Fusion protein for selectively depleting antigen-specific antibodies

Abstract

To provide means for selectively depleting target antigen-specific antibodies from the body.SOLUTION: The present disclosure includes a fusion protein, called a "Seldeg", including a targeting component that specifically binds to a cell surface receptor or other cell surface molecule at near-neutral pH, and an antigen component fused directly or indirectly to the targeting component. The antigen component is configured to specifically bind a target antigen-specific antibody. The present disclosure also includes a method of depleting a target antigen-specific antibody from a patient by administering to the patient a Seldeg having an antigen component configured to specifically bind the target antigen-specific antibody.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This disclosure relates to engineered proteins, and more specifically, fusion proteins ("Seldegs") that selectively deplete target antigen-specific antibodies from the body. [Background technology]

[0002] Antibodies are Y-shaped proteins found in the blood and other bodily fluids of humans and mammals. Antibodies are an important component of the body's immune system. They function by recognizing specific parts of heterologous targets called antigens. Antibodies can selectively recognize antigens through their two antigen-binding sites and trigger an immune response against them. Each antigen-binding site is located at the respective ends of the upper tip of the antibody's Y-shape. A target antigen may bind to one or both antigen-binding sites. The base of the antibody's Y-shape is called the Fc fragment. When an antibody binds to its target, the Fc region can lead to target clearance through its antibody effector function. Such a reaction may involve cellular processes to destroy the antigen. In certain autoimmune diseases and other illnesses, pathogenic antibodies that target autoantigens in the body may be produced and contribute to disease development. Antibodies can exist in one of two physical forms: a soluble form secreted from cells and free in the plasma, or a membrane-bound form bound to the outer membrane of B cells. Secreted antibodies cause pathology in diseases that include autoreactive antibodies. These may also contribute to transplant rejection or the elimination of protein-based therapies.

[0003] Due to their ability to specifically bind to target molecules, antibodies can be used to treat diseases such as cancer and autoimmune disorders. They also have applications in detecting tumors during whole-body imaging using radiolabeled antibodies in positron emission tomography (PET), for example. However, their relatively long in vivo persistence can lead to high background in non-tumor tissues, resulting in poor contrast and undesirable off-target effects in tumor imaging. [Overview of the project]

[0004] This disclosure includes fusion proteins referred to herein as “Seldegs,” which are configured to enable selective clearance of antigen-specific antibodies. Seldeg comprises a target component configured to specifically bind to cell surface receptors or other cell surface molecules and an antigen component configured to specifically bind to antigen-specific antibodies or variants thereof.

[0005] The target component of Seldeg includes a protein or protein fragment configured to specifically bind to a cell surface receptor or other cell surface molecule. The antigen component of Seldeg includes one molecule of an antigen, antigen fragment, or antigen analog configured to specifically bind to a target antigen-specific antibody. The antigen component is fused directly or indirectly to the target component.

[0006] The disclosure also includes a method for depleting target antigen-specific antibodies from a patient by administering Seldeg to the patient in an amount sufficient to clear at least 50% of the target antigen-specific antibodies from the patient's circulation or target tissue.

[0007] The above Seldegs and methods may further include the following details, which may be combined with each other unless they are clearly not mutually exclusive: i) The target component binds to a cell surface receptor or cell surface molecule with a dissociation constant of less than 10 μM at near-neutral pH. ii) The pH near neutral may be higher than 6.8 and less than 7.5; iii) Seldeg may contain at least one first target component and a second target component, where the protein or protein fragment of the first target component is configured to bind to a different cell surface receptor or a different cell surface molecule than the protein or protein fragment of the second target component; iv) The target component may contain a heterodimer of two immunoglobulin Fc fragments, where one immunoglobulin Fc fragment of the heterodimer is fused to the antigen component and the other immunoglobulin Fc fragment is not fused; v) The immunoglobulin Fc fragment may have substantially reduced binding to the Fc gamma receptor or not have detectable binding; vi) The immunoglobulin Fc fragment may be derived from a class or isotype of immunoglobulin that does not bind to the Fc gamma receptor or complement; vii) The immunoglobulin Fc fragment may be configured to bind to the Fc gamma receptor and complement; viiii) The immunoglobulin Fc fragment At least one of the components may be modified to have a higher binding affinity for FcRn than the unmodified immunoglobulin Fc fragment at near-neutral pH; ix) The antigen component may be fused to one immunoglobulin Fc fragment at the N-terminus or C-terminus of the hinge-CH2-CH3 domain of the immunoglobulin Fc fragment; x) The immunoglobulin Fc fragment may be modified to have no binding affinity to the Fc gamma receptor and / or complement (C1q) or to have a lower binding affinity to the Fc gamma receptor and / or complement (C1q) than the unmodified immunoglobulin Fc fragment; xi) The target component may include one or more antibody variable regions or fragments thereof configured to specifically bind to cell surface receptors or cell surface molecules; xii) The antibody variable region or fragments thereof may include at least one nanobody; xiii) The nanobody may be a nanobody multimer, where one nanobody is fused to the antigen component and all other nanobodies in the nanobody multimer are not fused;xiv) The target component may be configured to dissociate from the cell surface receptor or cell surface molecule following entry into the endosome of a complex containing Seldeg and the cell surface receptor or cell surface molecule; xv) The antigen component may be fused to the N-terminal or C-terminal position on the target component; xvi) The antigen component may be fused to the non-terminal position on the target component; xvii) The antigen component may be fused via a chemical reaction, via a linker, or during the formation of a single combined antigen component-targeting component fusion protein. (xviii) The target component may fuse with the protein; the target component may be a mutant albumin variant configured to specifically bind to one or more albumin molecules, albumin fragments, or FcRn; the target component may include one or more antibody variable domains or nanobodies configured to bind to the transferrin receptor; the target component may include one or more protein molecules or protein domains configured to bind to the transferrin receptor; the target component may include one or more protein molecules or protein domains configured to bind to the transferrin receptor; the target component may include one or more protein molecules or protein domains configured to bind to phosphatidylserine. xxii) The target protein component may include one or more antibody-variable domains or nanobodies configured to bind to phosphatidylserine; xxiii) One or more protein molecules or protein domains may be configured to bind to phosphatidylserine via a calcium-dependent mechanism; xxiv) The target component may include the C2A domain of synaptotagmin 1; xxv) Seldeg may include at least one first antigen component and a second antigen component, where one molecule of the antigen, antigen fragment, or antigen analog of the first antigen component is different from one molecule of the antigen molecule, antigen fragment, or antigen analog of the second antigen component;xxvi)Seldeg may contain at least one first antigenic component and a second antigenic component, where one molecule of the antigen, antigenic fragment, or antigen analog of the first antigenic component is the same as one molecule of the antigenic molecule, antigenic fragment, or antigen analog of the second antigenic component; xxvii) The method may include administering Seldeg in an amount sufficient to clear at least 50% of the target antigen-specific antibodies from the circulating or target tissue in the patient within 5 hours of administration;xxviii) The method may include administering Seldeg having a target component comprising a protein or protein fragment configured to bind to a cell surface receptor or other cell surface molecule with a dissociation constant of less than 10 μM at near-neutral pH;xxix) The sufficient amount of Seldeg administered may be at least equimolar to the amount of target antigen-specific antibodies to be depleted;xxx) The method may include administering Seldeg in an amount sufficient to clear at least 90% of the target antigen-specific antibodies from the circulating or target tissue in the patient within 2 hours of administration;xxxi) The amount of Seldeg may be sufficient to clear at least 50% of the target antigen-specific antibodies from the circulating or target tissue in the patient within 1 hour of administration;xxxii) Whenever 50% of the patients are expected to have regenerated a threshold amount of target antigen-specific antibodies in the circulating or target tissue Seldeg may be readjusted;xxxiii) Seldeg may clear less than 10% of non-target antibodies in circulation or in tissues targeted by target antigen-specific antibodies;xxxiv) Seldeg may clear untargeted antibodies in the patient's circulation or in target tissues in amounts that do not cause clinically adverse effects in the patient;xxxv) Seldeg may clear less than 1% of non-target antibodies in circulation or in tissues targeted by target antigen-specific antibodies;xxxvi) Seldeg may cause degradation of target antigen-specific antibodies by cells expressing cell surface receptors or cell surface molecules;xxxvii) Seldeg may be administered to patients with autoimmune diseases, and target antigen-specific antibodies may specifically bind to autoantigens;xxxviii) Seldeg may be administered to patients who have received transplanted organs, and target antigen-specific antibodies may specifically bind to antigens on the transplanted organs;xxxix) Seldeg may be administered to enhance contrast during tumor imaging, and target antigen-specific antibodies may specifically bind to tumor antigens;xl) Seldeg may be administered to patients who have received a biological agent, and the target antigen-specific antibody may be the biological agent; xli) If a patient has antibodies specific to the therapeutic agent, Seldeg may be administered to the patient before delivery of the therapeutic agent, and Seldeg is configured to target antibodies specific to the therapeutic agent; xlii) Seldeg may be administered to provide a PET imaging contrast agent; xliii) The target antigen-specific antibody may be an anti-MOG antibody; xliv) The target antigen-specific antibody may be an anti-HER2 antibody; xlv) Seldeg is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 may contain proteins having at least one amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34 or their homologous sequences; xlvi)Seldeg may contain heterodimers of proteins having amino acid sequences of SEQ ID NO: 2 plus SEQ ID NO: 6, SEQ ID NO: 4 plus SEQ ID NO: 6, SEQ ID NO: 8 plus SEQ ID NO: 10, SEQ ID NO: 12 plus SEQ ID NO: 14, SEQ ID NO: 16 plus SEQ ID NO: 18 plus SEQ ID NO: 20, SEQ ID NO: 20 plus SEQ ID NO: 22 plus SEQ ID NO: 24, SEQ ID NO: 26 plus SEQ ID NO: 28, SEQ ID NO: 30 plus SEQ ID NO: 6, SEQ ID NO: 32 plus SEQ ID NO: 6, or SEQ ID NO: 34 plus SEQ ID NO: 6 or their homologous sequences.

[0008] To further fully understand the present invention and its features and advantages, the following description, as understood together with the accompanying drawings, refers to the drawings not to scale and the same numbers in the drawings refer to the same features. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram of selected cell events that lead to the degradation of antigen-specific antibodies in the presence of Seldeg; [Figure 2A] This is a schematic diagram of Seldeg containing an antigen fused to the N-terminal position of an Fc fragment; [Figure 2B] This is a schematic diagram of Seldeg containing an antigen fused to the C-terminal position of an Fc fragment; [Figure 2C] This is a schematic diagram of Seldeg containing an antigen fused to the non-terminal position of an Fc fragment; [Figure 2D] This is a schematic diagram of Seldeg containing an antigen fused to the terminal position of a protein or protein fragment that binds to a cell surface receptor or cell surface molecule; [Figure 2E] This is a schematic diagram of Seldeg containing an antigen fused to the non-terminal position of a protein or protein fragment that binds to a cell surface receptor or cell surface molecule; [Figure 2F] This is a schematic diagram of Seldeg, which includes an antigen fused to the N-terminus of an Fc fragment and a cell surface protein or cell surface receptor-binding protein or protein fragment fused to the C-terminus of an Fc fragment; [Figure 2G] This is a schematic diagram of Seldeg, which includes an antigen fused to the C-terminal position of the Fc fragment and a cell surface protein or cell surface receptor-binding protein or protein fragment fused to the N-terminus of the Fc fragment; [Figure 2H] This is a schematic diagram of Seldeg containing an antigen fused to the C-terminal position of an antibody that binds to a cell surface protein or cell surface receptor; [Figure 2I] This is a schematic diagram of Seldeg, which includes an antigen fused to the C-terminal position of the Fc fragment and an scFv fragment that binds to a cell surface protein or cell surface receptor fused to the N-terminus of the Fc fragment; [Figure 2J] This is a schematic diagram of Seldeg, which includes an antigen fused to the N-terminal position of the Fc fragment and an scFv fragment that binds to a cell surface protein or cell surface receptor fused to the C-terminus of the Fc fragment; [Figure 2K] This is a schematic diagram of Seldeg containing two antigens fused to the N-terminal position of an Fc fragment; [Figure 2L]Schematic of Seldeg containing two antigens fused to the N-terminal position of the Fc fragment and a protein or protein fragment that binds to a cell surface protein or cell surface receptor fused to the C-terminal of the Fc fragment; [Figure 2M] Schematic of Seldeg containing two antigen molecules fused to the N-terminal position of the Fc fragment; [Figure 2N] Schematic of Seldeg containing two antigen molecules fused to the N-terminal position of the Fc fragment and a protein or protein fragment that binds to a cell surface protein or cell surface receptor fused to the C-terminal of the Fc fragment; [Figure 3A] Schematic of two representative FcRn-targeted Seldegs, human epidermal growth factor receptor 2 Seldeg (“HER2-Seldeg”) and myelin oligodendrocyte glycoprotein Seldeg (“MOG-Seldeg”); [Figure 3B] Shows the increased binding of a representative FcRn-targeted Seldeg to FcRn at pH 6.0 and 7.4. [Figure 3C] Shows the HPLC analysis of two representative FcRn-targeted Seldegs after incubation at 4 °C (30 days) and 37 °C (5 days) to evaluate the storage stability of FcRn-targeted Seldeg. [Figure 3D] Shows a graph reporting representative normalized body counts versus time, showing clearance of antigen-specific antibodies by a representative FcRn-targeted Seldeg; [Figure 3E] Shows an additional graph reporting representative normalized blood and body counts versus time, showing clearance of antigen-specific antibodies by a representative FcRn-targeted Seldeg; [Figure 3F] Shows an additional graph reporting representative normalized blood and body counts versus time, showing clearance of antigen-specific antibodies by a representative FcRn-targeted Seldeg; [Figure 4A]In the upper left panel, a representative Seldeg called MOG-Seldeg-PS is shown, which contains an antigen fused to a target protein (C2A domain of synaptotagmin 1, Syt1) that binds to phosphatidylserine (PS); in the upper right panel, FIG. 4A shows a representative SDS-PAGE gel (reduced and non-reduced states) of MOG-Seldeg-PS, MOG-Seldeg-PS (DN) having a mutation with substantially reduced binding to PS, and a recombinant protein of Fc-Syt1 to which the antigen (MOG) is not bound; in the lower panel, FIG. 4A shows a representative HPLC profile of the recombinant proteins shown in the upper right FIG. 4A, MOG-Seldeg-PS, MOG-Seldeg-PS (DN), and Fc-Syt1; [Figure 4B] Additional graphs reporting representative normalized blood and body counts versus time are shown, demonstrating antigen-specific antibody clearance by a representative PS-targeted Seldeg; [Figure 5A] A graph reporting representative data showing the deposition of antigen-specific antibodies in cells in the presence of representative FcRn-targeted Seldegs and a reference standard protein is shown; [Figure 5B] A representative series of microscopic images of a representative FcRn-targeted Seldeg and a reference standard protein in the presence of a target antigen-specific antibody, with a representative microscopic image of an endosome cropped out, enlarged, and shown in the inset in the upper right corner; [Figure 5C] Another representative series of microscopic images of a representative FcRn-targeted Seldeg and a reference standard protein in the presence of a target antigen-specific antibody, with a representative microscopic image of an endosome cropped out, enlarged, and shown in the inset in the upper right corner; [Figure 6A] Another representative series of microscopic images of a representative FcRn-targeted Seldeg and a reference standard protein in the presence of a target antigen-specific antibody, with a representative microscopic image of a lysosome cropped out, enlarged, and shown in the inset in the upper right corner; [Figure 6B]This is a representative microscopic image of another series of representative FcRn-targeted Seldeg in the presence of antigen-specific antibodies that do not recognize the antigen targeted by the Seldeg. A representative lysosome microscopic image has been cropped, enlarged, and shown in the inset in the upper right corner. [Figure 7] This graph shows representative data illustrating the deposition of antigen-specific antibodies in cells in the presence of typical PS-targeted Seldegs and reference standard proteins. [Figure 8] This is a schematic diagram of a typical Seldeg containing an antigen fused to a target protein (antibody) that binds to the transferrin receptor (TfR); it shows the HPLC distribution of recombinant proteins, including the analysis of target proteins (antibodies) that are not bound to the antigen (MOG); [Figure 9] This graph reports representative data showing the deposition of antigen-specific antibodies in cells in the presence of representative TfR-targeted Seldeg and reference standard proteins; [Figure 10A] This is a representative series of positron emission tomography (PET) analyses of tumors in mice after delivery of radiolabeled HER2-specific antibodies and treatment with representative FcRn-targeted Seldeg, reference standard protein, or vehicle reference standard. [Figure 10B] This graph shows contrast measures in the tumor:thoracic region of tumor-carrying mice after delivery of radiolabeled HER2-specific antibodies and treatment with representative FcRn-targeted Seldeg, reference standard protein, or vehicle reference standard.

[0010] Detailed description of the invention This disclosure relates to artificially modified proteins, and more specifically, Seldegs, which are fusion proteins configured to selectively target antigen-specific antibodies for depletion from the body. Seldegs cause selective degradation of targeted antigen-specific antibodies by binding to antigen-specific antibodies and directing them to late endosomes or lysosomes containing degrading enzymes. Seldegs are fusion proteins comprising at least one target component and an antigen component. The target component is a protein or molecule. The target component includes a protein or protein fragment or other molecule configured to bind to a cell surface receptor or other cell surface molecule. The antigen component includes one molecule of an antigen, antigen fragment, or antigen analog recognized by the targeted antigen-specific antibody.

[0011] When an antigen-specific antibody binds to an antigen component, a complex is formed containing Seldeg and the antigen-specific antibody. The complex is also configured to bind to cell surface receptors or other cell surface molecules, enabling the internalization of the complex containing Seldeg, the antigen-specific antibody, and the targeted cell surface receptor or other cell surface molecule (see Figure 1). Upon entering the endosome, the targeted cell surface receptor or cell surface molecule may dissociate from the complex due to the acidic pH, low calcium concentration, and / or other conditions that distinguish the endosomal environment from the extracellular environment. Transition into the endosome and lysosome leads to the selective degradation of the complex.

[0012] As used herein, the term “antigen-specific antibody” refers to an antibody that binds to a specific antigen, antigen fragment, or antigen analog.

[0013] As used herein, the term “antigen fragment” refers to a portion of an antigen that can be recognized by an antigen-specific antibody.

[0014] As used herein, the term “antigen analog” refers to a protein, protein fragment, peptide, or other molecule that has the same overall shape and properties as a portion of an antigen recognized by an antigen-specific antibody.

[0015] As used herein, the term “cell surface molecule” refers to proteins or other biomolecules (e.g., phospholipids, carbohydrates) exposed on the plasma membrane of a cell.

[0016] Seldeg may include an antigen fused to an IgG antibody Fc fragment (also referred herein as “immunoglobulin Fc fragment”), an FcRn-specific nanobody-antigen fusion molecule, an FcRn-specific antibody that binds to FcRn via its variable region and is fused to the antigen, an albumin-antigen fusion protein, a PS-binding protein, a TfR-specific antibody, or other proteins, protein fragments, or other molecules configured to bind to cell surface receptors or other cell surface molecules that can be identified by those skilled in the art by reading this disclosure.

[0017] Examples of Seldegs described herein include target components configured to bind to cell surface molecules such as human FcRn, exposed phosphatidylserine (PS), or transferrin receptor (TfR) with an affinity (dissociation constant) of less than 10 μM at near-neutral pH.

[0018] FcRn and TfR are proteins, and PS is a phospholipid; they may be found on the surface and within various cell types in the body. The present invention is not limited to targeting these receptors or cell surface molecules, and may encompass many other targets, including low-density lipoprotein receptors, high-density lipoprotein receptors, asialoglycoprotein receptors, inhibitory Fc-gamma receptors, T cell receptors, B cell receptors, G protein-coupled receptors, insulin receptors, glucagon receptors, galactose receptors, mannose receptors, VEGF receptors, and others, which can be identified by those skilled in the art. Other targets can be identified, for example, in the following publication or database: Cell surface receptor atlas (Bausch-Fluck, D., Hofmann, A., Bock, T., Frei, AP, Cerciello). ,F.,Jacobs,A.,Moest,H.,Omasits,U.,Gundry,RL,Yoon,C.,Schiess,R.,Schmidt,A.,Mirkowska,P.,Haertlova,A.,Van Eyk, JE, Bourquin, JP., Aebersold, R., Boheler, KR, Zandstra, P., Wollscheid, B. (2015) A mass spertometric-derived cell surface protein atlas.PLoS One 10:e0121314) and the Human protein atlas (https: / / www.proteinatlas.org / humanproteome / secretome).

[0019] The target component can bind to cell surface receptors or other cell surface molecules with an affinity (dissociation constant) of less than 10 μM at a pH near neutral.

[0020] Therefore, the target components of Seldeg may include any type of molecule configured to specifically bind to cell surface receptors or other cell surface molecules. Such molecules may include proteins, protein fragments, polynucleotides such as ribonucleic acid or deoxyribonucleic acid, polypeptides, polysaccharides, lipids, amino acids, peptides, sugars, and / or other small or large molecules and / or polymers that can be identified by those skilled in the art by reading this disclosure. For example, the target components of Seldeg may include polynucleotides that are ligands for cell receptors.

[0021] Seldeg may include at least one first target component and a second target component, where the protein or protein fragment of the first target component is configured to bind to a different cell surface receptor or a different cell surface molecule than the protein or protein fragment of the second target component.

[0022] As shown in Figure 1, Seldeg 20 may reversibly bind to cell surface receptors or other molecules 30 on the surface of cell 10. Target antigen-specific antibodies 40 present in the extracellular space 50 may reversibly bind to Seldeg 20. This binding typically occurs at near-neutral pH levels, such as pH above 6.8 and below 7.5, because this is the typical pH of the extracellular space 50. Non-target antibodies 60 either do not bind to Seldeg 20 or bind with such low affinity that any binding is nonspecific. Cell surface receptors or molecules 30 bound to Seldeg 20 and target antigen-specific antibodies 40 are transported into cell 10 via pathway A, through receptor-mediated absorption into endosomes 70. The receptors or other molecules can be recirculated back to the cell surface. Therefore, via pathway B, the receptors or molecules 30 bound to Seldeg 20 and target antigen-specific antibodies 40 are recirculated back to the surface of cell 10. Subsequently, the target antigen-specific antibody 40 may be released and may rebind from Seldeg 20 to the same or another Seldeg, or it may remain bound. Similarly, Seldeg 20 may then be released from the receptor or molecule 30 and may rebind to the same or another cell surface receptor or molecule. In some Seldegs, the Seldeg 20-antibody 40 complex may be released in the early or late endosome in an acidic or low-Ca compartment within this area. -2 Due to their concentration, Seldeg 20 may dissociate from the receptor or other molecules 30 (pathway C). Therefore, in pathway D, the receptor or cell surface molecule may recirculate back to the cell surface, but Seldeg 20 bound to the antibody 40 enters the lysosome and is broken down into fragments 80 in pathway E. In some cases of Seldegs, at some point following their transition into the cell 10, the receptor or molecule 30 bound to Seldeg 20 and the target antigen-specific antibody 40 enters late endosomes / lysosomes in pathway F, where at least the target antigen-specific antibody 40 is broken down into fragments 80. This entry into lysosomes appears to increase when Seldeg 20 crosslinks the receptor or molecule 30 into dimers or higher-order aggregates.

[0023] Through this selective depletion mechanism, Seldeg targets antigen-specific antibodies and selectively depletes them from the body without adversely affecting the levels of non-targeted, specific antibodies.

[0024] In particular, Seldeg as described herein can target and selectively deplete antigen-specific antibodies from the body without having adverse clinical effects in patients due to the depletion of untargeted, specific antibodies. Such adverse clinical effects include, for example, immunosuppression and symptoms of epidemic conjunctivitis, bronchitis, ear infections, sinus infections, colds, diarrhea, pneumonia, candidiasis, meningitis, skin infections and other opportunistic infections, especially opportunistic infections that are usually suppressed through antibody-mediated immune responses; and low platelet count. This includes blood disorders such as anemia (counts) and hypogammaglobulinemia, as well as symptoms such as abdominal pain, bloating, nausea, vomiting, diarrhea, or weight loss.

[0025] Generally, Seldegs according to this disclosure are configured to specifically bind to cell surface receptors / molecules via a target component at near-neutral pH and to specifically bind to antigen-specific antibodies at near-neutral pH via an antigen component directly or indirectly fused to the target component. As used herein, the term “specifically bind” refers to a detectable selective intermolecular interaction between the target component and the cell surface receptor / molecule or between the antigen component and the antigen-specific antibody. For example, to specifically bind, the antigen must exhibit a detectable interaction with the targeted antibody, while not exhibiting a detectable interaction with other antibodies. Methods for detecting specific binding are known in the art, such as ELISA, surface plasmon resonance analysis, and other methods that can be identified by those skilled in the art.

[0026] Therefore, Seldeg allows at least a portion of the patient's circulating antigen-specific antibodies to migrate into cells expressing the targeted cell surface receptor or other targeted cell surface molecule, and subsequently be degraded within the cell.

[0027] Seldegs according to this disclosure may avoid an immune response by containing one copy per Seldeg of each type of antigen, antigen fragment, or antigen analog, otherwise referred to herein as "one molecule," which, combined with the insertion of mutations that reduce or eliminate Fc gamma receptor binding and / or complement binding, is thought to reduce antibody crosslinking and possibly the formation of inflammatory immune complexes. In particular, at least 99%, at least 99.5%, or at least 99.9% of Seldegs may contain only one copy per Seldeg of antigen, antigen fragment, or antigen analog at near-neutral pH. Other Seldegs according to this disclosure may contain more than one molecule of antigen, antigen fragment, or antigen analog. The bivalent nature of the antibody to which a Seldeg containing one antigen molecule per Seldeg molecule binds may result in a complex of two Seldeg molecules per antibody, which is thought to improve the effectiveness of lysosomal delivery of the Seldeg-antibody complex via target receptor dimerization.

[0028] Seldeg can contain at least one first antigenic component and a second antigenic component, where one molecule of the antigen, antigen fragment, or antigen analog of the first antigenic component is different from one molecule of the antigen, antigen fragment, or antigen analog of the second antigenic component. Thus, Seldegs containing at least one first antigenic component and a second antigenic component enable the clearance of antigen-specific antibodies with more than one specificity.

[0029] Seldeg may contain at least one first antigenic component and a second antigenic component, where one molecule of the antigen, antigenic fragment, or antigen analog of the first antigenic component is the same as one molecule of the antigenic molecule, antigenic fragment, or antigen analog of the second antigenic component.

[0030] Therefore, Seldegs may include, for example, one or more antigenic components fused to the C-terminus and / or N-terminus of a target component, where one molecule of the antigen, antigenic fragment, or antigen analog of each antigenic component may be the same or different.

[0031] Furthermore, Seldegs may contain human or humanized proteins or protein fragments to avoid or reduce the possibility of an immune response to Seldeg when administered to humans. The antigen, antigen fragment, or antigen analog of the antigen component is preferably a human protein or protein fragment for the administration of Seldeg to humans. The target component is also preferably a human protein or protein fragment such as a human antibody fragment or human albumin or albumin fragment, or a humanized antibody or humanized antibody fragment for the administration of Seldeg to humans. When Seldeg is developed for use in animals other than humans, proteins or protein fragments derived from or modified to be immunologically compatible with those animals may be used instead.

[0032] Figure 2A is a schematic diagram of the activity of Seldeg20a containing antigen 100 fused to a target component having IgG Fc fragment 110. As those skilled in the art will understand, the IgG Fc fragment is the entirety of the Y-shaped lower base of the antibody, which consists of a sulfhydryl-bridged hinge region and CH2 and CH3 domains. Seldeg may have an Fc fragment without a hinge region, or an Fc fragment whose hinge region does not have sulfhydryl bridges. Fc fragment 110 allows Seldeg20a to bind to an FcRn molecule on an FcRn-expressing cell. In the example shown in Figure 2A, antigen 100 may be fused to Fc fragment 110a at the N-terminus of the hinge-CH2-CH3120. Antigen 100 is fused to Fc fragment 110a, and the resulting antigen-Fc fragment is used in a knobs-into-holes strategy (e.g., Moore, GL, Bautista, C., Pong, E., Nguyen, DH, Jacinto, J., Eivazi, A., Muchhal, US, Karki, S., Chu, SY, Lazar, GA, (2011). A novel bispecific antibody format enables simultaneous bivalent and Dimerization with another antigen-free Fc fragment 110b using monovalent co-engagement of distinct target antigens (as described in MAbs 3,546-557) produces a heterodimeric Seldeg molecule 20a, as shown. Seldeg 20a has an Fc fragment with antigen 100 monomerically represented, which avoids the formation of a multimeric immune complex that could cause inflammation or other adverse effects. Seldegs containing only antigen 100 fused to Fc fragment 110a are produced and may be used in some situations, but due to the tendency of Fc fragments to dimerize, dimers will typically be produced. To avoid the formation of Fc fragment dimers with fusion antigen 100, both Fc fragment 110a, which can lead to the formation of multimeric immune complexes, Seldegs employs Knob-in-Towholes mutations and / or electrostatic steering mutations (e.g., Gunasekaran, K., Pentony, M., Shen, M., Garrett, L., Forte, C., Woodward, A., Ng, SB, Born, T., Retter, M., Manchulenko, K., Sweet, H., Foltz, IN, Wittekind, M., Yan, W. (2010) Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. Designed to promote heterodimer formation using (as described in J Biol Chem 285, 19637-19646), so that there is only one Fc fused to one antigen. For heterodimer formation, (G4S) between the C-terminus of the antigen-Fc fusion and the N-terminus of the second Fc fragment. 13Other methods may also be used, such as the insertion of linker peptides (as described in Zhou, L., Wang, HY., Tong, S., Okamono, CT, Shen, WC., Zaro, JL (2016) Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery. Biomaterials, 117, 24-31). DNA and protein sequences of several examples of Seldegs, including Knobs-in-Towholes mutations, electrostatic steering mutations and / or arginine mutations or other mutations that reduce Fc gamma receptor and complement binding, are described in Example 10.

[0033] Examples of additional Knobs-in-Two-Halls mutations include Y349T / T394F:S364H / F405A and Y349T / F405F:S364H / T394F (e.g., Moore, GL, Bautista, C., Pong, E., Nguyen, DH, Jacinto, J., Eivazi, A., Muchhal, US, Karki, S., Chu, SY, Lazar, GA, (2011) A novel bispecific antibody format) This includes mutations that enable simultaneous bivalent and monovalent co-engagement of distinct target antibodies (as described in MAbs 3,546-557) and T366W:T366S:L368A / Y407V (for example, as described in Atwell, S., Ridgway, JBB, Wells, JA, Carter, P (1997) Stabel heterodimers from remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol., 270, 26-35). The residue numbering of these representative Knobs-in-Two-Halls mutations refers to the EU antibody numbering system, as will be understood by those skilled in the art.

[0034] Additional examples of electrostatic steering mutations include E356K / D399K:K392D / K409D and K409D / K370D:D357K / D399K (as described, for example, in Gunasekaran, K., Pentony, M., Shen, M., Garrett, L., Forte, C., Woodward, A., Ng, SB, Born, T., Retter, M., Manchulenko, K., Sweet, H., Foltz, IN, Wittekind, M., Yan, W. (2010) Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J Biol Chem 285, 19637-19646). The residue numbering of these representative electrostatic steering mutations refers to the EU antibody numbering system, as those skilled in the art will understand.

[0035] Additional examples of arginine mutations or other mutations that reduce binding to the Fc gamma receptor and complement (C1q) include G236R / L328R (e.g., Horton, HM, Bernett, MJ, Pong, E., Peipp, M., Karki, S., Chu, SY, Richards, JO, Vostia, I., Joyce, PF, Repp, R., Dasjarlais), among others that can be identified by those skilled in the art. JR, Zhukosky, E. (2010) Potent in vitro and in vivo activity of an Fc-engineered anti-CD19 monoclonal antibody against lymphoma and leukemia.Cancer Res.,68,8049-8057;Moore,GL,Bautista,C.,Pong,E.,Nguyen,DH,Jacinto,J.,Eivazi,A.,Muchhal,US,Karki,S.,Chu,SY,Lazar,GA,(2011) A novel bispecific antibody format enables Simultaneous bivalent and monovalent co-engagement of distinct target anatigens (as described in MAbs 3,546-557), N297A, or N297Q (e.g., Tao, MH., Morrison, SL (1989) Studies). of aglycosylated chimeric mouse-human IgG:role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J.Immunol., 143, 2595-2601; Lux, A., Yu, X., Scanlan, CN, Nimmerjahn, F. (2013) Impact of immune complex size and glycosylation on IgG binding to human FcγRs. J.Immunol., 190, 4315-4323, as described), D265A (for example, Lux, A., Yu, X., Scanlan, CN, Nimmerjahn, F. (2013) Impact of immune complex size and glycosylation on IgG binding to human FcγRs. J.Immunol., 190, 4315-4323; Clynes, RA, Towers, TL, Presta, LG, Ravetch, JV (2000) Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor As described in targets.Nat.Med.6,443-446, L234A / L235A (e.g., Wins, BD, Powell, MS, Parren, PWHI, Barnes, N., Hogarth, PM, (2000) The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors FcγRI and FcγRIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein. As described in AJImmunol., 164, 5313-5318) and L234A / L235A / P329G (e.g., Schlothauer, T., Herter, S., Koller, CF, Grau-Richards, S., Steinhart, V., Spick, C., Kubbies, M., Klein, C., Umana, P., Mossner, E. (2016) Novel human IgG1 This includes IgG4 Fc-engineered antibodies with completely abolished effector functions (as described in PEDS, 29, 457-466). The numbering of residues of these representative arginine mutations or other mutations that reduce binding to the Fc gamma receptor and complement (C1q) refers to the EU antibody numbering system, as those skilled in the art will understand.

[0036] Other mutations can be used to ablate FcγR and / or complement binding by targeting residues at or near the FcγR site and complement binding sites. These sites on the Fc region of IgG are localized (e.g., Jefferi s,R.,Lund,J.(2002) Interaction sites on human IgG-Fc for FcγR:current models.Immunol. Letts.,82,57-65;Duncan,AR,Winter,G.(1988) The binding site for C1q on IgG.Nature,332,738-740;Idusogie,EE,Presta,LG,Gazzano-Santoro,H.,Totpal,K.,Wong,PY,Ultsch,M.,Meng,G.,Mulkerrin,MG(2000) Mapping of the C1q binding site on rituxan, a chimeric human antibody with a (As described in human IgG1 Fc.J.Immunol.,164,4178-4184; Hogarth, PM, Anania, J., Wines, BD (2014) The FcγR of humans and non-human primates and their interaction with IgG: implications for induction of inflammation, resistance to infection and the use of therapeutic monoclonal antibodies. Curr.Top.Microbiol.Immunol.,382,321-352).

[0037] Seldegs may contain Fc plugments derived from a class or isotype of immunoglobulin that does not bind to or has very weak binding to the Fc gamma receptor or complement, such as human IgG2 or human IgG4.

[0038] For several applications, such as diagnostic imaging, Seldegs may contain Fc fragments that have binding sites for Fc gamma receptors and / or complement to enhance the inflammatory response to antigens present in the Seldeg.

[0039] Fc fragment 110 may be modified to substantially improve its binding affinity to FcRn at near-neutral pH compared to the unmodified Fc fragment. For example, the dissociation constant between Fc fragment 110 and FcRn at pH above 6.8 and below 7.5 may be less than 10 μM, as determined by surface plasmon resonance or other biophysical methods. However, Fc fragment 110 may have similar or improved affinity to FcRn at acidic endosomal pH (approximately 6.0) compared to the unmodified Fc fragment, or it may be modified to have a much lower or negligible binding affinity to FcRn at endosomal pH compared to the unmodified Fc fragment. This improvement in binding affinity at near-neutral pH allows each Seldeg to effectively transfer its bound target antigen-specific antibody into late endosomes or lysosomes in FcRn-expressing cells. Enhancement of the binding affinity of the Fc fragment to FcRn may be achieved by insertion of mutations. Naturally occurring IgGs exhibit substantially higher binding affinity to FcRn at acidic pH levels, as opposed to near-neutral pH. This property is essential for the recirculation and transport of IgG within FcRn-expressing cells. In contrast, the increased binding affinity to FcRn at pH 7.4 leads to receptor-mediated translocation into cells and lysosomal delivery, for example.

[0040] The Fc fragment 110 may also be modified to remove or substantially reduce its binding affinity to the Fc gamma receptor and complement (C1q). This modification prevents inflammatory responses caused by the formation of multimeric immune complexes. For example, as described in Example 10, the Fc region can be mutated, also referred to herein as an "arginine mutation," to prevent it from binding to the Fc gamma receptor (G236R / L328R;E U-numbering) (e.g., Moore, GL, Bautista, C., Pong, E., Nguyen, DH, Jacinto, J., Eivazi A., Muchhal, US Karki, S., Chu, SY, Lazar, GA (2011). A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. As described in MAbs 3,546-557) (EU-numbering). In Example 10, these mutations correspond to residues 22 and 114 of Fc-Syt1 (see SEQ ID NO: 10) and residues 144 and 236 of MOG-Seldeg-PS (see SEQ ID NO: 8). Other examples of mutations that substantially reduce or eliminate binding to the Fc gamma receptor and complement include N297A or N297Q (EU numbering; e.g., Tao, MH., Morrison, SL (1989) Studies of aglycosylated Chimeric mouse-human IgG: role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J.Immunol., 143, 2595-2601; as described in Lux, A., Yu, X., Scanlan, CN, Nimmerjahn, F. (2013) Impact of immune complex size and glycosylation on IgG binding to human FcγRs. J.Immunol., 190, 4315-4323), D265A (EU numbering; for example, Lux, A., Yu, X., Scanlan, CN, Nimmerjahn, F. (2013) Impact of immune complex size and glycosylation on IgG binding to human FcγRs. J.Immunol., 190, 4315-4323; Clynes, RA, Towers, TL, Presta, LG, Ravetch, JV (2000) Inhibitory Fc receptors As described in modulate in vivo cytotoxicity against tumor targets. Nat. Med. 6, 443-446, L234A / L235A (EU numbering; e.g., Wins, BD, Powell, MS, Parren, PWHI, Barnes, N., Hogarth, PM, (2000) The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors FcγRI and FcγRIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and This includes L234A / L235A / P329G (as described in protein.AJImmunol., 164, 5313-5318, and L234A / L235A / P329G (EU numbering; for example, as described in Schlothauer, T., Herter, S., Koller, CF, Grau-Richards, S., Steinhart, V., Spick, C., Kubbies, M., Klein, C., Umana, P., Mossner, E. (2016) Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished effector functions. PEDS, 29, 457-466). A reduction of at least 10-fold in binding affinity to the Fc gamma receptor is preferred.

[0041] As shown in Figure 2B, antigen 100 may bind to Fc fragment 110a at a different terminal position in Seldeg20b, or as shown in Figure 2C, antigen 100 may bind at a non-terminal position in Seldeg20c. This interferes with specific FcRn binding. Any of the above positions are suitable. Such positions include amino acid residues that are sufficiently far from the FcRn interaction site (including residues 252-256, 309-311, and 433-436 at the CH2-CH3 domain interface; EU numbering) so as can be identified by those skilled in the art, that FcRn binding is not directly or sterically blocked.

[0042] Antigen 100 may be fused to Fc fragment 110 by any suitable method, including bonding via chemical reaction, bonding via a linker, or during the formation of a combined antigen-Fc fragment protein. Examples of usable chemical bonds include: amine-to-amine (NHS ester), sulfhydryl-to-sulfhydryl (maleimide), amine-to-sulfhydryl (NHS ester / maleimide), sulfhydryl-to-carbohydrate (maleimide / hydrazide), or bonding via a non-natural amino acid with the desired chemical reactivity. This non-natural amino acid can be inserted during recombinant production of the Fc fragment and / or antigen. Polyethylene glycol (PEG) spacers can also be inserted between chemically coupled proteins, protein fragments, or other molecules. Possible linkers include repeats of glycine-serine linker peptides or other more rigid linker peptides, which are encoded in the recombinant expression plasmid for antigen-Fc fusion. The chemistry of linkage, the linkage site, and the selection of peptides can be derived by molecular modeling and, as those skilled in the art will understand, can be designed to minimize the loss of binding activity of proteins / protein fragments targeting antigens or cell surface molecules.

[0043] Figure 2D is a schematic diagram of Seldeg20d with antigen 100 bound to antibody variable region 130. Antibody variable region 130 specifically binds to cell surface receptors or cell surface molecules. Antibody variable region 130 may be the entire variable region or a fragment thereof, insofar as it specifically binds to cell surface receptors or cell surface molecules. Antibody variable region 130 may include a portion of the non-variable region of the antibody configured to bind to cell surface receptors or cell surface molecules. For example, antibody variable region 130 may be a single-domain antibody (sdAb) or a camel-derived VHH domain (also commonly called a nanobody). Such a variable region has an overall fold of an immunoglobulin domain containing two antiparallel β-sheets and may also include domains from other members of the immunoglobulin superfamily, such as the T cell receptor variable domain, the constant region domain of an antibody, or a coreceptor, or the CD4 domain, among others that can be identified by those skilled in the art. Antibody variable region 130 may exist as a monomer or as a polymer, as shown in Figure 2D. For example, if the antibody variable region 130 exists as a nanobody, it may be modified using a linker peptide such as GSSGGSGGGGS between the C-terminus of the first nanobody and the N-terminus of the second nanobody to form a dimer and improve binding activity to the target receptor / molecule. If the antibody variable region 130 is another protein modified to form a nanobody or multimer, variants without antigen 100 may be included during seldeg formation so that the multimer contains only one copy of antigen 100, just as described above with respect to seldegs containing an Fc fragment. The antibody variable region may also contain a heterodimer of a heavy chain variable (VH) domain linked to a light chain variable (VL) domain by a peptide linker, forming an scFv fragment. Linker sequences used to link the VH and VL domains are well known to those skilled in the art and include the GGGGSGGGGSGGGGS[(G4S)3] sequence which links the C-terminus of the VH domain to the N-terminus of the VL domain.In some embodiments, the C-terminus of the VL domain can be ligated to the N-terminus of the VH domain using a similar linker sequence. scFvs that bind to cell surface receptors or other cell surface molecules can be isolated from a library of scFvs using phage display, yeast display, or other antibody display methods. The target protein component of Seldeg is isolated from a library of Fab fragments using phage display, yeast display, etc. The resulting antibody may also include a Fab fragment. For nanobodies, scFvs, and Fab fragments, affinity for binding to cell surface receptors or cell surface molecules can be improved by randomly muting residues in the complementarity-determining regions (CDRs) or by forming a library of mutated nanobodies or variable domains using an error-prone polymerase chain reaction. Representative target CDR residues are those in CDR3 (residues 89-97; Kabat numbering) of the light chain variable domain and CDR3 (residues 95-102; Kabat numbering) of the heavy chain variable domain. These libraries can be displayed on phages or yeast, and mutants with higher affinity can be selected using methods known to those skilled in the art.

[0044] Figure 2D shows antigen 100 at the terminal position of antibody variable region 130, although it may instead be at a non-terminal position. Antigen 100 may fuse to antibody variable region 110 in any suitable way, including through chemical bonding, linker bonding, or the formation of a single combined antigen-antibody variable region fusion protein.

[0045] Seldeg may contain an antigen component fused to a target component that includes a protein other than an antibody or antibody fragment, provided that this protein is configured to bind to a cell surface receptor or other cell surface molecule. For example, as shown in Figure 2E, Seldeg20e contains antigen 100 fused to albumin or albumin fragment 140 that can bind to FcRn. Albumin or albumin fragments may be mutated or modified to bind to FcRn with improved affinity. For example, a mutation can be inserted into the FcRn-binding domain (DIII) of (human serum) albumin using error-prone PCR, followed by displaying a library of mutated albumin variants on yeast or phage to select variants with higher affinity. Alternatively, higher affinity variants can be produced by mutating residues at or near the albumin:FcRn interface and selecting or screening for albumin variants with improved binding affinity. Figure 2E shows antigen 100 at a non-terminal position on albumin or albumin fragment 140, although it may instead be at a terminal position. Antigen 100 may fuse to albumin or albumin fragment 140 in any suitable way, including chemical bonding, linker-mediated bonding, or during the formation of a single combined antigen-FcRn binding protein.

[0046] Figure 2F is a schematic diagram of a typical Seldeg 20f containing antigen 100 bound to the N-terminus of Fc fragment 150. In the example shown in Figure 2F, a protein or protein fragment 160 that binds to a cell surface receptor or cell surface molecule is bound to the C-terminus of Fc fragment 150a. For example, the protein or protein fragment may be the C2A domain of synaptotagmin that binds to phosphatidylserine (PS). Fc fragment 150 can be modified to bind to FcRn with improved affinity, and it may be mutated to bind to the Fc gamma receptor and complement with very low or undetectable binding affinity. An Fc fragment homodimer having two Fc fragments 150a with fused antigen 100 can lead to the formation of a multimeric immune complex. To avoid this, the Seldegs shown in Figure 2F are designed to promote heterodimer formation using knobs-into-holes and / or electrostatic steering mutations, so that there is only one Fc with one Fc-antigen. In Figure 2F, both Fc fragments 150a and 150b have a protein or protein fragment fused to them that binds to a cell surface protein or other cell surface molecule; or, there may be only one such protein or protein fragment. In a representative Seldeg shown in Figure 2G, antigen 100 and protein or protein fragment 160 that binds to a cell surface receptor or cell surface molecule are fused to the C-terminus and N-terminus of Fc fragments 150a and 150b, respectively, forming Seldeg 20g. .

[0047] Figure 2H is a schematic diagram of a representative Seldeg 20h containing antigen 100 bound to the C-terminus of antibody 170, which binds to a cell surface protein or cell surface molecule. The Fc fragment (Fc) in the antibody can be modified to bind to FcRn with improved affinity, and it may be mutated to bind to the Fc gamma receptor and complement with very low or undetectable binding affinity. To avoid antibody homodimers in which antigen 100 is fused to both Fc fragments, which could lead to the formation of a multimeric immune complex, the Seldegs shown in Figure 2H are designed to promote heterodimer formation using knobs-in-to-holes and / or electrostatic steering mutations, so that there is only one antibody heavy chain bound to antigen 100 per antibody molecule. Both Fab fragments of the antibody may bind to the same cell surface protein or other cell surface molecule, or they may bind to two or more different cell surface proteins or molecules.

[0048] Figure 2I is a schematic diagram of a representative Seldeg20i containing antigen 100 bound to the C-terminus of an scFv(180)-Fc fusion that binds to a cell surface protein or cell surface molecule. The Fc fragment (Fc) in the antibody can be modified to bind to FcRn with improved affinity, and it may be mutated to bind to the Fc gamma receptor and complement with very low or undetectable binding affinity. To avoid antibody homodimers where antigen 100 is fused to both Fc fragments, which could lead to the formation of a multimeric immune complex, the Seldegs shown in Figure 2I are designed to promote heterodimer formation using knobs-in-to-holes and / or electrostatic steering mutations, so that there is only one antibody heavy chain-scFv fusion with antigen 100 bound per molecule. Both scFv fragments of the antibody may bind to the same cell surface protein or other cell surface molecule, or they may bind to two or more different cell surface proteins or molecules.

[0049] Figure 2J is a schematic diagram of a representative Seldeg20j containing antigen 100 bound to the N-terminus of a -Fc-scFv(180) fusion that binds to a cell surface protein or cell surface molecule. The Fc fragment (Fc) in the antibody can be modified to bind to FcRn with improved affinity, and it may be mutated to bind to the Fc gamma receptor and complement with very low or undetectable binding affinity. To avoid antibody homodimers in which antigen 100 is fused to both Fc fragments, which could lead to the formation of a multimeric immune complex, the Seldegs shown in Figure 2J are designed to promote heterodimer formation using knobs-into-holes and / or electrostatic steering mutations, so that there is only one antibody heavy chain -scFv fusion with antigen 100 bound per molecule. Both scFv fragments of the antibody may bind to the same cell surface protein or other cell surface molecule, or they may bind to two or more different cell surface proteins or molecules.

[0050] As shown in Figure 2K, in a typical Seldeg20k, two antigenic components (100, 190) may bind to target components, such as Fc fragment 110a and Fc fragment 110b, at the N-terminus or other positions, forming a Seldeg capable of clearing antigen-specific antibodies of different specificities. The Seldegs shown in Figure 2K are designed to promote heterodimer formation using knob-in-to-hole and / or electrostatic steering mutations, and therefore each Seldeg molecule contains the respective type of antigenic molecule (100, 190).

[0051] Figure 2L is a schematic diagram of a typical Seldeg20l containing antigens 100 and 190 bound to the N-terminus of Fc fragment 150. In the typical embodiment shown in Figure 2L, the protein or protein fragment 160 that binds to a cell surface receptor or cell surface molecule is bound to the C-terminus of Fc fragments 150a and 150b. gs is designed to promote heterodimer formation using knob-in-to-hole and / or electrostatic steering mutations, so that each Seldeg molecule contains the respective type of antigen molecule (100, 190). In Figure 2L, both Fc fragments 150a and 150b have a protein or protein fragment fused to them, which binds to a cell surface protein or other cell surface molecule, although in other embodiments, only one such protein or protein fragment may be present.

[0052] As shown in Figure 2M, in a typical Seldeg20m, two molecules of the same antigen (100) may bind to the Fc fragment 200 at the N-terminus or other positions, forming a typical Seldeg20m. This typical Seldeg is a homodimer containing mutations that enhance binding to FcRn and does not contain knobs-in-to-holes and / or electrostatic steering mutations.

[0053] Figure 2N is a schematic diagram of a typical Seldeg 20n containing two molecules of the same antigen (100) bound to the N-terminus of Fc fragment 210. In the typical embodiment shown in Figure 2N, the protein or protein fragment 160 that binds to a cell surface receptor or cell surface molecule is bound to the C-terminus of Fc fragment 210. This typical Seldeg is homodimer and does not contain knobs-in-to-holes and / or electrostatic steering mutations. In Figure 2N, the homodimeric Fc fragment 200 has protein or protein fragments fused to both polypeptide chains, which bind to cell surface proteins or other cell surface molecules, although in other embodiments only one such protein or protein fragment may be present.

[0054] For FcRn-targeted Seldegs, similar principles may be applied to other proteins capable of binding to FcRn. Furthermore, the FcRn-targeted Seldeg or the method of its formation may be influenced by the properties of the FcRn-binding protein. Albumin tends not to form dimers or other multimers, but other FcRn-binding proteins may, in which case the final Seldeg may be formed in a manner similar to that of containing antibody fragments so that each Seldeg contains only one copy of the antigen. In the examples shown in Figures 2A, 2B, 2C, 2F, 2G, 2H, 2I, 2J, 2K, and 2L, the Seldeg has two antibody Fc fragments modified to drive the formation of a heterodimer containing one antigen linked to one Fc fragment and one Fc fragment without an antigen bound, using knobs-in-to-holes and / or electrostatic steering mutations. The Fc fragment can be modified to bind to FcRn with improved affinity at near-neutral pH (Figures 2A, 2B, 2C, 2K, 2M), or it can be ligated to one or more proteins, scFv fragments, Fab fragments, or other molecules that target one or more cell surface receptors or molecules (Figures 2F, 2G, 2H, 2I, 2J, 2L, 2N). The Fc fragments in the examples shown in Figures 2F, 2G, 2H, 2I, 2J, 2L, 2N can also be modified to bind to FcRn with higher affinity, so that they target both FcRn and one or more cell surface receptors or molecules.

[0055] Albumin binds more strongly to FcRn at neutral pH than at acidic pH. However, modifying albumin can alter its binding affinity at near-neutral pH or endosomal pH, promoting the degradation of target antigen-specific antibodies and the recirculation of Seldeg. Similarly, antibody variable region FcRn-binding proteins may be affected by pH in a protein-specific way, but they can still be modified to alter their binding affinity at near-neutral pH or endosomal pH, promoting the degradation of target antigen-specific antibodies. These FcRn-binding proteins have immunoglobulin variable domains, and scFv(VH and VL domains) can be linked by linker peptides such as GGGGSGGGGSGGGGS. Libraries of interconnected VH:VL heterodimers or Fab fragments can be isolated using phage display, yeast display, or other methods known to those skilled in the art. These libraries may be derived from naturally occurring antibody-variable genes or may be prepared using methods that produce “semisynthetic” libraries in which complementarity-determining regions (CDRs) are made using randomized oligonucleotide sequences. Further improvements in their affinity can be achieved, for example, by inserting random mutations into the CDRs using error-prone PCR, followed by selection using phage display or yeast display. Representative CDR residues targeted are those in CDR3 of the light chain variable domain (residues 89-97; Kabat numbering) and CDR3 of the heavy chain variable domain (residues 95-102; Kabat numbering). Similar methods can be used to isolate antibody-based proteins or scaffold-based proteins that bind to other cell surface receptors / molecules.

[0056] Seldegs may also contain any target component configured to specifically bind to a receptor or other molecule on the cell surface (Figures 2D, 2E, 2F, 2G, 2H, 2I, 2J, 2L, or 2N). The target component is fused directly or indirectly (e.g., via a linker) to an antigen component having one molecule of each type of antigen, antigen fragment, or antigen analogue to reduce antibody-mediated crosslinking. As used herein, the term “antigen type” refers to an antigen that binds to a specific antibody. Thus, a Seldeg may contain more than one antigen type, where each Seldeg has only one molecule of each antigen type. If the target protein contains an immunoglobulin-derived Fc fragment, the Fc region may be mutated so that it does not bind to the Fc gamma receptor and complement, or binds to them at substantially reduced levels. Several different possible configurations of Seldegs are shown in Figures 2A–N; these are shown as examples and not limitations, as numerous other configurations can also be conceived by those skilled in the art.

[0057] Seldegs may contain Fc fragments that bind to the Fc gamma receptor and complement. The presence of Fc gamma and complement binding sites may be desirable in certain application contexts where an immune response to an antigen in the Seldeg is desired (e.g., in tumor imaging). In such applications, Seldegs configured to bind to the Fc gamma receptor and complement may be preferred. Seldegs containing Fc fragments without modified mutations that reduce or eliminate binding affinity to the Fc gamma receptor and / or complement (C1q) as described herein, such as arginine mutations, may be configured to elicit such an immune response. Seldegs containing Fc fragments with mutations known to those skilled in the art to improve binding to the Fc gamma receptor and / or complement (C1q) may also be configured to elicit such an immune response. Such Seldegs may also be configured to contain more than one of the same type of antigen molecules per Seldeg (Figure 2K, 2L, 2M, or 2N) to enhance immune complex formation.

[0058] For example, Seldegs can vary in the number of target domains or antibody fragments (e.g., Fab fragments or scFv fragments), ranging from 1 to 3 target domains or antibody fragments (Figure 2). These target domains or antibody fragments can be linked to immunoglobulin Fc fragments, or in other cases, the target domains or antibody fragments can be linked to each other; antigens and antibody fragments can be fused to or to Fc fragments in various orientations (Figure 2); Seldegs can contain linker sequences, which vary in length and composition among the fusion proteins, and can use domains or fragments, e.g., IEGRMD, GGGGS, or 2-3 repeats of this linker; antigen analogs such as small molecules or peptides can be used; Fc fragments in Seldegs The fragment may be mutated to have substantially reduced binding affinity to the Fc gamma receptor and complement, and improved affinity to binding to FcRn; Seldeg's Fc fragment may have mutations such as knobs-into-holes and / or electrostatic steering mutations to form a heterodimer of antigen-bound Fc fragment and non-antigen-bound Fc fragment or a heterodimer containing two antigens.

[0059] Seldegs may contain the following antigens, including proteins, glycoproteins, and nucleic acids associated with autoimmune diseases, including autoimmune encephalitis: myelin oligodendrocyte glycoprotein (MOG), myelin basic protein, proteolipid protein, myelin-associated glycoprotein, myelin-associated oligodendrocyte basic protein, transaldolase, acetylcholine receptor, muscle-specific kinase, low-density lipoprotein receptor-associated protein 4, insulin, islet antigen 2, glutamate decarboxylase 65, and zinc transporter 8, among others that can be identified by those skilled in the art. Citrullinated antigen, carbamylated antigen, collagen, cartilage gp39, gp130-RAPS, 65kDa heat shock protein, fibrillarin, micronucleus protein (snoRNP), aquaporin 4, thyroid-stimulating factor receptor, nuclear antigen, DNA, histone, glycoprotein gp70, ribosome, pyruvate dehydrogenase dehydrolyamide acetyltransferase, hair follicle antigen, human tropomyosin isoform 5, N-methyl-D-aspartate (NMDA) receptor, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, GABA A and GABA B Receptors, glycine receptor, dipeptidyl-peptidase-like protein 6 (DPPX), glutamate receptor (GluR5), voltage-gated potassium channel, Hu, thyroid peroxidase, thyroglobulin, thyroid-stimulating hormone (TSH) receptor, thyroid hormones T3 and T4, desmoglein 1 and 3. The following antigens are examples of tumor-associated antigens that can be introduced into Seldegs to clear tumor-specific antibodies during diagnostic imaging: HER2, prostate-specific membrane antigen (PSMA), prostate stem cell-associated antigen (PSCA), c-Met, EpCAM, carcinoembryonic antigen (CEA). Other antigens include therapeutic agents for which the patient has specific antibodies, or graft antigens recognized by antibodies in the graft recipient. Furthermore, molecular analogues of antigens (synthesized, protein fragments, etc.) can be produced and used in the preparation of Seldegs. The above antigens are examples and do not limit additional types of possible antigens that a person skilled in the art could identify after reading this disclosure.

[0060] In some examples described herein, Seldeg may be a heterodimer of a fusion protein containing the amino acid sequences of SEQ ID NO: 2+SEQ ID NO: 6, SEQ ID NO: 4+SEQ ID NO: 6, SEQ ID NO: 8+SEQ ID NO: 10, SEQ ID NO: 12+SEQ ID NO: 14, SEQ ID NO: 16+SEQ ID NO: 18+Antibody Light Chain SEQ ID NO: 20, SEQ ID NO: 22+SEQ ID NO: 24+Antibody Light Chain SEQ ID NO: 20, SEQ ID NO: 26+SEQ ID NO: 28, SEQ ID NO: 30+SEQ ID NO: 6, SEQ ID NO: 32+SEQ ID NO: 6, or SEQ ID NO: 34+SEQ ID NO: 6, or their homologs.

[0061] Seldeg can be a fusion protein containing an amino acid sequence that has at least 50% identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34.

[0062] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences means with respect to the maximum correspondence across a specified comparison window. A reference refers to a nucleotide base or residue in two sequences that are identical when placed side by side. When percentages of sequence identity or similarity are used in reference to proteins, it is recognized that the positions of non-identical residues are often different due to homogeneous amino acid substitutions, in which an amino acid residue is replaced by a functionally equivalent residue of an amino acid residue with similar physicochemical properties, and therefore does not change the functional properties of the molecule.

[0063] As used herein, functionally equivalent amino acid residues typically refer to other amino acid residues having substantially similar physicochemical and stereochemical properties to the first amino acid. Physicochemical properties include water solubility (hydrophobic or hydrophilic), dielectric and electrochemical properties, physiological pH, partial charge of the side chain (positive, negative, or neutral), and other properties identifiable to those skilled in the art. Stereochemical properties include the spatial and conformational arrangement of amino acids and their chirality. For example, glutamic acid is considered a functionally equivalent residue to aspartic acid in the sense of this disclosure. Tyrosine and tryptophan are considered functionally equivalent residues to phenylalanine. Arginine and lysine are considered functionally equivalent residues to histidine.

[0064] Those skilled in the art will understand that similarity between sequences is typically measured by a method that involves aligning two polypeptide or polynucleotide sequences to form aligned sequences, then detecting the number of matching characters, i.e., similar or identical characters, between the two aligned sequences, and calculating the total number of matching characters divided by the total number of aligned characters in each polypeptide or polynucleotide sequence, including gaps. The similarity result is expressed as a percentage of identity.

[0065] As used herein, “percentage of sequence identity” means a value determined by comparing two optimally aligned sequences across a comparison window, where the portion of the polynucleotide sequence within the comparison window may contain additions or deletions (gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences, giving the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0066] As used herein, “reference sequence” is a defined sequence used as the basis for sequence comparison. A reference sequence may be a subset or the whole of a sequence identified, for example, as a full-length protein or a section of a protein fragment. A reference sequence may be a sequence that can be identified in a database, such as GenBank, Uniprot, and other databases that can be identified by those skilled in the art.

[0067] As those skilled in the art will understand, the determination of percentage identity between any two sequences can be performed using mathematical algorithms. A suitable computer implementation of a mathematical algorithm can be used for comparing sequences to determine sequence identity. Such implementations include, but are not limited to, CLUSTAL, ALIGN, GAP, BESTFIT, BLAST, and FASTA, among others that those skilled in the art can identify.

[0068] For example, Seldegs according to this disclosure have at least 50% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 50%, compared to SEQ ID NOs: 2, 4, 6, or 8, or SEQ ID NOs: 10, 12, 14, 16, or 18, 20, 22, 24, 26, 28, 30, 32, or 34. Both can have amino acid sequences with 95% sequence identity.

[0069] The antigen-specific antibodies targeted by Seldegs may include autoantibodies present in the patient, antibodies that bind to therapeutic agents, antibodies that recognize grafts, and modified (e.g., radiolabeled) antibodies or fragments / modified forms used in diagnostic imaging, among others that can be identified by those skilled in the art.

[0070] As shown in the examples below, Seldegs can selectively deplete target antigen-specific antibodies that have specificity for their fused antigens, such as the target antigen-specific antibody HER2-specific trastuzumab or pertuzumab ("TZB" or "PZB") and MOG-specific antibody ("8-18C5"). As shown in the examples below, target antigen-specific antibodies can be selectively depleted without negatively affecting overall IgG levels or eliciting an unfavorable immune response. These findings are in contrast to other methods in which the treatment results in overall IgG depletion via the use of FcRn inhibitors or antibodies that destroy B cells. Such methods unfavorably affect antibody specificity or B cell function because they lack the selectivity provided by Seldegs.

[0071] Based on this unique selectivity, the Seldeg platform has many applications, as it can be used to create Seldegs with many other target proteins and antigens. Examples of such applications include the treatment of autoimmune diseases, the treatment of antibody-mediated rejection before or during transplantation, enhancing contrast during systemic imaging of tumors (for example, tumor antigens PSMA, EpCAM, and CEA may be used as antigens for the development of additional Seldegs), depletion of body concentrations of specific biologics if an adverse reaction is observed after administration, and antibody clearance to clear antibodies that recognize therapeutic agents before delivery of the therapeutic agent.

[0072] Seldegs may be administered by any method that can deliver them to cells expressing the target cell surface receptor or other molecule, particularly by injection, especially intravenous, subcutaneous, or intramuscular injection, or by injection into the tissue targeted by the antigen-specific antibody to be depleted. Seldegs can also be expressed in genetically modified cells that contain an expression construct encoding Seldegs. In particular, cells can be genetically modified by introducing an expression construct encoding a Seldeg protein, which includes a protein or peptide that enables the genetically modified cells to secrete Seldegs in situ. For example, patient-derived cells can be transfected with an expression construct encoding Seldegs, and the transfected cells can be delivered back into the patient using a method similar to that described for chimeric antigen-receptor (CAR) T cell therapy. The expression construct may contain an expression vector known to those skilled in the art, and may contain, for example, the gene encoding MOG-Seldeg (SEQ ID NOs: 1 and 5) together with a secretory leader peptide, such as one derived from an immunoglobulin gene ligated to the 5' end of the coding sequences for mature MOG-Seldeg (SEQ ID NOs: 1) and Fc (SEQ ID NOs: 5).

[0073] Seldegs may be administered in amounts that do not block all targeted receptors / cell surface molecules, allowing for normal function of these receptors / molecules. The dose of Seldeg used may be similar to the amount of targeted antibody for clearance, and will depend, for example, on the specifics of the antibody-mediated disease or whether Seldegs are used to enhance contrast in diagnostic imaging. The amount of Seldeg used is thought to be less than the total number of targeted receptor types, so that the normal function of the targeted receptors is not adversely affected. Furthermore, for example, IgG binding By using nanobodies (VHHs) that bind to FcRn at sites that do not overlap with the target site, Seldegs can be designed so that they do not compete for binding to the cell surface receptor or the natural ligand of the cell surface molecule (as described, for example, in Andersen, JT, Gonzalez-Pajuelo, M., Foss, S., Landsverk, OJB, Pinto, D., Szyroki, A., de Haard, HJ, Saunders, M., Vanlandshoot, P., Sandlie, I. (2012) Selection of nanobodies that target human neonatal receptor. Sci. Rep., 3, 1118). Furthermore, Seldegs may clear less than 10%, less than 5%, less than 1%, or less than 0.1% of untargeted antibodies in the tissue targeted by antigen-specific antibodies that are circulating or depleted. The retention of untargeted antibodies during and after Seleg treatment may be important in maintaining normal immune function and avoiding infection, among other effects described herein.

[0074] Seldegs may be administered daily, weekly, or whenever 50% of patients are expected to have regenerated a threshold level of the targeted antigen-specific antibody in the circulating tissue or in tissues recognized by the targeted antigen-specific antibody. The level of the targeted antigen-specific antibody can be determined by using enzyme-linked immunosorbent assays (ELISAs) to analyze serum samples. Alternatively, other methods well known to those skilled in the art may be used.

[0075] In transplant patients at risk of antibody-mediated rejection, Seldegs may be administered before or after transplantation. An emergency dose of Seldegs may be administered when the target antigen-specific antibody reaches a threshold in circulation or in tissues recognized by the target antigen-specific antibody. The level of the targeted antigen-specific antibody can be determined by enzyme-linked immunosorbent assays (ELISAs) for analyzing serum samples. Alternatively, other methods well known to those skilled in the art may be used.

[0076] In patients with antibodies specific to therapeutic drugs, such as protein-based therapies, Seldegs may be administered prior to drug delivery to deplete such antibodies. This is thought to overcome problems associated with rapid antibody-mediated clearance of the drug if the drug has elicited an immune response during the previous delivery, or if pre-existing antibodies specific to the drug are present in the patient. Pre-existing or induced antibodies specific to protein-based therapeutic drugs can be detected using several different methods (e.g., as described in Xue, L., Clements-Egan, A., Amaravadi, L., Birchler, M., Gorovits, B., Liang, M., Myler, H., Purushothama, S., Manning, MS, Sung, C. (2017) Recommendations for the assessment and management of pre-existing drug-reactive antibodies during biotherapeutic development. AAPS, 19, 1576-1586).

[0077] In diagnostic / theranostic imaging, there is likely to be a period of time during which the delivery of radiolabeled imaging antibodies allows for tumor localization. Subsequently, Seldegs are used to clear the radiolabeled antibodies from off-target sites (e.g., circulation), resulting in improved contrast. For example, this method may include the following steps: First, a radiolabeled (or otherwise labeled) antibody that binds to the tumor antigen is injected into the patient. Second, after a period of time (e.g., 16-24 hours) to allow the radiolabeled antibody to bind to the tumor, Seldegs are injected in a molar amount equal to the dose of the injected imaging agent. After the clearance period (e.g., 4-24 hours), positron emission tomography (PEM) The patient is imaged using thoracic tomography or another whole-body imaging method.

[0078] Seldegs may be administered in amounts sufficient to deplete at least 50%, at least 80%, or at least 90% of the concentration of target antigen-specific antibodies in circulation or in tissues recognized by the target antigen-specific antibodies within 1 hour, 2 hours, 5 hours, 24 hours, 48 ​​hours, or longer. The persistence of Seldegs in the body will be a determinant of how long they remain active in depleting antigen-specific antibodies. Seldegs can be designed to have varying in vivo half-lives depending on the behavior of the cell surface receptors or cell surface molecules they target. The affinity of Seldegs for these cell surface receptors or cell surface molecules can also be modified using mutagenesis and methods known to those skilled in the art to produce Seldegs with varying persistences in circulation and / or tissues. In particular, Seldegs may be administered in amounts at least equimolar to the amount of antigen-specific antibodies to be depleted. For example, Seldeg can be administered in amounts with a molar ratio of approximately 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or higher relative to the target antigen-specific antibody. In particular, if Seldeg targets an antibody administered to the patient (e.g., anti-MOG or anti-HER2 antibody), Seldeg can be administered in doses with a molar ratio of approximately 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or higher relative to the administered target antigen-specific antibody. [Modes for carrying out the invention]

[0079] The following examples are given to further illustrate specific aspects of the present disclosure. They are not intended, and should not be understood, to disclose or describe in full detail each and all aspects of the present disclosure. Unless otherwise noted, the names of cell systems and compositions are used consistently throughout these examples. [Examples]

[0080] Expression and purification of Seldegs that bind to -FcRn Seldegs targeting FcRn on cells contain recombinant antigens as monomers linked to a dimeric human IgG1-derived Fc fragment (Figure 3A) that has mutations to eliminate interaction with human FcγRs at near-neutral pH and enhance the binding affinity of the SeldegFc fragment to FcRn. Heterodimer formation of these two Seldegs is achieved by inserting a knobs-into-holes mutation in the CH3 domain.

[0081] Expression constructs for representative HER2-Seldegs (SEQ ID NOs: 3, 4, 5, and 6) were prepared as follows: To express a polypeptide chain containing HER2 fused to a modified Fc fragment (SEQ ID NO: 3), genes encoding the HER2 leader peptide and the extracellular domain (ECD consisting of 630 residues) were isolated from a HER2-overexpressing breast cancer cell line (BT-474) using standard molecular biological methods. This gene was fused to the N-terminus of the hinge region of the gene encoding the human IgG1-derived Fc fragment via the IEGRMD linker peptide using overlap extension splicing. Mutations were inserted into the Fc fragment gene using standard methods to eliminate binding to FcγRs (G236R / L328R; EU numbering), to strengthen binding to FcRn (MST-HN; M252Y / S254T / T256E / H433K / N434F; EU numbering), and to create 'knobs in to holes' (Y349T / T394F; EU numbering). The cysteine ​​in the hinge region that cross-links with the cysteine ​​in the light chain constant region (C220; EU numbering) was also mutated to serine. The Fc fragment gene, which has an FcRn strengthening mutation and a mutation to eliminate FcγRs binding, and lacks a fusion antigen, was then combined with a complementary nobzui... Created using the Ntohols mutation (S364H / F405A; EU numbering) (Sequence ID 5). Gibco Expi293 TMRecombinant proteins were expressed in HEK-293F (Life Technologies) cells after transient transfection using an expression kit (Life Technologies). HER2-Seldeg was purified from the culture supernatant using an anion exchange column (SOURCE-15Q, GE Healthcare) at pH 8.0 and a linear salt gradient (0-0.5 M NaCl). Alternatively, HER2-Seldeg could be purified using protein A-Sepharose and standard methods. After elution from the column (ion exchange or protein A-Sepharose), HER2-Seldeg was dialyzed against phosphate-buffered saline (PBS). HER2-Seldeg was further purified using size exclusion chromatography (SEC) (GE Healthcare) in PBS (Lonza) before experimental use. Expression plasmids for other Seldegs were constructed using similar methods and recombinant proteins expressed in transfected HEK-293F cells. Seldegs without FcRn-enhancing mutations (e.g., MOG-Seldeg-PS, MOG-Seldeg-TfR; SEQ ID NOs. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20) were purified using protein G-Sepharose.

[0082] Seldegs targeting antibodies specific to two antigens were created: (1) HER2-Seldeg and (2) MOG-Seldeg. The antigen HER2 is a well-defined target for therapies using HER2-specific antibodies such as trastuzumab (TZB) or pertuzumab (PZB), and for diagnostic imaging of HER2-overexpressing tumors. The antigen MOG is recognized by autoreactive antibodies in both animal models of multiple sclerosis (MS) and in human MS.

[0083] HER2-Seldeg and MOG-Seldeg retain significantly higher binding affinity for FcRn at neutral and acidic pH levels, in contrast to recombinant fusion proteins containing HER2 or MOG fused to an Fc fragment to create similar constructs ("HER2-WT" and "MOG-WT," respectively) to HER2-Seldeg and MOG-Seldeg, except for the absence of FcRn-enhanced MST-HN mutations (M252Y, S254T, T256E, H433K, N434F; EU numbering) that improve binding affinity at near-neutral pH (Figure 3B). Surface plasmon resonance experiments were performed using BIAcore T200 (GE Healthcare) to analyze the interaction between recombinant proteins and FcRn. The binding of Seldeg / WT to recombinant mouse FcRn was analyzed by injecting 100 nM MOG / HER2-Seldeg or MOG / HER2-WT at a flow rate of 10 μl / min onto immobilized FcRn (bound to ~600 RU on a CM5 sensor tip) in PBS (pH 6.0 or 7.4) plus 0.01% v / vTween-20. Flow cells were regenerated using 0.15 M NaCl, 0.1 M sodium bicarbonate, pH 8.5 following each injection and dissociation step. Data were zeroed and background was subtracted (background was obtained by injecting onto flow cells bound only to buffer during the binding reaction).

[0084] Mutations that enhance FcRn binding, such as MST-HN, were identified using the following method: random mutations were introduced in Fc fragment genes at residues adjacent to amino acids known to be essential for FcRn binding (e.g., 253, 435), and a library of mutated Fc fragments was displayed on phages. (Phage display method) (Ghetie, V., Popov, S., Borvak, J., Radu, C., Matesoi, D., Medesan, C., Ober, RJ, Ward, ES (1997) Increasing the serum persistence of Using an IgG fragment by random mutagenesis (Nature Biotech., 15, 637-640; Dall'Acqua, WF, Woods, RM, Ward, ES, Palaszynski, SR, Patel, NK, Brrewah, YA, Wu, H., Kiener, PA, Langermann, S. (2001) Increasing the affinity of a human IgG1 for the neonatal receptor: biological consquences, J.Immunol., 169, 5171-5180), we selected Fc fragments with improved binding affinity to FcRn. Alternatively, these residues can be mutated to all other possible amino acids, and Fc fragments with higher affinity to FcRn can be identified using methods such as ELISA or surface plasmon resonance coupling analysis.

[0085] Size exclusion analysis shows that recombinant proteins containing Seldegs do not form aggregates after incubation in phosphate-buffered saline when incubated at up to 4°C for 30 days or 37°C for 5 days (Figure 3C). [Examples]

[0086] -Seldegs targeting FcRn have the ability to deplete target antigen-specific antibodies in mice that express human FcγRs through transformation (huFcγR mice). To determine the ability of Seldegs to deplete target antigen-specific antibodies, mice expressing human FcγRs through exogenous gene transfer (huFcγR mice; Smith, P., DiLillo, DJ, Bournazos, S., Li, F., Ravetch, JV (2012). Mouse model recapitulating human Fcγ receptor structural and functional diversity. Proc. Natl. Acad. Sci. USA 109, 6181-6186. http: / / dx.doi.org / 10.1073 / pnas.1203954109) were subjected to 15 μg of radiolabeled ( 125 I-labeled) MOG-specific antibody 8-18C5 was injected. 24 hours after injecting 8-18C5 into huFcγR mice, 125 μg or 31 μg of MOG-Seldeg or reference standard protein was administered by injection. In Figure 3D, “MOG-Seldeg-High” corresponds to a 125 μg dose, and “MOG-Seldeg-Low” corresponds to a 31 μg dose. The reference standards are phosphate-buffered saline ("PBS") and unmodified MOG-Fc fusion protein ("MOG-WT"). MOG-WT is a construct similar to MOG-Seldeg except that it lacks the FcRn-enhanced MST-HN mutation that improves binding affinity at near-neutral pH.

[0087] To create the data for Figure 3D, whole-body counts of radiolabeled 8-18C5 were taken at the indicated times. 24 hours after 8-18C5 administration, the count per minute ("CPM") obtained immediately before Seldeg or reference standard delivery was considered 100% for each mouse, and subsequent whole-body CPMs were normalized to this point in time. Error bars indicate the standard deviation, and two-way ANOVA with Tukey's multiple comparisons was used to determine statistically significant differences at p<0.05 and n=6 mice per group.

[0088] The 125 μg and 31 μg doses of MOG-Seldeg are approximately 2 to 8 times lower (based on moles) than the 500 μg dose of MST-HN Abdeg, which has been shown to eliminate IgG levels overall in mice. Such relatively low doses of MOG-Seldeg minimize the impact on IgGs that are not specific to the fused antigen through competition with Seldeg for FcRn binding. The doses used were 125 μg and 31 μg, respectively, representing a 16-fold and 4-fold molar excess compared to the 8-18C5 target antigen-specific antibody.

[0089] Figure 3D shows a graph of normalized body counts of radiolabeled 8-18C5 (target antigen-specific antibody) against time. Administration of MOG-Seldeg resulted in a rapid dose-dependent decrease of 8-18C5 in normalized body counts (Figure 3D), demonstrating MOG-Seldeg's ability to selectively deplete 8-18C5 from the body. In contrast, infusion of reference standard MOG-WT lacking the FcRn-enhanced MST-HN mutation had no effect on 8-18C5 antibody depletion. This highlights the importance of the MST-HN mutation for conferring high binding affinity for FcRn at pH 6.0–7.4. [Examples]

[0090] - The specificity of Seldegs targeting FcRn and their ability to induce a significant decrease in target antigen-specific antibody levels both in the blood and throughout the body. To analyze the specificity of Seldegs and their effects on antibodies with various antigen recognition capabilities, the behavior of the HER2-specific antibody TZB was investigated in the presence of both HER2-Seldeg and MOG-Seldeg (Figure 3E). To prepare the data for Figure 3E, huFcγR mice were given 15 μg of radiolabeled ( 125I-labeled TZBs were intravenously injected, followed by intravenous injection of 4x molar excess HER2-Seldeg or MOG-Seldeg as a reference standard for antigen specificity. Additional reference standards included phosphate-buffered saline ("PBS"), Abdeg, and recombinant fusion proteins containing HER2 fused to an Fc fragment to create a construct similar to HER2-Seldeg ("HER2-WT") except for the lack of an FcRn-enhanced MST-HN mutation that improves binding affinity at near-neutral pH. In contrast to Seldeg, "Abdeg" is a human IgG1-derived antibody with an MST-HN mutation, which non-selectively depletes the antibody. Such proteins are called Abdegs because they are antibodies that commonly cause IgG degradation.

[0091] To prepare the data for Figure 3E, mice were induced to bleed, and blood and whole-body counts were taken at the instructed times. Counts per minute ("CPM") obtained 24 hours after TZB administration, immediately before Seldeg or reference standard delivery, were considered 100% for each mouse, and subsequent blood and whole-body CPMs were normalized to this point in time. Error bars indicate standard deviation, and statistically significant differences were determined using two-way ANOVA with Tukey's multiple comparisons, with p<0.05 and n=6 mice per group.

[0092] Figure 3E shows graphs of normalized blood and body counts of radiolabeled TZBs (target antigen-specific antibodies) against time. Administration of HER2-Seldeg caused a significant decrease in normalized TZBs in body counts in both blood and body counts, demonstrating HER2-Seldeg's ability to selectively deplete TZBs from the body. In contrast, the reference standard protein exhibited behavior similar to that observed with respect to the PBS reference standard (Figure 3E). [Examples]

[0093] - The ability of Seldegs targeting FcRn to rapidly deplete target antigen-specific antibodies Mice that were radiolabeled ( 125I-labeled TZB (15 μg) was administered intravenously, and HER2-Seldeg (51 μg; 4-fold molar excess of TZB) was delivered intravenously 24 hours later. Additional reference standards were phosphate-buffered saline ("PBS") and Abdeg (60 μg).

[0094] To create the data shown in Figure 3F, mice were induced to bleed, and blood and whole-body counts were instructed. The data was collected at the appropriate time. 24 hours after TZB administration, the count per minute ("CPM") obtained immediately before Seldeg or reference standard delivery was considered 100% for each mouse, and subsequent blood and systemic CPMs were normalized to this point in time. Error bars indicate standard deviation, and statistically significant differences were determined using two-way ANOVA with Tukey's multiple comparisons, with p<0.05 and n=5-6 mice per group.

[0095] Figure 3F shows graphs of normalized blood and body counts of radiolabeled TZBs (target antigen-specific antibodies) against time. Administration of HER2-Seldeg causes a rapid decrease in normalized body and blood counts of TZBs, demonstrating HER2-Seldeg's ability to rapidly deplete TZBs from the body. Notably, blood counts of TZBs decreased to near background levels within 2 hours of Seldeg administration (Figure 3F), further supporting HER2-Seldeg's ability to rapidly deplete target antigen-specific antibodies from the body. In contrast, delivery of Abdeg at a dose of 60 μg / mouse showed behavior similar to that observed with respect to a PBS reference standard (Figure 3F). [Examples]

[0096] -Seldegs activity targeting exposed phosphatidylserine (PS) on the cell surface We also examined the ability of Seldeg, in which the target protein is the C2 domain of synaptotagmin 1 (Syt1), to selectively deplete MOG-specific antibodies in huFcγR mice. Syt1 binds to exposed PS on the cell surface, and MOG-Seldeg-PS was designed for this purpose and is schematically shown (Figure 4A). MOG-Seldeg-PS(DN), which does not bind to PS due to the presence of the ‘DN’ mutations (D173N, D179N, D231N, D233N and D239N), was also generated. MOG-Seldeg-PS and MOG-Seldeg-PS(DN) were purified from the culture supernatants of transfected HEK-293F cells using protein G-sepharose and standard methods. Heterodimer formation was achieved by insertion of knobs-into-holes and electrostatic steering mutations into the Fc region, and size exclusion analysis indicates that the recombinant proteins behave well (Figure 4A).

[0097] For generation of the data shown in Figure 4B, mice were intravenously injected with radioactively labeled 125 I) chimeric 8-18C5 (human constant / mouse variable domains; MOG-specific; 15-20 μg), and 24 hours later, phosphate-buffered saline (PBS), 40 μg of MOG-Seldeg-PS or 34 μg of Fc-Syt1 (unbound MOG) as a reference standard or 40 μg of MOG-Seldeg-PS(DN) were delivered intravenously. Radioactivity levels were determined at the indicated times. The whole body CPM or blood CPM levels obtained immediately prior to MOG-Seldeg-PS or reference standard delivery were considered 100%, and all subsequent CPMs obtained were normalized to these CPM levels. Error bars indicate standard error of the mean (SEM), and statistically significant differences between the MOG-Seldeg-PS treatment group and reference standard groups (Fc-Syt1, MOG-Seldeg-PS(DN) and PBS) were * indicated by (p < 0.05, two-way ANOVA using Tukey's multiple comparisons; n = 5-6 mice per group).

[0098] Administration of MOG-Seldeg-PS induced a significant decrease in normalized body counts of 8-18C5 in both blood and whole-body counts, demonstrating the ability of MOG-Seldeg-PS to selectively deplete 8-18C5 from the body. In contrast, the reference standard protein Fc-Syt1 exhibited similar behavior to that observed with respect to the PBS reference standard, and while MOG-Seldeg-PS(DN) induced a decrease in 8-18C5 (due to residual binding to PS), the effect was much less significant than that of MOG-Seldeg-PS (Figure 4B). [Examples]

[0099] -Seldegs that target FcRn have the ability to effectively transport target antigen-specific antibodies into endosomes and induce degradation via delivery to lysosomes. To determine the mechanism of Seldegs activity at the cellular level, flow cytometry and fluorescence microscopy were performed to analyze the effects of Seldegs on the translocation and deposition of target antigen-specific antibodies TZB, PZB, and 8-18C5. Human endothelial cells (HMEC-1) were initially transfected with a human FcRn-GFP expression construct mutated to have binding similar to mouse FcRn.

[0100] Seldegs effectively translocates bound target antigen-specific antibodies into intracellular endosomes, and after 8 hours of incubation, the target antigen-specific antibodies are delivered to lysosomes.

[0101] Figure 5A is a graph of mean fluorescence intensity ("MFI"), and Figures 5B and 5C are microscopic images of Seldeg activity in the presence of target antigen-specific antibodies. Representative endosome microscopic images are cropped, enlarged, and shown in the inset in the upper right corner. Figure 5A shows greater MFI in the presence of Alexa 647-labeled TZB with HER2-Seldegs and Alexa 647-labeled 8-18C5 with MOG-Seldegs, respectively. This greater MFI indicates that co-incubation of HER2- and MOG-Seldegs with TZB and 8-18C5 (400 nM Seldeg or WT reference standard plus 100 nM antigen-specific antibody; 30 min pulse, no follow-up or 60 min follow-up, labeled with 30'P or 30'P,60'C in Figures 5A, 5B, and 5C) results in higher levels of TZB and 8-18C5 entry cells. Similar results were obtained when the HER2-specific antibody pertuzumab (PZB) was used instead of TZB in the presence of HER2-Seldeg (Figure 5A). Furthermore, the majority of each target antigen-specific antibody was retained by cells during the 60-minute follow-up period. In contrast, intracellular accumulation of target antigen-specific antibodies was substantially lower in the presence of negative reference standards HER2-WT (selective for TZB or PZB) and MOG-WT (selective for 8-18C5).

[0102] Figures 5B and 5C show that antigen-specific antibodies accumulate in cells and are associated with FcRn in endosomes in the presence of Seldegs. To prepare the microscopic images in Figure 5B, HMEC-1 cells were pulsed for 30 minutes with 100 nM Alexa 647-labeled TZB (HER2-specific) in a complex with 400 nM Alexa 555-labeled HER2-Seldeg or HER2-WT (as shown), washed, and immediately fixed, or washed, tracked in medium for 60 minutes, and then fixed. Fixed cells were imaged using fluorescence microscopy. The data in Figure 5C were prepared using the same method except that 100 nM Alexa 647-labeled 8-18C5 (MOG-specific) in a complex with 400 nM Alexa 555-labeled MOG-Seldeg or MOG-WT. The bars in each microscopic image are 5 μm, and the bars in each microscopic image insert are 0.25 μm. The data shown in Figures 5B and 5C demonstrate that antigen-specific antibodies accumulate in FcRn-expressing cells to substantially higher levels in the presence of Seldegs that bind to FcRn compared to the presence of a reference standard (WT) protein.

[0103] Figure 6A shows microscopic images of Seldeg activity in the presence of target antigen-specific antibodies, with representative lysosome microscopic images cropped and enlarged, shown in the inset in the upper right corner. For Figure 6A, HMEC-1 cells were pre-pulsed and washed for 2 hours with Alexa 555-labeled dextran, and then complexed with 100 nM Alexa 647-labeled 8- 100 nM in complex with 400 nM MOG-Seldeg or MOG-WT (as shown). Cells were pulsed for 30 minutes with 18C5 (MOG-specific), followed by 8 hours of tracking, washing, fixation, and imaging. For the preparation of the microscopic images in Figure 6B, HMEC-1 cells were pre-pulsed for 2 hours with Alexa 555-labeled dextran, washed, and then pulsed for 30 minutes with 100 nM Alexa 647-labeled TZB or 8-18C5 antibody and 400 nM MOG-Seldeg or HER2-Seldeg (as shown), followed by 8 hours of tracking, washing, fixation, and imaging. For each overlay image (shown in the right column), GFP, Alexa 555, and Alexa 647 were pseudo-colored to green, red, and blue, respectively. The bars in each microscopic image are 5 μm, and the bars in each microscopic image insert are 0.25 μm. Of particular note is that Figure 6B shows that cells do not deposit TZB and 8-18C5 in lysosomes in the presence of MOG-Seldeg or HER2-Seldeg, respectively, which demonstrates the antigen specificity of their effects. [Examples]

[0104] - The ability of phosphatidylserine-targeting Seldegs to effectively deliver target antigen-specific antibodies into cells. Figure 7 shows that MOG-Seldeg-PS effectively transfers MOG-specific antibodies (chimeric 8-18C5) into endothelial cells and macrophages, and that absorption is PS-binding dependent. PS-exposed endothelial cells (2H11) or macrophages (RAW264.7) were incubated for 2 hours with 10 nM Alexa 647-labeled chimeric 8-18C5 mixed with 20 nM MOG-Seldeg-PS, MOG-Seldeg-PS(DN), or Fc-Syt1 (without MOG). The mean fluorescence intensity (MFI) of Alexa 647-labeled chimeric 8-18C5 (prepared by fusing the variable domain of 8-18C5, a mouse antibody, to the constant region of human IgG1, Cκ, using a method well known to those skilled in the art) for the three samples was determined by flow cytometry.

[0105] This typical PS-binding Seldeg is calcium-dependent and therefore dissociates in endosomes where calcium concentration is much lower. As will be understood by those skilled in the art, not all PS-targeted Seldegs are likely to be calcium-dependent, but some, such as those containing annexin V, will have this property. Furthermore, some antibodies can bind to beta-2 glycoprotein 1, which in turn can bind to PS. [Examples]

[0106] - The ability of Seldegs targeting transferrin receptors to effectively deliver target antigen-specific antibodies into cells expressing human transferrin receptors. Seldegs containing an antigen (MOG) fused to an antibody targeting the transferrin receptor (MOG) were constructed (MOG-Seldeg-TfR), which are schematically shown in Figure 8. MOG-Seldeg-TfR, a reference standard antibody, and Ab-TfR (without MOG) were purified from the culture supernatant of transfected HEK-293F cells using protein G-Sepharose and standard methods. MOG-Seldeg-TfR effectively translocates the MOG-specific antibody (chimeric 8-18C5) into endothelial (HMEC-1) cells, and absorption is dependent on the presence of MOG in the Seldeg (Figure 9). Human TfR-expressing HMEC-1 cells were incubated for 30 minutes with 50 nM Alexa 647-labeled chimeric 8-18C5 (specific to MOG) mixed with 200 nM MOG-Seldeg-TfR or TfR-specific antibody (Ab-TfR; without MOG), followed by washing and no follow-up (30'P) or washing followed by 30 minutes and follow-up for 60 minutes (30'P, 60'C). The mean fluorescence intensity (MFI) of Alexa 647-labeled 8-18C5 for the triple samples was determined by flow cytometry (Figure 9). [Examples]

[0107] -Seldegs targeting FcRn have the ability to clear background during positron emission tomography analysis of tumors using radiolabeled antibodies. Female BALB / cSCID mice were transplanted with HER2-positive breast cancer cell line, HCC1954, in the breast fat pad (0.5 x 10⁶ mice). 6 Individual cells (cells suspended in RPMI-1640 / Matrigel before injection). After 6-7 days, mice were injected with 124-I labeled TZB (60 μg / mouse) and analyzed by positron emission tomography (PET) 22 hours later. Mice were injected with molar equivalents of HER2-Seldeg (51 μg / mouse) or MOG-Seldeg (31 μg / mouse) or a PBS vehicle as a reference standard (n = 3 mice per group). Mice were analyzed by PET 4 hours after injection of Seldegs or PBS. Siemens Mouse images were obtained using the Inveon PET-computed tomography (CT) Multimodality System.

[0108] To create the data shown in Figure 10A, mice were imaged 22 hours after injection of 124-I labeled TZB ('22 hours') and 4.5–5 hours after injection of HER2-Seldeg, MOG-Seldeg, or PBS ('4.5-hour clearance'). PET and CT images were co-registered in the AMIDE software package. Figure 10B shows graphs of contrast measurements for radiolabeling intensity 22 hours after injection of 124-I labeled TZB and 4.5–5 hours after injection of HER2-Seldeg, MOG-Seldeg, or PBS. For determining the contrast measurements, two regions of concern were identified: the chest region (background) and the tumor. The mean ratio of mean intensity in the tumor to mean intensity in the chest region (n = 3 mice per group) is plotted. Error bars indicate the standard error. The study showed a statistically significant difference (p<0.05) between mice treated with HER2-Seldeg and mice treated with MOG-Seldeg or PBS. The data indicate that HER2-Seldeg delivery reduces background in the thoracic region of mice. [Examples]

[0109] - Representative Seldegs and reference standards, as well as DNA and protein sequences of their variants with mutations such as knobs-in-two-hole mutations, arginine mutations, and electrostatic steering mutations. Table 1 shows the DNA sequences of polynucleotides encoding representative proteins described herein, and Table 2 shows the amino acid sequences of representative proteins encoded by the polynucleotides shown in Table 1, where the DNA sequence of SEQ ID NO: 1 encodes the protein of SEQ ID NO: 2, the DNA sequence of SEQ ID NO: 3 encodes the protein of SEQ ID NO: 4, the DNA sequence of SEQ ID NO: 5 encodes the protein of SEQ ID NO: 6, the DNA sequence of SEQ ID NO: 7 encodes the protein of SEQ ID NO: 8, the DNA sequence of SEQ ID NO: 9 encodes the protein of SEQ ID NO: 10, the DNA sequence of SEQ ID NO: 11 encodes the protein of SEQ ID NO: 12, the DNA sequence of SEQ ID NO: 13 encodes the protein of SEQ ID NO: 14, and sequence number The DNA sequence of sequence 15 codes for the protein of sequence number 16, the DNA sequence of sequence number 17 codes for the protein of sequence number 18, the DNA sequence of sequence number 19 codes for the protein of sequence number 20, the DNA sequence of sequence number 21 codes for the protein of sequence number 22, the DNA sequence of sequence number 23 codes for the protein of sequence number 24, the DNA sequence of sequence number 25 codes for the protein of sequence number 26, the DNA sequence of sequence number 27 codes for the protein of sequence number 28, the DNA sequence of sequence number 29 codes for the protein of sequence number 30, the DNA sequence of sequence number 31 codes for the protein of sequence number 32, and the DNA sequence of sequence number 33 codes for the protein of sequence number 34.

[0110] [Table 1-1]

[0111] [Table 1-2]

[0112] [Table 1-3]

[0113] [Table 1-4]

[0114] Table 1-5

[0115] Table 1-6

[0116] Table 1-7

[0117] Table 1-8

[0118] Table 1-9

[0119] Table 1-10

[0120] Table 1-11

[0121] Table 1-12

[0122] Table 1-13

[0123] Table 1-14

[0124] Table 1-15

[0125] Table 1-16

[0126] Table 1-17

[0127] Table 1-18

[0128] Table 1-19

[0129] Table 1-20

[0130] Table 1-21

[0131] Table 1-22

[0132] Table 1-23

[0133] Table 1-24

[0134] Table 1-25

[0135] Table 1-26

[0136] Table 1-27

[0137] Table 2-1

[0138] Table 2-2

[0139] Table 2-3

[0140] Table 2-4

[0141] Table 2-5

[0142] Table 2-6

[0143] Table 2-7

[0144] [Table 2-8]

[0145] [Table 2-9]

[0146] The DNA sequence of Sequence ID No. 1 is a polynucleotide encoding a representative MOG-Seldeg fusion protein (Sequence ID No. 2) that has mutations that enhance FcRn binding, knobs-into-holes, and arginine mutations. The representative DNA and amino acid sequences of MOG in Sequence ID No. 1 and No. 2 are of mouse origin.

[0147] In particular, the amino acid sequence of the representative MOG-Seldeg of Sequence ID No. 2 forms a fusion protein with the first linker at residues 1-117 and 118-122 of mouse (m)MOG in N-terminus to C-terminus, an immunoglobulin hinge (derived from human IgG1) at residues 123-138, an immunoglobulin CH2 domain (derived from human IgG1) at residues 139-248, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 249-355. The representative MOG-Seldeg of Sequence ID No. 2 has mutations that improve FcRn binding at residues 160, 162, 164, 341 and 342, arginine mutations at residues 144 and 236, and 'knobs in to holes' mutations at residues 257 and 302. The cysteine ​​residue (128) that normally pairs with the immunoglobulin light chain has been mutated to serine. The amino acid residue numbers mentioned in Sequence ID No. 2 refer to protein sequence numbers and do not refer to the EU numbering convention.

[0148] The DNA sequence of Sequence ID No. 3 is a polynucleotide that encodes a representative HER2-Seldeg fusion protein (Sequence ID No. 4) that has mutations that improve FcRn binding, knobs-in-to-holes, and arginine mutations.

[0149] In particular, the amino acid sequence of the representative HER2-Seldeg of Sequence ID No. 4 forms a fusion protein with HER2 residues 1-630, a first linker at residues 631-636, an immunoglobulin hinge (derived from human IgG1) at residues 637-650, an immunoglobulin CH2 domain (derived from human IgG1) at residues 651-760, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 761-876, in N-terminus to C-terminus. The representative HER2-Seldeg of Sequence ID No. 4 has mutations that improve FcRn binding at residues 672, 674, 676, 853 and 854, arginine mutations at residues 656 and 748, and 'knobs in to holes' mutations at residues 769 and 814. The cysteine ​​residue (640), which normally pairs with the immunoglobulin light chain, has been mutated to serine. The amino acid residue numbers mentioned in Sequence ID No. 4 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0150] The DNA sequence of Sequence ID No. 5 is a polynucleotide encoding a representative Fc fragment (Sequence ID No. 6) that has mutations that enhance FcRn binding, knobs-in-to-holes, and arginine mutations. The fusion protein of Sequence ID No. 6 is configured for heterodimer formation with, for example, MOG-Seldeg (Sequence ID No. 2) or HER2-Seldeg (Sequence ID No. 4) fusions.

[0151] In particular, the amino acid sequence of the representative Fc fragment of SEQ ID NO: 6 has an immunoglobulin hinge (derived from human IgG1) at residues 1-16, an immunoglobulin CH2 domain (derived from human IgG1) at residues 17-126, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 127-233, in order from the N-terminus to the C-terminus. The representative Fc fragment of SEQ ID NO: 6 has mutations that improve FcRn binding at residues 38, 40, 42, 219, and 220, arginine mutations at residues 22 and 114, and 'knobs in to holes' mutations at residues 150 and 191. The cysteine ​​residue (6), which normally pairs with the immunoglobulin light chain, has been mutated to serine. The amino acid residue numbers mentioned in SEQ ID NO: 6 refer to protein sequence numbers and do not refer to the EU numbering agreement.

[0152] The DNA sequence of Sequence ID No. 7 is a polynucleotide encoding a representative MOG-Seldeg-PS fusion with a knobs-in-to-holes mutation and an arginine mutation, and the corresponding amino acid sequence of the encoded fusion protein is Sequence ID No. 8.

[0153] In particular, the amino acid sequence of the representative MOG-Seldeg-PS fusion compound of Sequence ID No. 8 is N-terminal From end to C-terminus, the mMOG has the first linker at residues 1-117 and 118-122, the immunoglobulin hinge (derived from human IgG1) at residues 123-138, the immunoglobulin CH2 domain (derived from human IgG1) at residues 139-248, and the immunoglobulin CH3 domain (derived from human IgG1) at residues 249-355. The representative MOG-Seldeg-PS of Sequence ID No. 8 has arginine mutations at residues 144 and 236 and 'knobs in to holes' mutations at residues 257 and 302. The cysteine ​​residue (128) paired with the immunoglobulin light chain has been mutated to serine. The C2A PS-binding domain of synaptotagmin, residues 141-266 (Syt1) (shown as residues 361-486), is fused to the C-terminus of the CH3 domain via the GGGGS linker peptide (residues 356-360). The amino acid residue numbers mentioned in Sequence ID No. 8 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0154] The DNA sequence of Sequence ID No. 9 is a polynucleotide encoding a representative Fc-Syt1 fusion (Sequence ID No. 10) that has a knobs-into-holes mutation and an arginine mutation, which are configured for heterodimer formation with, for example, MOG-Seldeg-PS (Sequence ID No. 8).

[0155] In particular, the amino acid sequence of the representative Fc-Syt1 fusion of Sequence ID No. 10, in order from the N-terminus to the C-terminus, has an immunoglobulin hinge (derived from human IgG1) at residues 1-16, an immunoglobulin CH2 domain (derived from human IgG1) at residues 17-126, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 126-233. The representative Fc-Syt1 fusion protein of Sequence ID No. 10 has arginine mutations at residues 22 and 114, and 'knobs in to holes' mutations at residues 150 and 191. The cysteine ​​residue (6) that normally pairs with the immunoglobulin light chain has been mutated to serine. The C2A PS-binding domain of synaptotagmin, residues 141-266 (Syt1) (shown as residues 239-364), is fused to the C-terminus of the CH3 domain via the GGGGS linker peptide (residues 234-238). The amino acid residue numbers mentioned in Sequence ID No. 10 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0156] The DNA sequence of Sequence ID No. 11 is a polynucleotide encoding a representative MOG-Seldeg-PS fusion (Sequence ID No. 12) that has a knobs-in-to-holes mutation, an electrostatic steering mutation, and an arginine mutation.

[0157] In particular, the amino acid sequence of the representative MOG-Seldeg-PS fusion of Sequence ID No. 12 has the first linker at residues 1-117 and 118-122 of the mMOG, in order from the N-terminus to the C-terminus, an immunoglobulin hinge (derived from human IgG1) at residues 123-138, an immunoglobulin CH2 domain (derived from human IgG1) at residues 139-248, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 249-355. The representative MOG-Seldeg-PS of Sequence ID No. 12 has arginine mutations at residues 144 and 236, electrostatic steering mutations at residues 300 and 317, and 'knobs in to holes' mutations at residues 257 and 302. The cysteine ​​residue (128) paired with the immunoglobulin light chain has been mutated to serine. The C2A PS-binding domain of synaptotagmin, residues 141-266 (Syt1) (shown as residues 361-486), is fused to the C-terminus of the CH3 domain via the GGGS linker peptide (residues 356-360). The amino acid residue numbers mentioned in Sequence ID No. 12 refer to protein sequence numbers and do not refer to the EU numbering agreement.

[0158] The DNA sequence of Sequence ID No. 13 is a representative Fc-Syt1 fusion (sequence) that has a knobs-into-holes mutation, an electrostatic steering mutation, and an arginine mutation, which are configured for heterodimer formation with, for example, MOG-Seldeg-PS (Sequence ID No. 12). This is the polynucleotide that codes for number 14).

[0159] In particular, the amino acid sequence of the representative Fc-Syt1 fusion protein of Sequence ID No. 14 has an immunoglobulin hinge (derived from human IgG1) at residues 1-16, an immunoglobulin CH2 domain (derived from human IgG1) at residues 17-126, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 127-233, in order from the N-terminus to the C-terminus. The representative MOG-Seldeg-PS of Sequence ID No. 14 has arginine mutations at residues 22 and 114, electrostatic steering mutations at residues 143 and 185, and 'knobs in to holes' mutations at residues 150 and 191. The cysteine ​​residue (6) paired with the immunoglobulin light chain has been mutated to serine. The C2A PS-binding domain of synaptotagmin, residues 141-266 (Syt1) (shown as residues 239-364), is fused to the C-terminus of the CH3 domain via the GGGGS linker peptide (residues 234-238). The amino acid residue numbers mentioned in Sequence ID No. 14 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0160] The DNA sequence of Sequence ID No. 15 is a polynucleotide encoding a representative MOG-Seldeg-TfR fusion protein (Sequence ID No. 16) that contains a TfR-specific antibody heavy chain with a knobs-in-two-holes mutation.

[0161] In particular, the amino acid sequence of the representative TfR-specific antibody heavy chain-MOG fusion (MOG-Seldeg-TfR) of Sequence ID No. 16 has, in order from the N-terminus to the C-terminus, a TfR-specific VH domain at residues 1-116, an immunoglobulin CH1 domain (derived from human IgG1) at residues 117-213, an immunoglobulin hinge (derived from human IgG1) at residues 214-229, an immunoglobulin CH2 domain (derived from human IgG1) at residues 230-339, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 340-446. The representative TfR-specific antibody heavy chain of Sequence ID No. 16 has 'knobs in to holes' mutations at residues 348 and 393. Remnants 1-117 of mMOG (shown as residues 452-568) are fused to the C-terminus of the CH3 domain via the GGGGS linker peptide (residues 447-451). The amino acid residue numbers mentioned in Sequence ID No. 16 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0162] The DNA sequence of Sequence ID No. 17 is a polynucleotide encoding a typical TfR-specific antibody heavy chain. The encoded fusion protein (Sequence ID No. 18) with a knobs-in-to-holes mutation is configured for heterodimer formation with, for example, MOG-Seldeg-TfR (Sequence ID No. 16).

[0163] In particular, the amino acid sequence of the representative TfR-specific antibody heavy chain of Sequence ID No. 18 for heterodimer formation with the TfR-specific antibody heavy chain-MOG fusion (SEQ ID No. 16) is, in N-terminus to C-terminus order, a TfR-specific VH domain at residues 1-116, an immunoglobulin CH1 domain (derived from human IgG1) at residues 117-213, an immunoglobulin hinge (derived from human IgG1) at residues 214-229, an immunoglobulin CH2 domain (derived from human IgG1) at residues 230-339, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 340-446. The representative TfR-specific antibody heavy chain of Sequence ID No. 18 has 'knobs in to holes' mutations at residues 363 and 404. The amino acid residue numbers mentioned in Sequence ID No. 18 are protein sequence numbers and do not refer to the EU Numbering Agreement.

[0164] The DNA sequence of SEQ ID NO: 19 is a polynucleotide that encodes the light chain (SEQ ID NO: 20) of a typical TfR-specific antibody, and is involved in heterodimer formation with, for example, the MOG-Seldeg-TfR fusion (SEQ ID NO: 16) and the TfR-specific antibody heavy chain (SEQ ID NO: 18). It is composed of the following.

[0165] In particular, the amino acid sequence of the representative TfR-specific antibody light chain of Sequence ID No. 20, in N-terminus to C-terminus order, contains a TfR-specific VL domain at residues 1-107 and an immunoglobulin CL domain (human Cκ) at residues 108-213. The amino acid residue numbers mentioned in Sequence ID No. 20 refer to protein sequence numbers and do not refer to the EU numbering agreement.

[0166] The DNA sequence of Sequence ID No. 21 is a polynucleotide encoding a representative MOG-Seldeg-TfR fusion protein (Sequence ID No. 22) that has a TfR-specific antibody heavy chain with arginine mutations and knobs-in-two-holes mutations.

[0167] In particular, the amino acid sequence of the representative TfR-specific antibody heavy chain-MOG fusion (MOG-Seldeg-TfR) of Sequence ID No. 22 has, in order from the N-terminus to the C-terminus, a TfR-specific VH domain at residues 1-116, an immunoglobulin CH1 domain (derived from human IgG1) at residues 117-213, an immunoglobulin hinge (derived from human IgG1) at residues 214-229, an immunoglobulin CH2 domain (derived from human IgG1) at residues 230-339, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 340-446. The representative TfR-specific antibody heavy chain of Sequence ID No. 22 has arginine mutations at residues 235 and 327 and 'knobs in to holes' mutations at residues 348 and 393. Remainders 1-117 of mMOG (shown as residues 452-568) are fused to the C-terminus of the CH3 domain via the GGGGS linker peptide (residues 447-451). The amino acid residue numbers mentioned in Sequence ID No. 22 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0168] The DNA sequence of Sequence ID No. 23 contains arginine mutations and knobs-into-holes mutations, and is a polynucleotide encoding a representative TfR-specific antibody heavy chain (Sequence ID No. 24) configured for heterodimer formation with, for example, the MOG-Seldeg-TfR fusion (Sequence ID No. 22).

[0169] In particular, the amino acid sequence of the representative TfR-specific antibody heavy chain of Sequence ID No. 24, in N-terminus to C-terminus order, contains the TfR-specific VH domain at residues 1-116, the immunoglobulin CH1 domain (derived from human IgG1) at residues 117-213, the immunoglobulin hinge (derived from human IgG1) at residues 214-229, the immunoglobulin CH2 domain (derived from human IgG1) at residues 230-339, and the immunoglobulin CH3 domain (derived from human IgG1) at residues 340-446. The TfR-specific antibody heavy chain of Sequence ID No. 24 has arginine mutations at residues 235 and 327 and 'knobs in to holes' mutations at residues 363 and 404. The amino acid residue numbers mentioned in Sequence ID No. 24 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0170] The DNA sequence of Sequence ID No. 25 is a polynucleotide encoding a representative HER2-Seldeg fusion protein (Sequence ID No. 26) that has mutations that enhance FcRn binding and knobs-in-to-holes mutations.

[0171] In particular, the amino acid sequence of the representative mutant HER2-Seldeg of SEQ ID NO: 26 forms a fusion protein with the first linker at residues 1-630 and 631-636 of HER2, the immunoglobulin hinge (derived from human IgG1) at residues 637-650, the immunoglobulin CH2 domain (derived from human IgG1) at residues 651-760, and the immunoglobulin CH3 domain (derived from human IgG1) at residues 761-867, in order from the N-terminus to the C-terminus. HER2-Seldeg of SEQ ID NO: 26 has residues 672, 674, 676, 853 and 854. It has mutations that improve FcRn binding and 'knobs-into-holes' mutations at residues 769 and 814. The cysteine ​​residue (640) that normally pairs with the immunoglobulin light chain is mutated to serine. The amino acid residue numbers mentioned in Sequence ID No. 26 refer to protein sequence numbers and do not refer to EU numbering agreements.

[0172] The DNA sequence of Sequence ID No. 27 contains mutations that enhance FcRn binding and knobs-into-holes mutations, and is a polynucleotide encoding a representative Fc fragment (Sequence ID No. 28) that is configured for heterodimer formation with, for example, HER2-Seldeg (Sequence ID No. 26).

[0173] In particular, the amino acid sequence of the representative mutant Fc fragment of SEQ ID NO: 28, in N-terminus to C-terminus order, contains an immunoglobulin hinge (derived from human IgG1) at residues 1-16, an immunoglobulin CH2 domain (derived from human IgG1) at residues 17-126, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 127-233. The mutant Fc fragment of SEQ ID NO: 28 has mutations that improve FcRn binding at residues 38, 40, 42, 219, and 220, as well as 'knobs in to holes' mutations at residues 150 and 191. The cysteine ​​residue (6) that normally pairs with the immunoglobulin light chain has been mutated to serine. The amino acid residue numbers mentioned in SEQ ID NO: 28 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0174] The DNA sequence of Sequence ID No. 29 is a polynucleotide encoding a representative prostate-specific membrane antigen (PSMA)-Seldeg fusion protein (Sequence ID No. 30) that has mutations that enhance FcRn binding, a knobs-in-to-holes mutation, and an arginine mutation. This fusion protein would form a heterodimer with, for example, a representative Fc fragment (Sequence ID No. 6).

[0175] In particular, the amino acid sequence of the representative mutant PSMA-Seldeg of SEQ ID NO: 30 has, in order from the N-terminus to the C-terminus, an immunoglobulin hinge (derived from human IgG1) at residues 1-16, an immunoglobulin CH2 domain (derived from human IgG1) at residues 17-126, and an immunoglobulin CH3 domain (derived from human IgG1) at residues 127-233. These are fused at the C-terminus to the extracellular domain of PSMA (residues 239-945) via a linker at residues 234-238, forming a fusion protein. The mutant PSMA-Seldeg of SEQ ID NO: 30 has mutations that improve FcRn binding at residues 38, 40, 42, 219, and 220, arginine mutations at residues 22 and 114, and 'knobs in to holes' mutations at residues 135 and 180. The cysteine ​​residue (6) that normally pairs with the immunoglobulin light chain has been mutated to serine. The amino acid residue numbers mentioned in Sequence ID No. 30 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0176] The DNA sequence of Sequence ID No. 31 is a polynucleotide encoding a representative GAD65-Seldeg fusion protein (Sequence ID No. 32) that has mutations that enhance FcRn binding, a knobs-in-to-holes mutation, and an arginine mutation. This fusion protein would form a heterodimer with, for example, a representative Fc fragment (Sequence ID No. 6).

[0177] In particular, the amino acid sequence of the representative mutant GAD65-Seldeg of Sequence ID No. 32, in order from the N-terminus to the C-terminus, is as follows: the first linker is located at residues 1-585 and 586-590 of human glutamate carboxylase 65 (GAD65), the immunoglobulin hinge (derived from human IgG1) is located at residues 591-606, the immunoglobulin CH2 domain (derived from human IgG1) is located at residues 607-716, and the immunoglobulin CH3 domain (derived from human Ig) is located at residues 717-823. It forms a fusion protein (derived from G1). The variant GAD65-Seldeg of SEQ ID NO: 32 has mutations that improve FcRn binding at residues 628, 630, 632, 809 and 810, arginine mutations at residues 612 and 704, and 'knobs in to holes' mutations at residues 725 and 770. The cysteine ​​residue (596) that normally pairs with the immunoglobulin light chain is mutated to serine. The amino acid residue numbers mentioned in SEQ ID NO: 32 are protein sequence numbers and do not refer to the EU Numbering Agreement.

[0178] The DNA sequence of Sequence ID No. 33 is a polynucleotide encoding a representative aquaporin 4-Seldeg fusion protein (Sequence ID No. 34) that has mutations that enhance FcRn binding, a knobs-in-to-holes mutation, and an arginine mutation. This fusion protein would form a heterodimer with, for example, a representative Fc fragment (Sequence ID No. 6).

[0179] In particular, the amino acid sequence of the representative mutant aquaporin 4 (AQP4)-Seldeg of Sequence ID No. 34 forms a fusion protein with the first linker at residues 1-323 and 324-328 of human aquaporin 4, the immunoglobulin hinge (derived from human IgG1) at residues 329-344, the immunoglobulin CH2 domain (derived from human IgG1) at residues 345-454, and the immunoglobulin CH3 domain (derived from human IgG1) at residues 455-561, in N-terminus to C-terminus. The mutant AQP4-Seldeg of Sequence ID No. 34 has mutations that improve FcRn binding at residues 366, 368, 370, 547, and 548, arginine mutations at residues 350 and 442, and 'knobs in to holes' mutations at residues 463 and 508. The cysteine ​​residue (334) that normally pairs with the immunoglobulin light chain has been mutated to serine. The amino acid residue numbers mentioned in Sequence ID No. 34 refer to protein sequence numbers and do not refer to the EU Numbering Agreement.

[0180] The subject matter disclosed above should be considered illustrative and not restrictive, and the attached claims are intended to encompass all modifications, emphasis, and other aspects that fall within the true spirit and scope of this disclosure. Thus, to the maximum extent permitted by law, the scope of this disclosure should be determined by the broadest possible interpretation of the following claims and their equivalents, and should not be limited or restricted by the foregoing detailed description.

[0181] Where used in this application and the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. The term “plurality” refers to two or more subjects unless the context clearly indicates otherwise. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as they would be commonly understood by a person of ordinary skill in the art relating to this disclosure.

Claims

1. A pharmaceutical composition containing Seldev for use in a method for depleting target antigen-specific antibodies from a patient, The method involves administering Seldev to the patient in an amount sufficient to clear at least 50% of the target antigen-specific antibodies from the patient's circulation or target tissue. This includes, where Seldeg is Target components having proteins or protein fragments configured to specifically bind to cell surface receptors or surface molecules; and An antigenic component having one molecule of antigen configured to specifically bind to a target antigen-specific antibody. A pharmaceutical composition comprising, wherein the target component comprises an antibody Fc fragment that binds to an Fc receptor, the C2 domain of synaptotagmin 1 that binds to phosphatidylserine on the cell surface, or an antibody that targets a transferrin receptor, and is directly or indirectly fused with the antigen component.

2. The pharmaceutical composition according to claim 1, wherein the method comprises administering Seldig in an amount sufficient to clear at least 50% of the target antigen-specific antibodies from the circulation or target tissue in the patient within 5 hours of administration.

3. The pharmaceutical composition according to claim 1, wherein the protein or protein fragment is configured to bind to a cell surface receptor or other cell surface molecule with a dissociation constant of less than 10 μM at a pH near neutral.

4. The pharmaceutical composition according to claim 1, wherein a sufficient amount of Seldev is equimolar to the amount of target antigen-specific antibody to be depleted.

5. The pharmaceutical composition according to claim 1, wherein the method comprises administering Seldig in an amount sufficient to clear at least 90% of the target antigen-specific antibodies from the circulation or target tissue in the patient within two hours of administration.

6. The pharmaceutical composition according to claim 1, wherein the method comprises administering Seldig in an amount sufficient to clear at least 50% of the target antigen-specific antibodies from the circulation or target tissue in the patient within one hour of administration.

7. The pharmaceutical composition according to claim 1, further comprising re-administering Seldev whenever it is expected that 50% of the patients have regenerated a threshold amount of target antigen-specific antibodies in circulation or target tissue.

8. The pharmaceutical composition according to claim 1, wherein Seldig clears less than 10% of non-target antibodies in the patient's circulation or target tissue.

9. The pharmaceutical composition according to claim 1, wherein Seldig clears less than 1% of non-target antibodies in the patient's circulation or target tissue.

10. The pharmaceutical composition according to claim 1, wherein, in the method described above, Seldev causes the degradation of the target antigen-specific antibody by cells expressing a cell surface receptor or cell surface molecule.

11. The pharmaceutical composition according to claim 1, wherein Seldeg is administered to a patient suffering from an autoimmune disease, and the target antigen-specific antibody specifically binds to the autoantigen.

12. The pharmaceutical composition according to claim 1, wherein, in the method described above, Seldig is administered to a patient who has received an organ transplant, and the target antigen-specific antibody specifically binds to the antigen on the transplanted organ.

13. The pharmaceutical composition according to claim 1, wherein, in the method described above, Seldeg is administered to increase contrast during tumor imaging, and the target antigen-specific antibody specifically binds to the tumor antigen.

14. The pharmaceutical composition according to claim 1, wherein, in the method described above, Seldev is administered to a patient who has received a biological preparation, and the target antigen-specific antibody is the biological preparation.

15. The pharmaceutical composition according to claim 1, wherein, in the method described above, if the patient has antibodies specific to the therapeutic agent, Seldev is administered to the patient before the delivery of the therapeutic agent, and Seldev targets these therapeutic agent-specific antibodies.

16. The pharmaceutical composition according to claim 1, wherein Seldev is administered in the above method to provide a PET image contrast agent.

Images