were added. * 10 3 The wells were replaced with 40 μL of Jarcut cells. The plates were then incubated at 37°C for 6 hours, after which 80 μL of Bio-Glo reagent (Promega) was added to each well, and luminescence was measured using a plate reader. Each antibody concentration and control was tested in three different wells. The luminescence signal was given as averaged and normalized luminescence across the three wells, with background (measured from wells that did not receive any antibody) subtracted, and the response was normalized to the highest measured luminescence from the assay (with background subtracted).

[0327] result Structure-dependent PD-L1 blockade by A2M-atezolizumab was investigated using a PD-1 / PD-L1 blockade bioassay developed by Promega. This bioassay represents human T cells using a human Jarcutt T cell line expressing human PD-1 and a luciferase reporter gene driven by an NFAT response element. PD-L1 blockade is performed using CHO-K1, which expresses an engineered surface protein that activates human PD-L1 and cognitive TCRs in an antigen-independent manner. + This represents the target cell. TCR-activated CHO-K1 is thought to activate Jurcut cells and induce an NFAT-driven luciferase response, except that this response is inhibited by PD-1-mediated signaling due to the association of PD-L1 on CHO-K1 cells with PD-1 on Jurcut cells. If PD-1 or PD-L1 is blocked by an antibody, the luciferase response is restored.

[0328] PD-L1 on CHO-K1 cells was blocked using a titration series (20 pM to 200 nM) of A2M-atezolizumab in its native and methylamine-treated degraded structures. IgG-like constructs prepared by fusing atezolizumab scFv to the human Fc region were included for comparison. Both A2M-atezolizumab and atezolizumab-hFc structures produced a concentration-dependent luminescence response (Figure 5). The maximum responses of methylamine-treated A2M-atezolizumab and atezolizumab-hFc were similar, and a saturation response to native A2M-atezolizumab was not achieved at the highest concentration measured (200 nM) (and its maximum response was therefore assumed to be the same as that for methylamine-treated A2M-atezolizumab). Both methylamine-treated A2M-atezolizumab and atezolizumab-hFc exhibited EC levels below nanomolar concentrations of 400 and 80 pM, respectively. 50 The values ​​shown were different, while native A2M-atezolizumab had an EC50 value of 216 nM. Therefore, there was approximately a 500-fold difference in the activity of A2M-atezolizumab between its native and activated structures.

[0329] conclusion A2M-atezolizumab is effective in cellular assays for immune checkpoint blockade, specifically against PD-1 + The study demonstrated a structure-dependent ability to block PD-L1 in T cells and restore NFκB signaling. This demonstrates that A2M-atezolizumab exhibits structure-dependent binding to cell surface PD-L1 and that A2M-atezolizumab retains the PD-L1 blocking functionality of the parental atezolizumab antibody. [Examples]

[0330] Modification of the A2M bait region to target specific proteases the purpose The sequence of the bait region of A2M determines whether that bait region can be cleaved by a given protease, thereby determining which proteases can activate (and be captured by) A2M. The bait region of wild-type A2M can be cleaved by almost all human proteases, and it is considered advantageous to restrict cleavage of the bait region to a specified protease, and more specifically to a target disease tissue. We first investigated whether the bait region could be replaced with a minimal sequence that does not contain cleavage sites for the majority of human proteases. Next, we investigated whether protease cleavage sites could be reintroduced into this minimal sequence with the aim of creating a bait region sequence with improved specificity for a single protease or a family of proteases (in this embodiment, matrix metalloproteinases (MMPs)).

[0331] material and method The proteins used The A2M proteins, which have modified bait region sequences, were expressed and purified in HEK292F cells as described for the A2M antibody in Example 2. The amino acid sequences of these A2M proteins are given in SEQ ID NOs. 65-73.

[0332] N-terminated StrepII-tagged proMMP2 (uniprot ID P08253, SEQ ID NOs. 61-62) was expressed and purified using StrepTactin affinity chromatography and size exclusion chromatography, as described for StrepII-tagged hFc fusion proteins in Example 3. proMMP2 was incubated at 37°C for 15 minutes and then activated with 1 mM APMA by desalting in HBS with 10 mM CaCl2 using a PD-10 column (GE Healthcare).

[0333] SDS-PAGE and Pore-Restricted Native PAGE Native pore-restricted PAGE was performed as previously described (36) using a homemade gel in TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA) with an acrylamide gradient of 5–10% for A2M analysis and 10–15% for C3 analysis. The pore-restricted electrophoresis gel was run overnight in TBE buffer at 100V. Modified SDS-PAGE was performed on homemade 5-15% acrylamide gradient gels using a discontinuous 2-amino-2-methyl-1,3-propanediol and glycine buffer system (37). Samples were reduced with 25 mM DTT at 95°C for 5 minutes.

[0334] Reaction of A2M with methylamine and protease To aminolyse the thiol ester of A2M, methylamine (pH 8) was added up to 250 mM and incubated at 37°C for at least 45 minutes. To evaluate the cleavage of A2M by trypsin and LysC, proteases were added to a 2.2:1 molar ratio of protease to A2M and incubated at 37°C for 5 minutes. Digestion was then inhibited using the serine protease inhibitor PMSF (2 mM, 15 minutes, room temperature). To evaluate the cleavage of A2M by MMP2, MMP2 was added to A2M in HBS with 10 mM CaCl2 up to a 6:1 molar ratio of MMP2 to A2M and incubated at 37°C for 15 minutes, followed by inhibition with 20 mM EDTA. When A2M was cleaved using other human proteases, incubation was performed for 1 hour at 37°C in HBS with 10 mM CaCl2, and serine proteases and metalloproteases were inhibited using PMSF or EDTA, respectively.

[0335] Determining the inhibition of A2M protein-substrate cleavage by MMP2. Inhibition of MMP by A2M was investigated using a fluorescently labeled gelatin substrate. 1.4 pmol (7.5 nM) of MMP was reacted with 0–2.7 pmol (0–15 nM) of A2M in 50 mM HEPES, 100 mM NaCl, and 5 mM CaCl2 pH 8 at 37°C for 15 minutes. Pig skin-derived DQ gelatin (Invitrogen) was added to a final concentration of 0.1 mg / ml. The fluorescence (excitation at 485 nm, emission at 520 nm) of the DQ gelatin unquenched digest product after 10 minutes at 37°C was measured using a FLUOstar Omega plate reader (BMG LABTECH). All reactions were performed in three different ways.

[0336] result Bait region substitution with 13 Gly-Gly-Ser triplets produces a tetramer, native, and inducible A2M. To essentially remove all protease cleavage sites from the bait region and determine the degree to which the bait region tolerates modification, we replaced the 39-residue wild-type A2M bait region sequence with 13 Gly-Gly-Ser repeats selected for their solubility and low sensitivity to proteolysis (Figure 6A). The resulting "tabula rasa" (TR) bait region was incorporated into recombinant A2M, which was primarily a tetramer and, when evaluated by native PAGE, yielded its native structure, A2M. The intact thiol ester was evident from the formation of characteristic thermally induced autolysis products on SDS-PAGE (Figures 6B-C). When its thiol ester was aminolyzed using methylamine, TR A2M underwent structural breakdown indistinguishable from that of wild-type A2M, as determined by pore-restricted native PAGE; however, TR A2M was not cleaved in its bait region by either trypsin or LysC, and retained its native structure even when proteolytically degraded by these proteases outside its bait region (Figure 6B-C). When a lysine residue was introduced at position 704 of the TR bait region, the resulting bait region could be cleaved by both trypsin and LysC, resulting in protease conjugation and characteristic structural changes of A2M (Figure 6A-C).

[0337] Identification of MMP2 cleavable bait region sequences with improved selectivity. Four TR bait regions incorporating the substrate sequence of human MMP2 were designed (Figure 7A). All four TR-based MMP2 substrate bait regions and the wild-type bait region were cleaved by MMP2, but the initial TR A2M was not cleaved (Figures 7B-D). Incomplete bait region cleavage and intermediate electrophoretic mobility of the A2M vs. MMP2 complex were observed for both wild-type A2M and the four MMP2 substrate TR A2M (Figures 7C-D).

[0338] Next, we used reduced SDS-PAGE to evaluate whether four MMP2 substrate bait regions were cleaved by nine additional human proteases (plasmin, cathepsin G, MMP1, MMP3, MMP8, MMP13, ADAMTS4, ADAMTS5, and ADAMTS13) to assess bait region cleavage and the formation of high-MW conjugation products. All evaluated proteases were able to cleave wild-type A2M except ADAMTS13 (which is highly specific to von Willebrand factor), but none were able to cleave TR A2M (Figure 7B). Incorporation of any of the four MMP2 substrate sequences into the TR bait region resulted in cleavage by all tested MMPs, while individual chamber sequences were cleaved differently by non-MMP proteases; for example, the C9 substrate was the only substrate containing an arginine residue and was the only A2M cleaved by plasmin (Figures 7A-B). The S1 substrate is only cleaved by MMPs (Figure 7A-B), and therefore, the A2M TR S1 protein was selected for further optimization as an A2M with improved MMP specificity compared to wild-type A2M.

[0339] The native content of Tabula Rasa-based A2M can be improved by shortening the bait region by 7 residues or restoring 10 C-terminal wild-type residues. The initial TR A2M protein was expressed with an increased amount of non-native A2M compared to wild-type A2M (Figure 6A, 7C). To address this issue, we tested two modified tabular survey regions: the first, TRΔ7, was shortened by 7 residues to a full length of 32 residues, and the second, TR QRT4, reintroduced the C-terminal quarter of the wild-type survey region (Figure 8A). Both TRΔ7 and TR QRT4 improved the native content of the resulting A2M to the same level as the native content of wild-type A2M (Figure 8B). The location of the S1 substrate sequence in the TRΔ7 survey region, which resulted in MMP2 inhibition efficiency indistinguishable from that of wild-type A2M, was identified (Figure 8C), demonstrating that this shortened survey region can produce fully functional A2M.

[0340] conclusion The bait region of A2M could be completely replaced with glycine and serine residues without compromising the structure and function of A2M, but a glycine-serine bait region shortened to 32 residues was found to improve the yield of native A2M. The glycine-serine bait region was not cleavable by 10 tested human proteases. When the S1 substrate for MMP2 was incorporated into the bait region, five human MMPs were able to cleave the bait region, while five non-MMPs remained uncleaved. This demonstrates that the glycine-serine bait region can be used as a basis for creating bait regions with improved specificity for proteases or families of proteases (such as MMPs). [Examples]

[0341] Modification of the bait region of A2M antibodies Research objectives In Example 6, a bait region sequence based on a "tabula rasa" (TR) bait region, in which the wild-type bait region is replaced with glycine and serine residues, was found to produce an A2M protein that is more specifically cleaved and activated by the target protease. Furthermore, a TR bait region shortened by 7 residues to a length of 32 residues (TRΔ7) was found to result in an increased yield of native A2M, and placing the S1 substrate for MMP2 at a specific position in TRΔ7 (TRΔ7 S1 I703) was found to result in MMP2 inhibition equivalent to that of the wild-type A2M bait region. Here, we investigated whether an A2M antibody incorporating a TR bait region with an MMP2 substrate site could be activated by MMP2 in the same way as an A2M antibody with a wild-type bait region.

[0342] material and method The proteins used A2M-atezolizumab containing the wild-type bait region (SEQ ID NOs: 7-8), the TRΔ7 S1 I703 bait region (SEQ ID NOs: 74-75), or the TRΔ7 S1 I703 P704 bait region (SEQ ID NOs: 76-77) was expressed and purified in HEK293F cells as described for the A2M-antibody in Example 2.

[0343] ProMMP2 was expressed, purified, and activated as described in Example 6.

[0344] Protease-mediated cleavage of A2M To cleave the A2M antibody with MMP2, MMP2 was added to HBS containing 10 mM CaCl2 at a ratio of 4:1 molar to A2M, incubated at 37°C for 15 minutes, and then inhibited with 20 mM EDTA. To cleave the A2M antibody with thermolysin, thermolysin was added to HBS containing 10 mM CaCl2 at a ratio of 2.2:1 molar to A2M, incubated at 37°C for 2 minutes, and then inhibited with 20 mM EDTA.

[0345] Biolayer Interferometry Biolayer interferometry was used to investigate the interaction between A2M-atezolizumab with different bait regions using the method described in Example 3.

[0346] SDS-PAGE and Pore-Restricted Native PAGE Native pore-restricted PAGE was performed as previously described (36) using a homemade gel in TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA) with an acrylamide gradient of 5–10% for A2M analysis and 10–15% for C3 analysis. The pore-restricted electrophoresis gel was run overnight in TBE buffer at 100V.

[0347] Modified SDS-PAGE was performed on homemade 5-15% acrylamide gradient gels using a discontinuous 2-amino-2-methyl-1,3-propanediol and glycine buffer system (37). Samples were reduced with 25 mM DTT at 95°C for 5 minutes.

[0348] result To evaluate the functionality of A2M-antibodies with modified bait regions, A2M-atezolizumab was expressed with either the wild-type bait region, the TRΔ7 S1 I703 bait region (the optimized MMP2 substrate bait region described in Example 6), or the TRΔ7 S1 I703 P704 bait region, which minimizes the MMP2 cleavage site and prevents serine protease cleavage of residue I703 by adding a proline residue at the P'1 position (Figure 9A). All three A2M-atezolizumab proteins were bait regions that were cleaved upon treatment with MMP2, as evaluated by their structural changes on native PAGE (Figure 9B) and cleavage of the A2M subunit on reduced SDS-PAGE (Figure 9C). These results demonstrate that A2M-antibodies with modified MMP2 substrate bait regions are cleaved at their bait region by MMP2.

[0349] The effect of MMP2 cleavage on A2M-atezolizumab protein binding to immobilized PD-L1 was investigated using biolayer interferometry. MMP2 cleavage was found to produce antigen binding similar to that induced by thermolysin cleavage in A2M-atezolizumab with a wild-type bait region (Figure 9D). Both A2M-atezolizumab proteins with modified bait regions showed similar antigen binding when cleaved using MMP2 (Figure 9D). These results indicate that MMP2-mediated bait region cleavage induces antigen binding in A2M antibodies, both in the wild-type bait region and the modified MMP2 substrate bait region.

[0350] conclusion The manipulated bait region can be incorporated into the A2M antibody without disrupting its structurally dependent antigen binding, as described in Example 5, and is still preferentially cleaved by a target protease such as MMP2. MMP2 cleavage can induce antigen binding in A2M antibodies having either a wild-type bait region or a manipulated bait region. [Examples]

[0351] Incorporation of the extracellular domain of the PD1 receptor into A2M Research objectives Here, we investigated whether the extracellular domain of the human PD1 receptor can be incorporated into A2M (in the same manner as the antibody, as shown in the previous example) and whether the resulting A2M-PD1 fusion protein binds to PD-L1, the ligand of the PD1 receptor, in a manner dependent on the structure of A2M.

[0352] material and method The proteins used The A2M-PD1 fusion construct was expressed and purified as described for the A2M antibody in Example 1. The extracellular region of PD1 incorporated into A2M was the same sequence used to test A2M-nivolumab in Example 2, i.e., residues 26-150 with uniprot ID Q15116, Cys93Ser mutation. The amino acid and nucleotide sequences of A2M-PD1 are given in SEQ ID NOs. 25-26. PD-L1 fused to the human Fc region was prepared as described in Example 2.

[0353] SDS-PAGE and Pore-Restricted Native PAGE A2M-PD1 was analyzed by reduced SDS-PAGE using the protocol described in Example 2.

[0354] Reaction of A2M antibody with methylamine and protease A2M-PD1 was treated with methylamine or the metalloproteinase thermolysin to alter its structure, as described in Example 3.

[0355] Biolayer Interferometry Using biolayer interferometry, the binding of A2M-PD1 to PD-L1 immobilized on the surface of the biosensor was investigated in untreated and methylamine- or thermolysin-treated structures, as described in Example 3.

[0356] result Using the same fusion strategy used to incorporate the antibody scFv and nanobodies into A2M, the extracellular region of human PD1 was incorporated into A2M, and the resulting A2M-PD1 was expressed and purified using the standard A2M protocol (Figure 10A).

[0357] Next, the structure dependence of PD-L1 binding by A2M-PD1 was evaluated by biolayer interferometry. PD-L1 binding by A2M-PD1 was strongly dependent on the structure of A2M (Figure 10B). The degree of binding shown by the control sample with untreated A2M-PD1 was minimal. This was because native A2M-PD1 had not been purified from non-native A2M-PD1 prior to this experiment, for example, using LRP1-based depletion as described in Example 3. In contrast, both methylamine- and thermolysin-treated A2M showed greatly enhanced binding responses, and these responses were very similar to each other. When A2M-PD1 in its native structure was enriched by three rounds of LRP1-based depletion of non-native A2M-PD1, binding by untreated A2M-PD1 was undetectable.

[0358] conclusion PD1 could be incorporated into A2M, resulting in a functional A2M capable of undergoing its typical methylamine- and thermolysin-induced structural changes, and PD1 capable of binding to its ligand, PD-L1. Furthermore, the binding of PD1 to PD-L1 was dependent on the structure of A2M, and the binding of A2M-PD1 to PD-L1 in its native structure could not be detected. [Examples]

[0359] Incorporation of IL2 cytokines into A2M the purpose Here, we investigated whether the IL2 cytokine could be incorporated into A2M (in the same manner as the antibody, as shown in the previous example) and whether the resulting A2M-IL2 fusion protein binds to the IL2 receptor, IL-2Rα, in a manner dependent on the structure of A2M.

[0360] material and method The proteins used The A2M-IL2 fusion construct was expressed and purified as described for the A2M antibody in Example 1. The IL2 cytokine incorporated into A2M was the wild-type human sequence (uniprot P60568, residues 21-153). The amino acid and nucleotide sequences of A2M-IL2 are given in SEQ ID NOs. 23-24.

[0361] The extracellular domain of the IL-2Rα receptor (uniprot P01589, residues 22-238, with Cys213Ala mutation, SEQ ID NO: 43) was expressed as a Strep-tagged human Fc fusion protein (SEQ ID NOs: 57-58) and purified in the same manner as described for other antigens in Example 2.

[0362] SDS-PAGE and Pore-Restricted Native PAGE A2M-IL2 was analyzed by reduced SDS-PAGE using the protocol described in Example 2.

[0363] Reaction of A2M antibody with methylamine and protease A2M-IL2 was treated with methylamine or the metalloproteinase thermolysin to alter its structure, as described in Example 3.

[0364] Biolayer Interferometry Using biolayer interferometry, the binding of A2M-IL2 to IL-2Rα immobilized on the surface of the biosensor was investigated in untreated and methylamine- or thermolysin-treated structures, as described in Example 3.

[0365] result Using the same fusion strategy that was used to incorporate the antibody scFv and nanobodies into A2M, human cytokine IL2 was incorporated into A2M, and the resulting A2M-IL2 was expressed and purified using the standard A2M protocol (Figure 11A).

[0366] Next, the structure-dependence of IL-2Rα binding by A2M-IL2 was evaluated by biolayer interferometry. IL-2Rα binding by A2M-IL2 was dependent on the structure of A2M (Figure 11B). Control samples with untreated A2M-IL2 showed moderate binding even after using LRP1-based depletion of non-native A2M-IL2. Cleavage of the A2M bait region with thermolysin resulted in an immediate increase in the association rate and saturation of binding, and aminolysis of the A2M thiol ester with methylamine resulted in a much greater increase in both the association rate and binding saturation. The degree of binding (when determined by the k obs value) is approximately 10-fold increased by methylamine treatment compared to A2M-IL2 in its native structure.

[0367] Conclusion IL2 can be incorporated into A2M, resulting in a functional A2M-IL2 that can cause its typical methylamine- and thermolysin-induced structural changes and bind to the receptor IL-2Rα. Furthermore, this receptor binding by A2M-IL2 is dependent on the structure of A2M, and an increase in receptor binding was observed when the A2M structure was disrupted by bait region cleavage or thiol ester aminolysis.

Example

[0368] Examine other fusion strategies for the incorporation of biopharmaceutical moieties into A2M Purpose The previous example before examining proteinaceous fusion constructs of A2M and biopharmaceutical moieties used the ciRBD approach, where the biopharmaceutical moiety was inserted between residues 1402 and 1403 of A2M. Here, we examined whether target binding that is dependent on the structure of A2M can be achieved by four other approaches: fusion, iRBD, miRBD, and tRBD.

[0369] Materials and Methods Proteins Used All A2M-and-biopharmaceutical fusion constructs were expressed and purified as described for the A2M-antibody fusion construct in Example 2. Non-native A2M depletion was not performed. The amino acid and nucleotide sequences of A2M-fusion-EgA1 (SEQ ID NOs. 94-95), A2M-iRBD-EgA1 (SEQ ID NOs. 84-85), A2M-miRBD-EgA1 (SEQ ID NOs. 86-87), A2M-miRBD-atezolizumab (SEQ ID NOs. 88-89), A2M-miRBD-KN035 (SEQ ID NOs. 90-91), and A2M-tRBD-EgA1 (SEQ ID NOs. 92-93) are given. EGFR and PD-L1 (SEQ ID NOs. 45-48) fused with the human FC region were prepared as described in Example 3.

[0370] Reaction of A2M antibody with methylamine and protease The A2M antibody was reacted with methylamine or protease as described in Example 3.

[0371] Biolayer Interferometry The biolayer interferometry was performed as described in Example 3.

[0372] result To determine whether shielding of the biopharmaceutical moiety within A2M can be achieved by incorporating the biopharmaceutical moiety into A2M in ways other than the previously used ciRBD approach, four novel fusion approaches were tested. First, an A2M-fusion-EgA1 protein was constructed by expressing an EgA1 nanobody immediately after the C-terminus of the RBD domain of A2M. A2M-fusion-EgA1 did not show structural dependence of FgA1 binding to EGFR (Figure 12A), indicating that not all positions adjacent to the RBD domain are shielded in the native structure of A2M.

[0373] In the second approach, the iRBD, EgA1 nanobody was inserted into the RBD domain replacing A2M residues 1392-1403. The resulting A2M-iRBD-EgA1 protein exhibited a high degree of structural dependence comparable to that of the ciRBD approach (Figure 12B) (see Example 3). This indicates that the region of the A2M RBD domain adjacent to residues 1393-1403 is a suitable site for incorporating the biopharmaceutical moiety to achieve structural dependence. Therefore, the third approach, miRBD, also achieved structural dependence by incorporating the EgA1 nanobody, KN035 nanobody, or atezolizumab scFv at a position replacing A2M residues 1393-1395 (Figures 12B-E).

[0374] In the fourth approach, tRBD, coiled-coil interactions were used to bring the incorporated biopharmaceutical moiety (EgA1) close to RBD residues 1393–1403. The EgA1 nanobody was incorporated at its C-terminus into an RBD domain having an alpha-helix sequence designed to be complementary to A2M residues 1393–1403 at its N-terminus. The alpha-helix sequence was bound to the RBD domain using a 15-residue linker to allow the alpha-helix to interact with residues 1393–1403. Furthermore, modifications were made to A2M residues 1393–1403 to enhance the designed complementary coiled-coil interaction. The resulting A2M-tRBD-EgA1 protein demonstrates the structure-dependent nature of the EgA1 / EGFR interaction (Figure 12F), showing that the coiled-coil interaction allows the EgA1 nanobody to be brought close to residues 1393-1403, and that this proximity results in at least partial shielding of the EgA1 nanobody in the native structure of A2M.

[0375] conclusion Investigations of fusion, iRBD, miRBD, and tRBD approaches to the construction of fusion constructs of A2M and biopharmaceutical moieties revealed that the position proximal to A2M RBD residues 1393–1403, through direct fusion at this site (as seen in the ciRBD, iRBD, and ciRBD approaches) or through other means (e.g., via coiled-coil interactions, as demonstrated by A2M-tRBD-EgA1), results in structure-dependent target binding for many different tested biopharmaceutical moieties (16 in total, examining all ciRBD, iRBD, miRBD, and tRBD fusion constructs). [Examples]

[0376] Research on insertion sites in the RBD region the purpose Identifying sites for drug insertion within A2M's RBD that provide structure-dependent reachability.

[0377] material and method The figures were created using PyMol Molecular Graphics System software (version 2.3.0).

[0378] result In the iRBD, miRBD, and ciRBD approaches to the creation of A2M-based prodrugs, residues 1392–1403 of the RBD domain of A2M are replaced with drug sequences (as well as N- and C-terminal linkers), or the drug is inserted between residues 1402–1403 without altering any residues of A2M. Residues 1391–405 or 1392–1404 contain loops or linker regions between the chains of the beta-sheet structure, and such loops are well-suited for modification, in contrast to beta-sheet sequences where modifications are more likely to affect domain folding.

[0379] Furthermore, loop orientation is also crucial for achieving structure-dependent drug loci, because drug fusion at position 1474 on the opposite side of the RBD (in the A2M-fusion-EgA1 construct) always produces an accessible drug.

[0380] Following structural evaluation of the RBD domain, three additional loop regions suitable for drug insertion were identified: in addition to the empirically tested region containing residues 1392–1404 (loop 2), regions containing residues 1368–1379 (loop 1), 1420–1426 (loop 3), and 1450–1457 (loop 4) (Figure 13). All three loops extend between the beta chains and are oriented in a direction similar to loop 2 (1392–1404), with these loops oriented inward toward the inside of the A2M tetramer.

[0381] conclusion In addition to the region containing residues 1392–1404 (loop 2), three additional loops were identified containing A2M residues 1368–1379 (loop 1), 1420–1426 (loop 3), and 1450–1457 (loop 4), which are considered usable for replacement or direct insertion by one or more drugs to result in structure-dependent binding of its therapeutic target. [Examples]

[0382] Production of furin-cleaved A2M having a releaseable MG8 domain Research objectives We investigated whether introducing a furin cleavage site between the CUB and MG8 (aka RBD) domains of A2M would enable the production of a native A2M protein containing the MG8 domain, which is released when A2M undergoes a structural change.

[0383] material and method Expression and purification of A2M antibody fusion constructs. A2M with furin cleavage sites (A2M furin RBD) was expressed in HEK293 FreeStyle cells using a standard transient transfection protocol. Briefly, 25 kDa linear polyethyleneimine (Polysciences) and plasmid DNA were incubated in antibiotic-free FreeStyle medium (Thermo Fisher Scientific) at a PEI to DNA ratio of 4:1 w / w for 10 minutes, and then slowly added dropwise to cell cultures at a density of 1 million cells per mL to a final DNA concentration of 1 μg per mL of culture. After 5 days, the supernatant was collected by centrifugation of the cells at 1500 x g.

[0384] The construct was purified using an established protocol for purifying A2M (1, 2). The supernatant was initially Zn 2+ The eluate was passed through a supported chelate HiTrap column (GE Healthcare) and eluted with 50 mM EDTA, 150 mM NaCl, 100 mM sodium acetate, and pH 7.4. The EDTA eluate was dialyzed against 20 mM HEPES at pH 7.4 and then loaded onto a HiTrap Q column (GE Healthcare), where it was eluted with a gradient of 0–400 mM NaCl (using a constant 20 mM HEPES at pH 7.4). The fraction containing A2M was pooled, concentrated by ultrafiltration, and purified by size exclusion chromatography on a Sephacryl S-300 HR (GE Healthcare) using 20 mM HEPES, 150 mM NaCl, and a running buffer (HEPES-buffered saline, HBS) at pH 7.4.

[0385] SDS-PAGE and Pore-Restricted Native PAGE Native pore-restricted PAGE was performed as previously described (3) using gels in homemade TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA) with an acrylamide gradient of 5–10%. The pore-restricted electrophoresis gels were run overnight in TBE buffer at 100 V.

[0386] Modified SDS-PAGE was performed on a homemade 5-15% acrylamide gradient gel using a discontinuous 2-amino-2-methyl-1,3-propanediol and glycine buffer system (4). Samples were reduced with 25 mM DTT at 95°C for 5 minutes.

[0387] Reaction of A2M with methylamine and protease To aminolyse the thiol ester of A2M, methylamine (pH 8) was added to 250 mM and incubated at 37°C for 2 hours. To evaluate the cleavage of A2M by thermolysin, thermolysin was added to a protease to A2M ratio of 2.2:1 mol / mol and incubated at 37°C for 5 minutes. Digestion was then inhibited using EDTA (10 mM, 15 minutes, room temperature).

[0388] Size exclusion chromatography Analytical size exclusion chromatography was performed using a Superdex 200 Increase (Cytiva Life Sciences) with 20 mM HEPES, 150 mM NaCl, pH 7.4 running buffer, and a flow rate of 0.4 mL per minute.

[0389] result Previous studies have suggested that while A2M proteins are in their native structure, the MG8 domain (aka. RBD) of many members of the A2M family forms strong non-covalent interactions with other domains (such as thiol ester domains), while these interactions are no longer present after aminolysis-or proteolysis-induced structural changes, with the MG8 domain remaining bound mostly due to the polypeptide backbone. We hypothesized that a furin cleavage could be introduced between the MG8 domain and its N-terminal domain (CUB domain), resulting in the release of MG8 following a structural change in A2M. Furin is an intracellular protease that processes many immature proteins in the endoplasmic reticulum: furin is highly specific to the R / KXXR / K sequence, where X can be any amino acid, but ideally it is arginine or lysine (5).

[0390] We constructed A2M (A2M furin RBD) by incorporating an ideal furin cleavage site, RRRR, and a short linker that ensures the furin reaches the cleavage site completely. The A2M furin RBD sequence is given as SEQ ID NO: 227-A2M_furin_RBD. The majority of A2M furin RBD was expressed and purified in its native structure with intact thiol esters, but a significant number (approximately 25%) were unable to form the native structure and were not cleavable by thermolysin (Figure 15A-B). A2M furin RBD showed heterogeneous untreated samples on native PAGE but typically disintegrated upon treatment with methylamine or thermolysin (Figure 15B). Native gels only showed the electrophoresis of relatively large proteins, and therefore the MG8 domain released by thermolysin was not visible on native gels (Figure 2B).

[0391] To investigate whether the MG8 domain of A2M furin RBD was released due to a structural change in A2M, purified A2M furin RBD was separated by size exclusion chromatography either without treatment or after methylamine treatment (Figure 16A). After methylamine treatment, the MG8 domain was separated from the remaining A2M and eluted into its own fraction, whereas in untreated A2M, the MG8 domain elutes co-eluted with the main A2M peak (Figure 16B). This indicates that the MG8 domain is released by a methylamine-induced structural change of A2M.

[0392] conclusion When a furin cleavage site was incorporated into the N-terminus of the MG8 domain in recombinant A2M, a predominantly native A2M protein was obtained that underwent complete furin processing and released its MG8 domain following the structural change. [Examples]

[0393] Production of TEV-protease cleavage A2M with a releaseable MG8 domain Research objectives We investigated whether introducing a tobacco etch (TEV) protease cleavage site between the CUB and MG8 (aka.RBD) domains of A2M would enable the production of a native A2M protein containing the MG8 domain, which is released when A2M undergoes a structural change.

[0394] material and method Expression and purification of A2M antibody fusion constructs. A2M (tevRBD) with a TEV protease cleavage site was expressed in HEK293 FreeStyle cells using a standard transient transfection protocol. Briefly, 25 kDa linear polyethyleneimine (Polysciences) and plasmid DNA were incubated in antibiotic-free FreeStyle medium (Thermo Fisher Scientific) at a PEI to DNA ratio of 4:1 w / w for 10 minutes, and then slowly added dropwise to cell cultures at a density of 1 million cells per mL to a final DNA concentration of 1 μg per mL of culture. After 5 days, the supernatant was collected by centrifugation of the cells at 1500 x g.

[0395] The construct was purified using an established protocol for purifying A2M (1, 2). The supernatant was initially Zn 2+ The eluate was passed through a supported chelate HiTrap column (GE Healthcare) and eluted with 50 mM EDTA, 150 mM NaCl, 100 mM sodium acetate, and pH 7.4. The EDTA eluate was dialyzed against 20 mM HEPES at pH 7.4 and then loaded onto a HiTrap Q column (GE Healthcare), where it was eluted with a gradient of 0–400 mM NaCl (using a constant 20 mM HEPES at pH 7.4). The fraction containing A2M was pooled, concentrated by ultrafiltration, and purified by size exclusion chromatography on a Sephacryl S-300 HR (GE Healthcare) using 20 mM HEPES, 150 mM NaCl, and a running buffer (HEPES-buffered saline, HBS) at pH 7.4.

[0396] SDS-PAGE and Pore-Restricted Native PAGE Native pore-restricted PAGE was performed as previously described (36) using gels in homemade TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA) with an acrylamide gradient of 5–10% for A2M analysis. The pore-restricted electrophoresis gels were run overnight in TBE buffer at 100 V.

[0397] Modified SDS-PAGE was performed on a homemade 5-15% acrylamide gradient gel using a discontinuous 2-amino-2-methyl-1,3-propanediol and glycine buffer system (37). Samples were reduced with 25 mM DTT at 95°C for 5 minutes.

[0398] Reaction of A2M with methylamine and protease To aminolyse the thiol ester of A2M, methylamine (pH 8) was added to 250 mM and incubated at 37°C for 2 hours. To evaluate the cleavage of A2M by thermolysin, thermolysin was added to a protease to A2M ratio of 2.2:1 mol / mol and incubated at 37°C for 5 minutes. Digestion was then inhibited using EDTA (10 mM, 15 minutes, room temperature). TEV protease cleavage of A2M was performed at room temperature for 24 hours using a TEV protease stock that had been desalted in HBS and contained no reducing agents.

[0399] Size exclusion chromatography Analytical size exclusion chromatography was performed using a Superdex 200 Increase (Cytiva Life Sciences) with 20 mM HEPES, 150 mM NaCl, pH 7.4 running buffer, and a flow rate of 0.4 mL per minute.

[0400] result Furin cleavage at the N-terminus of the MG8 domain of A2M functioned as intended, producing a completely cleaved A2M construct, which largely retained its native structure and thiol esters and released its MG8 domain during the structural change. However, intracellular furin processing was not readily controllable or regulated, resulting in heterogeneous A2M furin RBD. This suggests that furin processing during translation and maturation of A2M is destructive to the formation of its thiol esters and native structure. Therefore, it may be potentially advantageous to delay cleavage at the N-terminus of the MG8 domain until A2M is fully folded and assembled into its native tetramer, for example, until A2M is secreted into the extracellular space and purified. Hence, we investigated whether a cleavage site for a highly specific TEV protease could be used in place of the furin site.

[0401] We constructed A2M (A2M tevRBD) by incorporating the TEV cleavage site ENLYFQS (SEQ ID NO: 226) and a short linker to ensure complete access to the cleavage site. The sequence of A2M tevRBD is given in SEQ ID NO: 229-A2M_tevRBD. A2M tevRBD was expressed and purified as a homogeneous native tetramer A2M with intact thiol esters (Figures 17B-C). A2M tevRBD was cleaved by the TEV protease at the predicted site, and the MG8 domain was able to non-covalently associate with the rest of A2M (Figure 17A). After TEV cleavage, A2M tevRBD exhibited a smeared electrophoretic pattern on native PAGE, but underwent normal structural changes upon methylamine or protease treatment (Figure 17C).

[0402] To investigate whether the MG8 domain of A2M tevRBD was released due to a structural change in A2M, TEV protease-cleaved A2M tevRBD (TEV protease removed by a previous purification step) was isolated by size exclusion chromatography either without any treatment or after methylamine treatment (Figure 18A). After methylamine treatment, the MG8 domain was separated from the remaining A2M and eluted in two structures depending on two different glycosylation patterns (Figures 18B-C). In untreated A2M tevRBD, the MG8 domain elutes co-eluted with the main A2M peak (Figures 18A-B).

[0403] This indicates that the MG8 domain is released via a methylamine-induced structural change of A2M after cleavage of the TEV protease site.

[0404] conclusion The TEV protease cleavage site could be used to produce an A2M construct containing an MG8 domain that can be released by a structural change in A2M. Furthermore, the TEV protease site exhibits a more uniform native A2M product compared to the use of the furin cleavage site. [Examples]

[0405] Addition of two antibodies to A2M that can be cleaved by TEV protease. Research objectives We investigated whether it was still possible to initially produce native A2M by simultaneously fusing two antibody-derived binding fragments (scFv and nanobody) to the A2M tevRBD protein described in Example 13, and whether cleavage of such a protein with a TEV protease would enable the release of the MG8 domain and the two antibodies fused to it.

[0406] material and method Expression and purification of A2M antibody fusion constructs. A2M, possessing a TEV protease cleavage site, an EGFR-specific C-terminal nanobody (EgA1), and anti-CD3 scFv (derived from foralumab) fused via a ciRBD approach, was expressed in HEK293 FreeStyle cells using a standard transient transfection protocol. Similar constructs possessing a modified TEV protease cleavage site, an EGFR-specific C-terminal nanobody (EgA1), and anti-CD3 scFv (derived from UCHT1-) were also used in this embodiment and expressed / purified in the same manner. Briefly, 25 kDa linear polyethyleneimine (Polysciences) and plasmid DNA were incubated in antibiotic-free FreeStyle medium (Thermo Fisher Scientific) at a PEI-to-DNA ratio of 4:1 w / w for 10 minutes, and then slowly added to cell cultures at a density of 1 million cells per mL to a final DNA concentration of 1 μg per mL of culture. After 5 days, the supernatant was collected by centrifugation of the cells at 1500xg.

[0407] The construct was purified using an established protocol for purifying A2M (1, 2). The supernatant was initially Zn 2+ The eluate was passed through a supported chelate HiTrap column (GE Healthcare) and eluted with 50 mM EDTA, 150 mM NaCl, 100 mM sodium acetate, and pH 7.4. The EDTA eluate was dialyzed against 20 mM HEPES at pH 7.4 and then loaded onto a HiTrap Q column (GE Healthcare), where it was eluted with a gradient of 0–400 mM NaCl (using a constant 20 mM HEPES at pH 7.4). The fraction containing A2M was pooled, concentrated by ultrafiltration, and purified by size exclusion chromatography on a Sephacryl S-300 HR (GE Healthcare) using 20 mM HEPES, 150 mM NaCl, and a running buffer (HEPES-buffered saline, HBS) at pH 7.4.

[0408] SDS-PAGE and Pore-Restricted Native PAGE Native pore-restricted PAGE was performed as previously described (36) using gels in homemade TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA) with an acrylamide gradient of 5–10% for A2M analysis. The pore-restricted electrophoresis gels were run overnight in TBE buffer at 100 V.

[0409] Modified SDS-PAGE was performed on a homemade 5-15% acrylamide gradient gel using a discontinuous 2-amino-2-methyl-1,3-propanediol and glycine buffer system (37). Samples were reduced with 25 mM DTT at 95°C for 5 minutes.

[0410] Reaction of A2M with methylamine and protease To aminolyze the thiol ester of A2M, methylamine (pH 8) was added to 250 mM and incubated at 37°C for 2 hours. To evaluate the cleavage of A2M by thermolysin, thermolysin was added to a protease to A2M ratio of 2.2:1 mol / mol and incubated at 37°C for 5 minutes. Digestion was then inhibited using EDTA (10 mM, 15 minutes, room temperature).

[0411] result We previously demonstrated that by fusing an antibody-derived binding fragment to an internal location within the MG8 domain (e.g., between residues 1402 and 1403 in the ciRBD approach), we could create a prodrug version of that antibody that requires proteolytic activation before it can reach its antigen (Examples 2-4). We also demonstrated that by fusing an antibody to the C-terminus of the MG8 domain, the antibody can reach its antigen independently of the structural state of A2M (in Example 10 and Figure 12A). Here, we investigated whether the fusion of two different antibody fragments at both of these locations, along with the insertion of a TEV protease site at the N-terminus of MG8 as tested in the tevRBD construct of Example 13, could create a native A2M protein in which the MG8 domain remains releaseable due to a structural change in A2M.

[0412] We constructed an A2M having a tevRBD sequence combined with anti-CD3 scFv (derived from foralmab) at the ciRBD site and anti-EGFR nanobody (clone EgA1) at the C-terminal site, which we refer to in this example as tevRBD+2xAb. Both the ability of these antibody fragments to bind to their antigen when fused in A2M at these specific sites has been previously demonstrated in Examples 2-4 and 13. The sequence of tevRBD+2xAb is given as SEQ ID NO: 231-A2M_tevRBD+2xAb. This protein was produced in its native structure with intact thiol esters, as determined by its autolysis product band on SDS-PAGE (Figure 19A). This protein assembled into a tetramer with a larger electrophoretic profile than wild-type A2M due to the C-terminal nanobody located outside A2M (Figure 19B). Structural changes upon methylamine treatment were not evident on native PAGE (Figure 19B). Thermolysin can be cleaved within the TEV protease site, which contains several hydrophobic residues that are thermolysin substrates (Figure 19A-B). Therefore, cleavage of the thermolysin-induced bait region of tevRBD+2xAb results in both structural changes in A2M and heterogeneous release of the MG8 domain (and its two bound antibodies), giving rise to five distinctly different bands in native PAGE, corresponding to 0-4 disintegrated A2M with thermolysin-releasing MG8 domains, i.e., 0 = disintegrated A2M, 1 = disintegrated A2M lacking one RBD+2xAb, 2 = disintegrated A2M lacking two RBD+2xAb, etc. (Figure 19B). The released RBD+2xAb domains are not visible in the native gel due to their small protein size.

[0413] We constructed an additional A2M with a slightly modified tevRBD sequence combined with another anti-CD3 scFv (derived from UCHT1) at the ciRBD position and an anti-EGFR nanobody (clone EgA1) at the C-terminus, which we refer to in this embodiment as tevRBD+2xAb_2. This A2M was initially native, as evidenced by its thiol ester-mediated degradation on SDS-PAGE, and remained native after TEV protease cleavage of the tevRBD site (Figure 19C). After altering the structure of A2M using methylamine, the MG8 domain and the two fused antibodies remained associated with A2M in native PAGE prior to TEV protease cleavage. In TEV protease-cleaved tevRBD+2xAb_2, the MG8 domain and the two antibodies were instead released from A2M, and its normal structural breakdown was detected in native PAGE (Figure 19D). The fact that when the antibody is exposed due to a structural change of A2M, the A2M structural change is not detected by native PAGE is detailed in Example 15 and is shown to be due to a decrease in the electrophoretic mobility of the disintegrated structure of A2M when A2M is associated with the exposed antibody.

[0414] conclusion From these results, we conclude that both ciRBD and C-terminal antibody fusion approaches, as well as the TEV protease site insertions used in tevRBD, can be combined and still produce native A2M. Importantly, the MG8 domain of this construct remains releaseable upon structural changes in A2M. A2M with two antibodies and a TEV site can be fully processed by the TEV protease while maintaining the native state of A2M. Upon inducing a structural change in A2M, the TEV protease-cleaved A2M releases its MG8 domain, even if the MG8 domain is simultaneously fused to two antibodies. [Examples]

[0415] Protease cleavage is required for the release of the MG8-antibody domain from A2M-BiTE. Research objectives Example 14 demonstrated that an A2M construct having an MG8 domain that can be released by a structural change of A2M can be produced by using the TEV protease cleavage site. In this example, we investigated a further optimized A2M-BiTE construct similar to the construct tested in Example 14 to confirm that digestion with TEV yields two fragments, namely an MG8 domain containing two antibodies and an N-terminal A2M domain, and that the addition of trypsin cleaves the A2M domain in the bait region, yielding two smaller fragments. A generalized representation of the A2M-BiTE construct is shown in Figure 20.

[0416] material and method A2M, possessing a modified TEV protease cleavage site, a C-terminal antigen that specifically binds to tumor-associated antigens, namely an EGFR-specific nanobody (EgA1), and anti-CD3 scFv (derived from Teventafusp) (SEQ ID NO: 252) fused via a ciRBD approach, was expressed in HEK293 FreeStyle cells using a standard transient transfection protocol and purified as described in Example 14. This A2M-BiTE was named BiTE2-TEV4 (SEQ ID NO: 258) and also included a TEV cleavage site with additional linkers on both sides to allow for more rapid cleavage by the TEV protease.

[0417] The A2M-BiTE prodrug was treated with TEV protease or HBS in a molar ratio of 4:1 (TEV vs. A2M-BiTE), followed by treatment with serial dilutions of trypsin (TR) for 10 minutes, methylamine (MA) for 45 minutes, or HEPES-buffered saline (HBS; 20 mM HEPES, 150 mM NaCl, pH 7.4) for 45 minutes at 37°C. The trypsin reaction was stopped with 2 mM phenylmethylsulfonyl fluoride (PMSF) for 15 minutes at room temperature.

[0418] Next, the samples were analyzed by pore restriction and SDS-PAGE gel analysis, as described in the previous example.

[0419] result In this example, we investigated the potential of providing a BiTE construct that cross-links cells expressing tumor-associated antigens (TAAs) with T cells via anti-TAA antibodies and anti-CD3 antibodies. The anti-CD3 antibody was placed inside the MG8 domain so that it could only be reached within the tumor, with the aim of reducing side effects resulting from BiTE-dependent activation of T cells outside the tumor tissue.

[0420] The C-terminal anti-TAA antibody is freely reachable, allowing the BiTE construct to be directed to target TAA-expressing cells. We used trypsin as a model of cancer-specific proteases that the BiTE construct is likely to encounter in the tumor microenvironment. The provided construct can be pre-cleaved with TEV, but it releases the MG8 domain containing two antibodies in the tumor microenvironment only if it is cleaved by a cancer-specific protease and its structure is altered. Therefore, the anti-CD3 antibody becomes reachable only if it is cleaved by a cancer-specific protease.

[0421] After digestion with TEV, the MG8-BiTE domain containing anti-CD3-MG8-anti-EGFR can be released by a structural change (induced by MA or trypsin), giving "empty" A2M that moves in the same way as unmodified A2M. These results are shown in Figure 21A, which shows a pore-limited 4–15% gel.

[0422] Following digestion with TEV, followed by trypsin digestion, three distinctly different bands were observed, as shown in Figure 21B: one representing the N-terminal A2M domain (annotated as a BR cleavage fragment), another containing the MG7-CUB2 domain (annotated as a BR cleavage fragment without MG8), and a third representing the MG8 domain (annotated as MG8+2xAb) connected to the EGFR nanobody and anti-CD3 scFv. Faint bait region cleavage products were visible even before trypsin treatment because small amounts of A2M are cleaved in the bait region by proteases present during HEK293F expression.

[0423] conclusion This embodiment demonstrates that the MG8 domain, to which two commonly preferred antibodies are fused, can be exposed to selected protease activity, such as TEV protease, and subsequently undergo a structural change before being released. [Examples]

[0424] A2M-BiTE activates T cells in a protease cleavage-dependent manner. Research objectives As described in Example 15 and shown in Figure 20B, the anti-CD3 scFv in A2M-BiTE is shielded by A2M until a structural change is induced, for example, by cleavage of the first cleavage site by trypsin. Trypsin serves as a model for proteases present in the tumor microenvironment. In this example, we investigate whether the anti-CD3-scFv in TEV-cleaved A2M-BiTE can bind to T cell receptors expressed on Jurcut cells and induce Erk 1 / 2 signaling in a trypsin-dependent manner.

[0425] material and method For proteolytic TEV cleavage of BiTE2-TEV4 (SEQ ID NO: 258) and BiTE3-TEV5 (SEQ ID NO: 259) constructs, 200–400 μg of purified BiTE constructs were digested with TEV protease in HBS buffer containing 0.5 mM EDTA and 50 μg / ml leupeptin in a final volume of 300 μl, at a molar ratio of 8:1 (TEV / A2M). After incubation at 24°C for 16 hours, 200 μl of HBS was added, and the sample was filtered through a 45 μm filter. The sample was loaded onto a HiPrep 16 / 60 Sephacryl S-300 (Cytiva #17116701), and 0.5 ml / min of HBS was passed through the column, collecting a 1 ml fraction. Fractions of 42 ml to 47 ml were pooled, concentrated to 200-400 μL using Amicon Ultra-15 30K (Millipore UFC903024), and then stored on ice until use.

[0426] TEV-cleaved BiTE2-TEV4 (SEQ ID NO: 258) or BiTE3-TEV5 (SEQ ID NO: 259) were treated with trypsin (TR) or HBS (0) in a 2:1 molar ratio (TR vs. BiTE) and incubated at 37°C for 10 minutes. The reaction was stopped with RPMI containing 10% FBS, and the samples were stored on ice. Trypsin-treated (TR) and control (0) A2M BiTE samples were serially diluted in RPMI 10% FBS and incubated at 37°C for 5 minutes at a final volume of 200 μl in the presence of 150,000 fluorescently labeled (using carboxyfluorescein succinimidyl ester; CFSE) target cells (CHO or HCT116 cells) and 300,000 effector cells (Jarcut) per well.

[0427] Cells were immobilized by adding 100 μl of 4% PFA to a final concentration of 1.3% paraformaldehyde for 10 minutes at room temperature. The immobilized cells were centrifuged at 250 × g at 24°C for 5 minutes, the supernatant was removed, and the cell pellet was resuspended in 500 μl of ice-cold 95% methanol while vortexing (2500 rpm) and incubated on ice for 15 minutes. After centrifugation at 500 g at room temperature for 5 minutes, the supernatant was removed. The cells were washed three times with 1 ml of PBS 0.1% BSA, then resuspended in 100 μl of staining medium and transferred to a round-bottom 96-well plate. 0.05 μl of Fluor647 conjugate anti-phospho Erk1 / 2 antibody (Biolegend #675503) per sample was added and incubated at room temperature for 30 minutes. The cells were washed twice with PBS and resuspended in 200 μl of 1% PFA for 10 minutes at room temperature for fixation. The samples were centrifuged, resuspended in 200 μl of staining medium, and stored at 4°C until analyzed using a Novocyte Quanteon flow cytometer (Agilent).

[0428] result When determined by flow cytometry-based measurements at the pERK1 / 2 level, the bispecific anti-CD3, anti-EGFR portion released from TEV cleavage A2M is EGFR + We tested whether the effector Jurcut cells were induced to activate in the presence of target cells (HCT116). This was tested using two different A2M-BiTEs; BiTE2-TEV4 (SEQ ID NO: 258, described in Example 15) and BiTE3-TEV5 (SEQ ID NO: 259), which have a linker extended around the TEV protease cleavage site to make them more reachable and incorporate Lys1393Ala and Lys1397Ala mutations that prevent A2M from binding to LRP1 and Grp78 receptors. The bispecific moiety was released from A2M-BiTE by proteolytic cleavage of its bait region using trypsin. The data are summarized in Figures 22A and 22B. EC50 values ​​were calculated from the data shown in these figures and are summarized in Table 5.

[0429] In BiTE2-TEV4, ERK1 / 2 phosphorylation was 30.8 times higher after treatment with trypsin than without, as shown in Table 5. In BiTE3-TEV5, although it had a longer linker, ERK1 / 2 phosphorylation was 61.5 times higher after treatment with trypsin than without, as shown in Table 5.

[0430] [Table 5]

[0431] T cell activation by BiTE antibody constructs depends on the presence of target cells expressing a tumor-associated antigen (TAA) to which one of the construct's antibodies is bound. To confirm that ERK1 / 2 phosphorylation and therefore T cell activation are strictly dependent on the presence of TAA-expressing target cells, we repeated the experiment using wild-type CHO cells (CHO-wt) and CHO cells expressing EGFR (CHO-EGFR). The data are summarized in Figure 22C.

[0432] CHO cell experiments confirmed that T cell activation depends on both trypsin-treated BiTE2-TEV4 and the presence of target cells expressing TAA(EGFR). No ERK1 / 2 phosphorylation was observed when incubated with CHO-wt cells, regardless of whether BiTE2-TEV4 was trypsin-cleaved. In contrast, an increase in pERK1 / 2 was observed when CHO-EGFR was used and BiTE2-TEV4 was trypsin-cleaved (but not when BiTE2-TEV4 was not treated). These results indicate that T cell activation by BiTE2-TEV4 depends on both TAA-expressing target cells and structural changes in A2M.

[0433] conclusion This embodiment demonstrates that T cell activation by A2M-BiTE depends on the structural change of A2M when the protease cleavage site located in the bait region is proteolytically cleaved, releasing a TEV-cleaved MG8 domain containing anti-CD3 scFv. The data confirm that anti-CD3 scFv is masked by A2M prior to the cleavage of the protease cleavage site in the bait region. The results further indicate that T cell activation depends on the presence of target cells expressing TAA to which the antibody fused to the C-terminus of the MG8 domain is bound.

[0434] We hypothesize that the protease cleavage site in the bait region is modified so that cleavage at this site in A2M-BiTE primarily occurs in tumors, and is selectively cleaved by one or more proteases expressed in the tumor environment, thereby limiting T cell activation and reducing the side effects associated with conventional BiTE. [Examples]

[0435] Protease-activated A2M-BiTE induces antigen-specific cytotoxicity. Research objectives In this example, we investigated whether the A2M-BiTE construct produced in Example 15, namely BiTE2-TEV4 (SEQ ID NO: 258), could induce specific killing of TAA-expressing target cells after thermolysin-based cleavage of both bait regions and the TEV cleavage site.

[0436] material and method A2M-BiTE was pretreated ("activated") with thermolysin as described in Example 14 or TEV as described in Example 16, or left untreated ("inactivated"). TEV-activated A2M-BiTE was purified by size exclusion chromatography (SEC) as described in Example 16. To alter the structure of A2M and make anti-CD3 scFv reachable, A2M-BiTE was incubated with trypsin or MA to induce cleavage of the protease moiety located in the bait region.

[0437] PBMCs were purified from fresh buffy coats using Histopaque 1077 (Sigma-Aldrich) according to the manufacturer's instructions, and frozen aliquots were stored in liquid nitrogen. PBMCs were thawed and activated with 50 ng / ml OKT3 and 100 IU / ml IL-2. After 3 days, the medium was replaced with RPMI 10% FBS containing 100 IU IL-2, and the cells were used for toxicity assays between days 7 and 14. In the cytotoxicity assay, 100 μl of target cells (HCT116, CHO-wt, or CHO-EGFR) in 100 μl of medium were seeded into 96-well plates and incubated at 37°C under a humidified atmosphere with 5% CO2 for 4 hours. After 4 hours, 2 × 10⁶ cells were induced in 50 μl of RPMI 10% FBS. 5 PBMC effector cells and 50 μl of BiTE sample were added. The plates were centrifuged at 250 g for 2 minutes and incubated at 37°C in a humid atmosphere with 5% CO2 for 16 hours. The PBMCs were washed away by three washes with 200 μl of PBS. 100 μl of 10 mM resazurin in DMEM 10% FBS was added to each well, and the plates were incubated at 37°C in a humid atmosphere with 5% CO2 for 3 hours. The percentage of viable cells was determined by measuring the reduction of resazurin fluorescence (quenching at 540 nm and emission at 590 nm).

[0438] result Wild-type CHO cells (CHO-wt) do not express any EGFR on their surface. Wild-type CHO cells can be induced to express EGFR by transient transfection using an EGFR expression vector, producing EGFR-expressing CHO cells (CHO-EGFR). Therefore, CHO-wt and CHO-EGFR provide suitable models to investigate whether EGFR-targeting A2M-BiTE can induce cytotoxicity in an antigen-dependent manner. Accordingly, we conducted a first set of experiments using CHO-wt or CHO-EGFR as target cells to study whether thermolysin-activated or inactivated EGFR-targeting A2M BiTE, here BiTE2-TEV4 (SEQ ID NO: 258), can induce a cytotoxic T cell response when PBMCs are used as effector cells. Thermolysin cleaves both the TEV site (which enables the release of the BiTE moiety from A2M) and the A2M bait region (which triggers structural changes and BiTE release). As shown in Figure 23A, CHO-EGFR was killed by effector cells only in the presence of activated EGFR-targeted A2M-BiTE, indicating that BiTE activity depends on the release of the antibody-containing MG8 domain from A2M. Inactivated A2M-BiTE targeting EGFR did not have a cytotoxic effect, but the anti-EGFR antibody was exposed at the C-terminus. As expected, CHO-wt was not killed regardless of whether A2M-BiTE was activated, indicating that T cell activation by A2M-BiTE depends on the presence of cells expressing tumor-associated antigens, in this case EGFR.

[0439] The human colon cancer cell line HCT116 has been previously used as a model system for EGFR-expressing colorectal cancer. Therefore, HCT116 represents a therapeutically more relevant target cell for studying EGFR-targeted A2M-BiTEs. Accordingly, we conducted a second set of experiments using HCT116 cells to test whether EGFR-targeting TEV-activated or inactivated A2M-BiTEs, here BiTE3-TEV5 (SEQ ID NO: 259), can induce a cytotoxic T cell response in these cells. As shown in Figure 23B, HCT116 cells were effectively killed by PBMC-derived cytotoxic T cells in the presence of TEV-activated BiTE3-TEV5, and increased potency was observed with MA-treated or trypsin-cleaved BiTE3-TEV5 compared to untreated HCT116 cells. These results indicate that structural changes in A2M-BiTE, which expose anti-CD3-scFv and release the MG8 domain from the N-terminal A2M domain, are necessary to induce an effective cytotoxic T cell response.

[0440] conclusion From these results, we conclude that activated A2M-BiTE targeting TAA can induce a cytotoxic T cell response specific to TAA-expressing cells. In other words, T cell activation depends on the presence of TAA-expressing cells targeted by A2M-BiTE. Furthermore, BiTE-induced cytotoxicity depends on structural changes in A2M and the release of the antibody-containing MG8 domain.

Claims

1. A proteinaceous prodrug construct comprising a complement 3- and pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein, (a) a bait region containing the first protease cleavage site; and (b) Second protease cleavage site at the N-terminus of the receptor-binding domain (RBD), The first drug placed inside the RBD, and A second drug fused to the C-terminus of RBD RBD including Includes, (i) The CPAMD protein blocks the first drug, while the second drug remains accessible; (ii) When the first protease cleavage site of the CPAMD protein is proteolytically cleaved, it changes its three-dimensional structure, releases RBD, and thereby makes the first drug reachable. Protein-based prodrug constructs.

2. The proteinaceous prodrug construct according to claim 1, wherein the first protease cleavage site is different from the second protease cleavage site, or the first and second cleavage sites are the same.

3. The protein prodrug construct according to claim 2, wherein the second protease cleavage site is different and can be specifically cleaved by furin, TEV, enterokinase, or thrombin.

4. The proteinaceous prodrug construct according to any one of claims 1 to 3, wherein the second protease cleavage site is introduced into one or more amino acids corresponding to residues 1334 to 1340 of human A2M.

5. A proteinaceous prodrug construct according to any one of claims 1 to 4, wherein a second protease cleavage site is located between two linkers, the linkers are sometimes 5 to 30 amino acid long, and the linkers are sometimes GS linkers.

6. A proteinaceous prodrug construct comprising a complement 3- and pregnancy-related protein-like alpha-2-macroglobulin domain-containing (CPAMD) protein, (a) a bait region containing the first protease cleavage site; and (b) A first drug placed inside the receptor-binding domain (RBD), and A second drug fused to the C-terminus of RBD Receptor-binding domain (RBD) including Includes, (i) The CPAMD protein blocks the first drug, while the second drug remains accessible; (ii) RBD is bound to the CPAMD protein by non-covalent interactions; (iii) When the first protease cleavage site of the CPAMD protein is proteolytically cleaved, it changes its three-dimensional structure and releases RBD, thereby making the first drug reachable. Protein-based prodrug constructs.

7. The first protease cleavage site is: It is specifically cleaved by proteases expressed by cancer; and / or A proteinaceous prodrug construct according to any one of claims 1 to 6, which is specific to serine-, cysteine-, aspartic acid-, and / or metalloproteinase.

8. The proteinaceous prodrug construct according to any one of claims 1 to 7, wherein the CPAM protein is human alpha-2-macroglobulin (A2M) or a functional homolog thereof, for example, mammalian A2M.

9. A proteinaceous prodrug construct according to any one of claims 1 to 8, wherein the first drug is inserted into loop 2 of RBD corresponding to amino acid residues 1391–1405 of human A2M, and optionally the first drug is inserted between amino acids corresponding to residues 1402–1403 of human A2M or replaces one or more amino acids corresponding to residues 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, and / or 1405 of human A2M.

10. The protein prodrug construct according to any one of claims 1 to 9, wherein the second drug is fused to an amino acid corresponding to residue 1474 of human A2M.

11. A proteinaceous prodrug construct according to any one of claims 1 to 10, wherein the first drug and / or the second drug is an antigen-targeting moiety such as an antibody, for example, a single-chain or single-domain antibody.

12. The first and / or second drug is a protein prodrug construct according to any one of claims 1 to 11, wherein the protein prodrug construct can be directed to a specific tissue, a specific cell type, and / or a specific receptor.

13. The first drug is a protein prodrug construct according to any one of claims 1 to 12, wherein the protein prodrug construct can be directed to immune cells, such as NK cells, macrophages, T cells, or dendritic cells.

14. The first drug is, (i) A T cell specific moiety, for example, a T cell specific moiety that targets receptors expressed at increased levels on T cells, such as CD3, CD4 and / or CD8, and optionally a T cell specific moiety that is an anti-CD3 antibody. (ii) An NK cell specific moiety, for example, an NK cell specific moiety that targets a receptor expressed at increased levels on NK cells, such as CD16, and which may be an anti-CD16 antibody. (iii) Macrophage-specific moiety, for example, a macrophage-specific moiety that targets a receptor or molecule expressed at increased levels on macrophages, such as SIRPα or DNGR1, and optionally a macrophage-specific moiety that is an inhibitory anti-SIRPα antibody or an antibody against the DNGR1 receptor that blocks CD47. The proteinaceous prodrug construct according to claim 12 or 13.

15. The first drug is a protein prodrug construct according to any one of claims 1 to 12, comprising a toxin, an enzyme, a protein conjugate containing a small molecule drug (e.g., a cytotoxin), a cytokine, an extracellular domain of a cell surface receptor, an extracellular domain of a cell surface ligand, or a receptor agonist.

16. The protein prodrug construct according to any one of claims 1 to 15, wherein the second drug is an antigen-targeting moiety that specifically binds to tumor cell surface antigens.

17. A nucleic acid, such as a plasmid, vector encoding a proteinaceous prodrug construct according to any one of claims 1 to 5 or 7 to 16.

18. A host cell containing the nucleic acid described in claim 17.

19. A pharmaceutical composition comprising a proteinaceous prodrug construct according to any one of claims 1 to 16, or a nucleic acid according to claim 17, and a pharmaceutically acceptable excipient.

20. A protein prodrug construct according to any one of claims 1 to 16, or a nucleic acid according to claim 17, for use as a pharmaceutical agent, for example, for use in a method of treating cancer.

21. A method for treating or preventing a disease or disorder in a subject that needs to be treated or prevented, comprising administering a therapeutically effective amount of a protein-based prodrug construct according to any one of claims 1 to 16, or a nucleic acid according to claim 17, to the subject.

22. (a) To provide a host cell containing the nucleic acid described in claim 17; and (b) Culturing host cells under conditions that enable the expression of proteinaceous prodrug constructs from nucleic acids. A method for producing a protein prodrug according to any one of claims 1 to 5 and 7 to 16, comprising the above.

23. (a) To provide a host cell containing the nucleic acid described in claim 17; (b) culturing host cells under conditions that enable the expression of nucleic acid-encoded proteinaceous prodrug constructs; and (c) Contacting the protein prodrug construct with a protease that specifically cleaves the second protease cleavage site. A method for producing the protein prodrug according to claim 6, comprising the above.

24. (a) Contacting the protein prodrug according to claim 6 with a protease that specifically cleaves a second protease cleavage site; and (b) Purifying the protein prodrug to remove the protease. A method for producing a protein-based prodrug containing [a specific substance].

25. The method according to claim 24, wherein the purification step includes chromatography, for example, size exclusion chromatography.

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