Biotin-orthogonal streptavidin system

Abstract

The present disclosure relates to an orthogonal system comprising a first bispecific polypeptide comprising D-streptavidin or a variant thereof covalently bound to an antibody or antibody fragment, and a conjugate comprising L-biotin covalently bound to a therapeutic or diagnostic agent. The system of the present disclosure can be useful, for example, in the treatment of a disease or condition (e.g., cancer, non-Hodgkin lymphoma, multiple sclerosis, Crohn's disease, rheumatoid arthritis, asthma, macular degeneration, psoriasis, Hodgkin lymphoma, paroxysmal nocturnal hemoglobinuria, X-linked hypophosphatemia). Peptides and polypeptides useful in the preparation of the disclosed bispecific polypeptides, and methods of making them, are also described. This summary is intended as a scanning tool for purposes of searching in a particular art and is not intended to limit the present invention.

Description

Technical Field

[0001] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant No. U54 AI170856 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Cross - Reference to Related Applications This application claims the benefit of U.S. Application No. 63 / 342,052, filed May 13, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to a Sequence Listing The Sequence Listing submitted on May 12, 2023, as an xml file named "21101.0438P1_Updated.xml" created on May 3, 2023 and having a size of 20,480 bytes, is incorporated herein by reference in accordance with 37 C.F.R.§1.52(e)(5).

Background Art

[0004] Streptavidin (SA) evolved in Streptomyces avidinii (S. avidinii) to prevent the growth of competing bacteria by sequestering biotin 24、25 . SA forms a complex network of hydrogen bonds and other non - covalent interactions with biotin 26、27 and forms the strongest known small - molecule - protein non - covalent interaction in nature with a K D (4.8×10 -14 M) in the femtomolar range. This extraordinary binding affinity has led to multiple applications of SA in biomedical research, including immunoassays 1 , affinity chromatography 2 , proximity labeling 28 , and phage display 4、5 . In addition, SA / biotin - based diagnostic tests 29 . 6is widely used (for example, for heart disease and thyroid disease).

[0005] SA / biotin has achieved great success in various biotechnology applications, but in particular in the therapeutic setting, several issues limit its usefulness. For example, biotin supplementation can cause interference in diagnostics using SA and biotin 30~33 . Similarly, proximity labeling is a powerful recent technique that uses non-specific biotin ligases to tag molecules in proximity to an ID-binding partner 4、5 . However, biotinylation by endogenous biotin ligases results in background noise that limits the identification of low-abundance targets 4 . Most importantly for therapeutic applications, streptavidin is a highly immunogenic foreign protein 3、20、34~36。

[0006] There is great interest in using SA in pretargeted immunotherapy (PTI) 7~19 . The concept of PTI evolved from targeted immunotherapy (TI), in which a radioactive payload is conjugated to an antibody targeting a tumor-specific antigen 19 . The antibody conjugate binds to tumor cells and concentrates radiation on the cells, while the unbound antibody irradiates and damages healthy tissue 9、14 and can remain in circulation for several days 9、10、20、35 . To overcome this problem, the PTI method involves conjugating high-affinity binding partners to the antibody and the drug. The antibody is administered first, which allows it to bind to tumor cells without irradiating healthy tissue. After the unbound antibody is removed from circulation, the drug is injected. The drug associates with the binding partner and concentrates at the tumor site. Since the drug is a small molecule, the excess is rapidly removed from circulation and does not harm healthy tissue.

[0007] PTI has been approached in four ways. (1) Using bispecific antibodies 34、36、37, where one arm of the antibody binds to tumor cells and the other arm binds to the drug, (2) using SA and biotin (SA PTI) 34、36、37 , where streptavidin is conjugated to the antibody and the drug is biotinylated, (3) using an oligonucleotide where one strand binds to the antibody and the complementary strand binds to the drug 36、37 , and (4) inverse electron demand Diels–Alder chemistry (IEDDAC) 35 using fast click reactions such as 20、34、36、37 . When directly compared to other methods, SA PTI was the least effective. SA is immunogenic and the antibody rapidly removes it from circulation 3、20、34~36 . Bispecific antibodies have higher therapeutic efficacy and lower immunogenicity 3 . IECCAC has fast kinetics (3×10 4 M -1 s -1 ) 35 and the advantage of forming covalent bonds. However, each of these methods has serious drawbacks. Bispecific antibodies are difficult to produce 3、35、36 and have weak affinity for the drugs they bind 36 . Oligonucleotides 35 and the IEDDAC method have low in vivo stability 35 .

[0008] The SA-biotin binding is up to 1000 times faster than IECCAC 35、38 . In addition, IECCAC forms covalent bonds, but due to the high affinity and low off-rate of SA, it results in an almost irreversible interaction 1、39 . In summary, SA PTI holds great promise, but its effectiveness is reduced by endogenous biotin interference 3、16、20、34、36 and extreme immunogenicity 3、20、34~36 .

[0009] D-proteins provide an elegant solution to the problems of SA PTI. D-proteins represent the mirror image or enantiomer of their naturally occurring L-counterpart. According to the law of mirror symmetry, the D-protein interaction with its mirror image ligand must have the same binding affinity as its naturally occurring L-counterpart. In addition, D-proteins cannot be proteolyzed for MHC presentation to the immune system 22 and significantly increase the half-life in circulation. Thus, systems based on L-biotin and D-SA have the potential to dramatically advance the field of PTI. Unfortunately, synthetic methods for accessing D-SA that are scalable and reproducible remain elusive. Accordingly, there is still a need for methods of synthesizing D-SA and its variants, conjugates comprising these D-proteins, and particularly methods of using the conjugates in PTI. These and other needs are met by the following disclosure. 21 SUMMARY OF THE INVENTION

[0010] According to the objects (s) of the invention embodied and broadly described herein, in one aspect, the invention relates to an orthogonal system comprising a first bispecific polypeptide comprising a D-SA or a variant thereof covalently linked to an antibody or an antibody fragment, and a conjugate comprising an L-biotin covalently linked to a therapeutic or diagnostic agent. The systems of the present disclosure can be useful, for example, in the treatment of diseases or conditions (e.g., cancer, non-Hodgkin lymphoma, multiple sclerosis, Crohn's disease, rheumatoid arthritis, asthma, macular degeneration, psoriasis, Hodgkin lymphoma, paroxysmal nocturnal hemoglobinuria, X-linked hypophosphatemia). Peptides and polypeptides useful in the preparation of the disclosed bispecific polypeptides, and methods of making them, are also described.

[0011] ​Thus, in one aspect, there is disclosed a peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYES (SEQ ID NO: 1), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid.

[0012] In one aspect, there is disclosed a peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS (SEQ ID NO: 3), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid.

[0013] In one aspect, there is disclosed a peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 5), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid.

[0014] In one aspect, the amino acid sequence X HH AEAGITGTWYNQLGSTFIVTAGADGALTGTYES-N 2 H 4 (SEQ ID NO: 6), there is disclosed a peptide comprising an amino acid sequence having at least 90% identity thereto, wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, and X HH is a linker.

[0015] In one aspect, the amino acid sequence KKKKKKX HH AEAGITGTWYNQLGSTFIVTAGADGALTGTYES-N 2 H 4A peptide comprising an amino acid sequence having at least 90% identity to (SEQ ID NO: 7), wherein each amino acid in the amino acid sequence is a D - amino acid, or each amino acid in the amino acid sequence is an L - amino acid, and KKKKKK and X HH both contain solubilizing residues, is disclosed.

[0016] In one aspect, a polypeptide comprising an amino acid sequence having 90% to 99% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 8), wherein each amino acid in the amino acid sequence is a D - amino acid, is disclosed.

[0017] In one aspect, the amino acid sequence KKKKKKX HH AEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS - N 2 H 4 A polypeptide comprising an amino acid sequence having at least 90% identity to (SEQ ID NO: 9), wherein each amino acid in the amino acid sequence is a D - amino acid, or each amino acid in the amino acid sequence is an L - amino acid, and KKKKKK and X HH both contain solubilizing residues, is disclosed.

[0018] In one aspect, the amino acid sequence KKKKKKX HHA polypeptide comprising an amino acid sequence having at least 90% identity to AEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 10), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, and KKKKKK and X HH are both solubilizing residues, is disclosed.

[0019] In one aspect, a method of making L-SA, D-SA, or a variant thereof, the method: (a) providing a first peptide having an amino acid sequence having at least 90% identity to AEAGITGTWYNQLGSTFIVTAGADGALTGTYES-N 2 H 4 (SEQ ID NO: 2), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; (b) providing a second peptide having an amino acid sequence having at least 90% identity to AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS-N 2 H 4 (SEQ ID NO: 4), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; (c) providing a third peptide having an amino acid sequence having at least 90% identity to ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 5), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; and (d) ligating the first, second, and third peptides, is disclosed.

[0020] In one aspect, a method of making L-SA, D-SA, or a variant thereof, the method comprising: (a) coupling a first peptide having an amino acid sequence having at least 90% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYES-N 2 H 4 (SEQ ID NO: 2), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, and a linker; (b) functionalizing the linker with a positively charged amino acid residue, thereby providing a solubilizing residue; (c) ligating the first peptide to a second peptide having an amino acid sequence having at least 90% identity to the amino acid sequence AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS-N 2 H 4 (SEQ ID NO: 4), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; (d) ligating the second peptide to a third peptide having an amino acid sequence having at least 90% identity to the amino acid sequence ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 5), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; and (e) cleaving the solubilized residue from the polypeptide. The method is disclosed.

[0021] In one aspect, the amino acid sequence KKKKKKX HHA method for producing a polypeptide comprising an amino acid sequence having at least 90% identity to AEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 10), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, and KKKKKK and X HH both contain solubilizing residues, and the method comprises: (a) providing a first polypeptide having an amino acid sequence having at least 90% identity to the amino acid sequence KKKKKKX HH AEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS-N 2 H 4 (SEQ ID NO: 9), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; (b) providing a third peptide having an amino acid sequence having at least 90% identity to the amino acid sequence ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 5), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; and (c) ligating the first polypeptide to the third peptide. The method is disclosed.

[0022] In one aspect, a method for producing L-SA, D-SA, or a variant thereof, the method comprising: (a) the amino acid sequence KKKKKKX HHTo provide a polypeptide having an amino acid sequence with at least 90% identity to AEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 10), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, and KKKKKK and X HH and both contain solubilizing residues, and to provide a method including (b) cleaving the solubilized residues from the polypeptide.

[0023] In one aspect, a bispecific polypeptide comprising a single-chain antibody (scFv) or F’ab fragment and the disclosed polypeptide is disclosed.

[0024] In one aspect, a bispecific polypeptide comprising an antibody or an antibody fragment covalently bound to D-SA or a variant thereof is disclosed.

[0025] In one aspect, a conjugate comprising L-biotin covalently bound to a therapeutic or diagnostic agent is disclosed.

[0026] In one aspect, a pharmaceutical composition comprising an effective amount of the disclosed bispecific polypeptide and a pharmaceutically acceptable carrier is disclosed.

[0027] In one aspect, a method of treating a disease or condition in a subject in need thereof, the method comprising (a) administering to the subject an effective amount of the disclosed bispecific polypeptide and (b) subsequently administering to the subject an effective amount of a composition comprising L-biotin covalently bound to a therapeutic agent, wherein D-SA or a variant thereof specifically binds to L-biotin, is disclosed.

[0028] In one aspect, a kit is disclosed that includes the disclosed bispecific polypeptide and one or more selected from (a) a therapeutic agent, (b) L-biotin, (c) instructions for administering the bispecific polypeptide in connection with the treatment of a disease or condition, and (d) instructions for treating a disease or condition.

[0029] Aspects of the invention may be described and claimed in specific statutory classes such as system statutory classes, but this is for convenience only, and those skilled in the art will understand that each aspect of the invention may be described and claimed in any statutory class. Unless otherwise specified, it is never intended that any method or aspect described herein be construed as requiring that its steps be performed in a particular order. Thus, method claims are never intended to imply an order in any respect where the steps are not specifically described in the claims or specification as being limited to a particular order. This is maintained for any possible implicit basis for interpretation, including the arrangement of steps or operation flow, the plain meaning derived from grammatical construction or punctuation, or the logical matter regarding the number or type of aspects described in the specification.

[0030] The accompanying figures, which are incorporated herein and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.

Brief Description of the Drawings

[0031]

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[0032] Further advantages of the present invention will be described in part in the following description, be apparent in part from the description, or can be learned by practice of the present invention. The advantages of the present invention will be realized and achieved by the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

Best Mode for Carrying Out the Invention

[0033] The present invention can be more easily understood by reference to the following detailed description of the invention and the examples included therein.

[0034] Before disclosing and describing these peptides, compositions, and methods, it should be understood that they are not limited to a particular synthesis method and are not limited to particular reagents, unless otherwise specified, as they may of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described herein.

[0035] Aspects of the present invention may be described and claimed in terms of specific statutory classes, such as system statutory classes, but this is for convenience only, and those skilled in the art will understand that each aspect of the present invention may be described and claimed in any statutory class. Unless otherwise specified, it is never intended that any method or aspect described herein be construed as requiring that its steps be performed in a particular order. Thus, method claims are never intended to imply an order in any respect where the steps are not specifically recited in the claims or the specification as being limited to a particular order. This is maintained for any possible implicit basis for interpretation, including the arrangement of steps or operation flows, the plain meaning derived from grammatical construction or punctuation, or the logic regarding the number or type of aspects described in the specification.

[0036] Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art relevant to this application. The disclosed references are also discussed in the context of the passages that rely on them, and the materials contained therein are hereby incorporated by reference individually and specifically into this specification. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of the publications provided herein may be different from the actual publication dates and can be independently verified.

[0037] A. Definitions As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, references to "functional group", "alkyl", or "residue" include mixtures of two or more such functional groups, alkyls, or residues.

[0038] As used in this specification and the claims, the term "comprising" can include the aspects of "consisting of" and "consisting essentially of".

[0039] In this specification, ranges can be expressed as from "about" one particular value to and / or "about" another particular value. When such a range is expressed, another aspect includes from one particular value to and / or to another particular value. Similarly, when a value is expressed as an approximation, it will be understood by use of the antecedent "about" that the particular value forms another aspect. It will be further understood that each endpoint of a range is significant both in relation to and independent of the other endpoint. Also, in this specification, several values are disclosed and each value is understood in this specification to be disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It is also understood that each unit between two particular units is disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0040] As used herein, the terms "about" and "approximately" mean that the quantity or value in question can be a value that is substantially or approximately the same as some other value. As used herein, generally, unless otherwise indicated or inferred, it is understood that it is a nominal value that exhibits a variation of ±10%. This term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, quantities, sizes, formulations, parameters, and other quantities and characteristics are not exact and need not be exact, but may be approximate and / or may be larger or smaller, as desired, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art. Generally, a quantity, size, formulation, parameter, or other quantity or characteristic is "about" or "approximate," whether or not explicitly stated to be so. When "about" is used before a quantitative value, the parameter is also understood to include the particular quantitative value itself, unless otherwise specified.

[0041] References in the specification and claims to the parts by weight of a particular element or component in a composition refer to the weight relationship between the element or component and any other element or component in the composition or product in which the parts by weight are expressed. Thus, in a composition containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5 and are present in such ratio regardless of whether additional components are contained in the composition.

[0042] The weight percent (wt%) of a component is based on the total weight of the formulation or composition in which the component is included, unless otherwise specifically stated.

[0043] As used herein, the terms "optional" or "optionally" mean that the event or circumstance described thereafter may or may not occur and that examples of the event or circumstance occurring and not occurring are included in the description.

[0044] As used herein, the term "subject" can be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. Thus, the subjects of the methods disclosed herein can be humans, non-human primates, horses, pigs, rabbits, dogs, sheep, goats, cows, cats, guinea pigs, or rodents. This term does not indicate a particular age or sex. Thus, it is intended to include adult and neonatal subjects, as well as fetuses regardless of sex. In one aspect, the subject is a mammal. A patient refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects.

[0045] As used herein, the term "treatment" refers to the medical management of a patient intended to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. This term includes active treatment, i.e., treatment specifically directed to the improvement of a disease, condition, or disorder, and causal treatment, i.e., treatment directed to the removal of the cause of the related disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than the cure of a disease, condition, or disorder, prophylactic treatment, i.e., treatment directed to minimizing or partially or completely inhibiting the onset of a related disease, condition, or disorder, and supportive treatment, i.e., treatment used to complement another specific therapy directed to the improvement of a related disease, condition, or disorder. In various aspects, this term encompasses any treatment of a subject, including a mammal (e.g., a human), that (i) prevents a disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (ii) inhibits a disease, i.e., prevents its onset, or (iii) alleviates a disease, i.e., causes regression of the disease. In one aspect, the subject is a mammal such as a primate, and in a further aspect, the subject is a human. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, Drosophila, etc.).

[0046] As used herein, the term "prevent" or "prevention" refers to preventing, avoiding, eliminating, forestalling, stopping, or hindering something from occurring, particularly by prior action. When "reduce", "inhibit", or "prevent" is used herein, the use of the other two terms is understood to be explicitly disclosed as well, unless otherwise specifically indicated.

[0047] As used herein, the term "diagnosed" means having been found, by a person skilled in the art, such as a physician, to have undergone a physical examination and to have a condition that can be diagnosed or treated by a fatty acid, composition, or method disclosed herein.

[0048] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration such as injectable administration including intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, the preparation can be therapeutically administered, i.e., administered to treat an existing disease or condition. In further various embodiments, the preparation can be prophylactically administered, i.e., administered for the prevention of a disease or condition.

[0049] As used herein, the terms "effective amount" and "effective quantity" refer to an amount sufficient to achieve a desired result or to affect an undesirable condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or to be effective against an undesirable symptom, but generally insufficient to cause adverse side effects. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors including the disorder being treated and the severity of the disorder, the specific composition being used, the age, weight, general health, sex, and diet of the patient, the time of administration, the route of administration, the rate of excretion of the specific compound being used, the duration of the treatment, drugs used in combination with or concurrently with the specific fatty acid being used, and like factors well known in the medical arts. For example, it is well within the skill of the art to initiate the dosage of the fatty acid at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dosage can be divided into multiple dosages for administration. Consequently, single-dose compositions can contain such an amount or an approximation thereof to constitute a daily dosage. In any event of contraindication, the dosage can be adjusted by the individual physician. The dosage can vary and can be administered in one or more doses per day for one or several days. Guidance can be found in the literature for the appropriate dosage of a given class of pharmaceuticals. In further various aspects, the preparation can be administered in a "prophylactically effective amount", i.e., an amount effective for the prevention of a disease or condition.

[0050] As used herein, "dosage form" means a pharmacologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. Dosage forms can include the disclosed fatty acids, products of the disclosed methods of making, or salts, solvates, or polymorphs thereof, in combination with pharmaceutically acceptable excipients such as preservatives, buffers, saline, or phosphate buffered saline. Dosage forms can be made using conventional pharmaceutical manufacturing and compounding techniques. Dosage forms can include inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH adjusters (e.g., hydrochloric acid, sodium hydroxide or potassium hydroxide, salts of citrate or acetate, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium desoxycholate), solutions and / or cryo / freezing stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsiloxane), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymer stabilizers and viscosity modifiers (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). Dosage forms formulated for injectable use can have the disclosed fatty acids, products of the disclosed methods of making, or salts, solvates, or polymorphs thereof, suspended in sterile saline for injection, together with a preservative.

[0051] As used herein, "kit" means an assembly of at least two components that make up the kit. Together, the components constitute a functional unit for a given purpose. The individual member components can be physically packaged together or separately. For example, a kit that includes instructions for using the kit may or may not physically include the instructions along with the other individual member components. Instead, the instructions can be provided in paper form, or on a computer-readable memory device, or in electronic form downloadable from an Internet website, or as a recorded presentation, and can be provided as a separate member component.

[0052] As used herein, "instructions(s)" means a document that describes relevant materials or methodology related to the kit. These materials can include, among other things, background information, a list of components and information about their availability (such as purchase information), a simple or detailed protocol for using the kit, troubleshooting, references, technical support, and any combination of any other relevant documents. The instructions can be provided in paper form, or on a computer-readable memory device, or in electronic form downloadable from an Internet website, or as a recorded presentation, either with the kit or as a separate member component. The instructions can include one or more documents and are meant to include future updates.

[0053] As used herein, the term "therapeutic agent" includes any synthetic or naturally-occurring composition of biologically active compounds or substances that, when administered to a living organism (human or non-human animal), induce the desired pharmacological, immunogenic, and / or physiological effects by local and / or systemic action. Thus, the term encompasses drugs, vaccines, and biopharmaceuticals, as well as compounds or chemical substances conventionally regarded as such, including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, etc. Examples of therapeutic agents are described in well-known references such as the Merck Index (14th Edition), Physicians’ Desk Reference (64th Edition), and The Pharmacological Basis of Therapeutics (12th Edition), which include agents; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or alleviation of diseases or disorders; substances that affect the structure or function of the body, or prodrugs that become biologically active or more active after being placed in a physiological environment, but are not limited thereto. For example, the term "therapeutic agent" includes compounds or compositions for use in all major therapeutic areas, including, but not limited to, adjuvants, anti-infective agents such as antibiotics and antiviral agents, anti-cancer and anti-tumor agents such as kinase inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors and other DNA damage response modifiers, epigenetic agents such as bromodomain and extra-terminal (BET) inhibitors, histone deacetylase (HDAc) inhibitors, iron chelators and other ribonucleotide reductase inhibitors, proteasome inhibitors and Nedd8-activating enzyme (NAE) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, conventional cytotoxic agents such as paclitaxel, doxorubicin, irinotecan, and platinum compounds, immune checkpoint blockers such as cytotoxic T lymphocyte antigen-4 (CTLA-4) monoclonal antibodies (mAbs), programmed cell death protein 1 (PD-1) / programmed cell death ligand 1 (PD-L1) mAbs, cluster of differentiation 47 (CD47) mAbs, toll-like receptor (TLR) agonists and other immunomodulators, cell therapy agents such as chimeric antigen receptor T cells (CAR-T) / chimeric antigen receptor natural killer (CAR-NK),Proteins such as interferon (IFN), interleukin (IL), and mAb, entry inhibitors, fusion inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI), nucleotide reverse transcriptase inhibitors, NCP7 inhibitors, protease inhibitors, and integrase inhibitors, anti-ALS agents, analgesics and combinations of analgesics, anorectic agents, anti-inflammatory agents, antiepileptic agents, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuronal blockers, anticholinergics and cholinergic agents, antimuscarinics and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensives, hormones and nutrients, antiarthritis agents, antiasthma agents, anticonvulsants, antihistamines, antiemetics, antitumor agents, antipruritics, antipyretics, antispasmodics, cardiovascular preparations (including calcium channel blockers, β-blockers, β-agonists and antiarrhythmics), antihypertensives, diuretics, vasodilators, central nervous system stimulants, cough and cold preparations, blood stasis removers, diagnostic agents, hormones, bone growth stimulants and bone resorption inhibitors, immunosuppressants, muscle relaxants, psychostimulants, sedatives, psychotropics, proteins, peptides, and fragments thereof (whether of natural origin, chemically synthesized, or recombinantly produced), and nucleic acid molecules (polymers of two or more nucleotides, deoxyribonucleotides (DNA) including both ribonucleotides (RNA) and double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin), and other biologically active macromolecules, e.g., proteins and enzymes. The agents can be biologically active agents used in medical applications, including veterinary medicine, and in agriculture such as for plants, and in other areas. The term "therapeutic agent" also includes, but is not limited to, agents, vitamins, mineral supplements, substances used for the treatment, prevention, diagnosis, cure, or alleviation of a disease or illness, or substances that affect the structure or function of the body, or prodrugs that become biologically active or more active after being placed in a given physiological environment.,

[0054] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, i.e., does not cause unacceptable levels of undesirable biological effects or interact in a harmful manner.

[0055] As used herein, the term "derivative" refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein), the structure of which is sufficiently similar to that disclosed herein such that, based on that similarity, it is expected to exhibit the same or similar activity and utility as the compound claimed by one of ordinary skill in the art or to induce the same or similar activity and utility as the compound claimed as a precursor. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound.

[0056] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents such as aluminum monostearate and gelatin which delay absorption. Injectable depot forms are prepared by forming a microcapsule matrix of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). The drug release rate can be controlled according to the ratio of the drug to the polymer and the nature of the particular polymer used. Depot injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0057] As used herein, the terms "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids or any unnatural analog that may be present in any of the disclosed antibodies, variants, or fragments. Thus, "amino acid" as used herein means both naturally occurring amino acids and synthetic amino acids. For example, homophenylalanine, citrulline, and norleucine are considered amino acids for the purposes of the present invention. "Amino acid" also includes amino acid residues such as proline and hydroxyproline. The side chain can be in either the (R) or (S) configuration. In some embodiments, the amino acid is in the D- or L-configuration further described herein. When non-naturally occurring side chains are used, non-amino acid substituents can be used, for example, to prevent or delay degradation.

[0058] As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues associated with naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof (i.e., peptide isosteres) linked via peptide bonds or modified peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, glycosylated polypeptides, and all "mimetic" and "peptide mimetic" polypeptide forms. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. This term can refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment, portion, or subunit thereof. The term "protein" typically refers to a large polypeptide. The term "peptide" typically refers to a short polypeptide. In various embodiments, each amino acid residue in a polypeptide or polymer can be, for example, a D-amino acid as in a D-protein (e.g., D-SA). Alternatively, in various further embodiments, each amino acid residue in a polypeptide or polymer can be, for example, a D-amino acid as in a D-protein (e.g., L-SA).

[0059] A "portion" of a polypeptide or protein means at least about 3 contiguous amino acid residues of the polypeptide. It is understood that a portion of a polypeptide can include all of the amino acid residues of the polypeptide.

[0060] As used herein, the terms "fragment" and "segment" can refer to a portion of a peptide (e.g., at least 5, 10, 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, or 500 amino acids or nucleic acids) that is substantially identical to a reference peptide and retains the biological activity of the reference peptide. In some embodiments, a fragment or portion of a peptide retains at least 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the biological activity of the reference peptide described herein. A fragment of a reference peptide can be a continuous or contiguous portion of the reference polypeptide (e.g., a fragment of a 10 - amino - acid - long reference peptide can be any 2 - 9 contiguous residues within that reference peptide).

[0061] "Variants", "derivatives", and "variants" of a polypeptide (or the nucleic acid encoding it) are polypeptides (or nucleic acids) that are modified or altered at one or more amino acids (or one or more nucleotides) such that the peptide (or nucleic acid) is not identical to the wild - type sequence but has homology to the wild - type polypeptide (or nucleic acid).

[0062] The term "variant" can refer to a peptide or gene product that shows a modification of sequence and / or functional characteristics (i.e., altered characteristics) when compared to a wild-type peptide or gene product. Generally, one way to define any known variants and derivatives of the genes and proteins disclosed herein, or those that may arise, is to define the variants and derivatives in terms of their homology to a specific known sequence. This identity of the specific sequences disclosed herein is also considered elsewhere in this specification. Generally, variants of the genes and proteins disclosed herein typically have at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the recited or native sequence. One of ordinary skill in the art can readily understand methods for determining the homology of two proteins or nucleic acids, e.g., genes. For example, homology can be calculated after aligning the two sequences such that the level of homology is at its highest. In some embodiments, the term "variant" can mean a difference in some way from a reference sequence other than a simple deletion of N-terminal and / or C-terminal amino acid residue(s). In one embodiment, a variant can include an amino acid residue substitution, which can be considered conservative or non-conservative. Conservative substitutions are those within the following groups: Ser, Thr, and Cys; Leu, Ile, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gin, Asn, Glu, Asp, and His. A variant can include at least one substitution and / or at least one addition, and there can also be at least one deletion. A variant can include one or more non-naturally occurring residues. For example, they can include selenocysteine (e.g., seleno-L-cysteine) at any position including the location of cysteine. Many other "non-natural" amino acid substitutions are known in the art and are available from commercial sources.Examples of non-naturally occurring amino acids include D-amino acids, amino acid residues having an acetylaminomethyl group bonded to the sulfur atom of cysteine, pegylated amino acids, and the formula NH. 2 (CH 2 ) n COOH (wherein n is from 2 to 6), omega amino acids such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and norleucine, and neutral non-polar amino acids. Phenylglycine may replace Trp, Tyr, or Phe, citrulline and methionine sulfoxide are neutral non-polar, cysteic acid is acidic, and ornithine is basic. Proline may be replaced with hydroxyproline and may retain the conformation conferring the properties of proline.

[0063] As used herein, the term "substituted" is intended to include all acceptable substituents of an organic compound. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of an organic compound. Exemplary substituents include, for example, those described hereinafter. There may be one or more acceptable substituents, which may be the same as or different from the appropriate organic compound. For the purposes of the present disclosure, a heteroatom such as nitrogen may have a hydrogen substituent and / or any acceptable substituent of the organic compounds described herein that satisfies the valence of the heteroatom. The present disclosure is not intended to be limited in any way by the acceptable substituents of an organic compound. Also, the terms "substituted" or "substituted with" imply the condition that such substitution results in a stable compound, i.e., a compound that does not undergo spontaneous transformation such as rearrangement, cyclization, elimination, etc., in accordance with the acceptable valences of the substituted atom and the substituent. Also, in certain embodiments, it is contemplated that, unless expressly indicated to the contrary otherwise, individual substituents may be further optionally substituted (i.e., may be further substituted or unsubstituted).

[0064] As used herein, the term "stability" refers to storage stability (e.g., stability at room temperature) and in vivo stability. The foregoing protecting groups can protect the peptides described herein from attack by proteolytic enzymes in vivo.

[0065] The peptides and fragments thereof disclosed herein may also include functional equivalents of the peptides described herein. As used herein, the term "functional equivalent" may refer to an amino acid sequence variant that has amino acid substitutions, additions, or deletions in a portion of the amino acid sequence of the peptide and, at the same time, has similar or improved biological activity as compared to the peptides described herein. In some embodiments, the amino acid substitutions can be conservative substitutions. Examples of conservative substitutions of naturally occurring amino acids include, for example, aliphatic amino acids (Gly, Ala, and Pro), hydrophobic amino acids (Ile, Leu, and Val), aromatic amino acids (Phe, Tyr, and Trp), acidic amino acids (Asp and Glu), basic amino acids (His, Lys, Arg, Gln, and Asn), and sulfur-containing amino acids (Cys and Met). In some embodiments, the amino acid deletions can be located in regions that are not directly involved in the activity of the peptides disclosed herein.

[0066] In some embodiments, the amino acid sequences of the peptides and fragments thereof disclosed herein may include peptide sequences that have substantial identity to any of the sequences of the peptides disclosed herein. As used herein, the term "substantial identity" means that two amino acid sequences, when optimally aligned and then analyzed by algorithms commonly used in the art such as BLAST, GAP, or BESTFIT, or by visual inspection, share at least about 60%, 70%, 80%, 85%, 90%, or 95% sequence identity. Methods of alignment for sequence comparison are known in the art.

[0067] In some embodiments, the amino acid sequences of the peptides and fragments thereof disclosed herein may include peptide sequences having a certain degree of identity or homology to any of the sequences of the peptides disclosed herein. The degree of identity can vary and be determined by methods known to those skilled in the art. The terms "homology" and "identity" each refer to sequence similarity between two polypeptide sequences. Homology and identity can each be determined by comparing the positions in each sequence that can be aligned for purposes of comparison. When the positions in the sequences being compared are occupied by the same amino acid residue, thus, the polypeptides can be said to be identical at that position, and when equivalent positions are occupied by the same amino acid (e.g., are identical) or a similar amino acid (e.g., having similar steric and / or electronic properties), the molecules can be said to be homologous at that position. The percentage of homology or identity between sequences is a function of the number of matching or homologous positions they share. The peptides described herein can have at least or about 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or homology to a peptide or polypeptide, and as further described herein, the peptide or polypeptide is, for example, one or more of SEQ ID NOs: 1-8, or the peptide or polypeptide is, for example, D-SA or L-SA.

[0068] Unless otherwise specified, it is never intended that any method shown herein be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim actually recites the order in which its steps are to be followed, or unless otherwise specifically recited in the claims or specification that the steps are to be limited to a particular order, no inference of order is ever intended in any respect. This holds for any possible non-explicit basis for interpretation, including the arrangement of steps or operational flow, the plain meaning derived from grammatical construction or punctuation, and the logical matter regarding the number or type of embodiments described in the specification.

[0069] Disclosed are the components used to prepare the compositions of the present invention, and the compositions themselves used within the methods disclosed herein. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, specific references to the various individual and collective combinations and permutations of each of these compounds cannot be explicitly disclosed, but each is understood to be specifically contemplated and described herein. For example, if a particular compound is disclosed, considered, and several modifications that can be made to several molecules containing that compound are considered, then each and every combination and permutation of that compound and the possible modifications are specifically contemplated, unless specifically indicated to the contrary. Thus, if classes of molecules A, B, and C, and classes of D, E, and F are disclosed, and an example of a combination molecule, A-D, is disclosed, then combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed, even if each is not individually listed, in the sense that each is individually and collectively contemplated. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups of A-E, B-F, and C-E are considered disclosed. This concept applies to all aspects of this application, including without limitation the steps in methods of making and using the compositions of the present invention. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any particular embodiment or combination of embodiments of the method of the present invention.

[0070] The compounds and compositions disclosed herein are understood to have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and there are various structures that can perform the same functions associated with the disclosed structures, and it is understood that these structures will typically achieve the same results.

[0071] B. Peptide In one aspect, peptides useful for the preparation of the disclosed polypeptides and / or the disclosed bispecific polypeptides are disclosed. Without wishing to be bound by theory, the disclosed peptides can be prepared by methods known to those of skill in the art and as described elsewhere herein. Exemplary peptides are shown in Table 1 below. [Table 1]

[0072] In various aspects, each amino acid in the amino acid sequence is a D - amino acid, or each amino acid in the amino acid sequence is an L - amino acid. For example, in one aspect, each amino acid in the amino acid sequence is a D - amino acid. As will be understood by those of skill in the art, a peptide in which each amino acid is a D - amino acid can be useful, for example, for the preparation of D - polypeptides (e.g., bispecific polypeptides in the D - configuration). In a further aspect, each amino acid in the amino acid sequence is an L - amino acid. A peptide in which each amino acid is an L - amino acid can be useful, for example, for the preparation of L - polypeptides (e.g., bispecific polypeptides in the L - configuration).

[0073] Accordingly, in various aspects, a peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYES (SEQ ID NO: 1), wherein each amino acid in the amino acid sequence is a D - amino acid or each amino acid in the amino acid sequence is an L - amino acid, is disclosed. In a further aspect, a peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYES - N 2 H 4 (SEQ ID NO: 2), wherein each amino acid in the amino acid sequence is a D - amino acid or each amino acid in the amino acid sequence is an L - amino acid, is disclosed.

[0074] In various embodiments, a peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS (SEQ ID NO: 3), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, is disclosed. In a further embodiment, the amino acid sequence X HH AEAGITGTWYNQLGSTFIVTAGADGALTGTYES-N 2 H 4 (SEQ ID NO: 4), a peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence, wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, is disclosed.

[0075] In various embodiments, a peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 5), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, is disclosed.

[0076] In various embodiments, the amino acid sequence X HH AEAGITGTWYNQLGSTFIVTAGADGALTGTYES-N 2 H 4 (SEQ ID NO: 6), a peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence, wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, and X HH is a linker, is disclosed. In a further embodiment, the amino acid sequence KKKKKKX HH AEAGITGTWYNQLGSTFIVTAGADGALTGTYES-N 2 H 4A peptide comprising an amino acid sequence having at least 90% identity to (SEQ ID NO: 7), wherein each amino acid in the amino acid sequence is a D - amino acid, or each amino acid in the amino acid sequence is an L - amino acid, and KKKKKK and X HH both contain solubilizing residues, and the peptide is disclosed.

[0077] C. Polypeptide In one aspect, a polypeptide useful for the preparation of the disclosed bispecific polypeptides is disclosed. Without wishing to be bound by theory, the disclosed peptides can be prepared by the methods described elsewhere in this specification. Exemplary polypeptides are shown in Table 2 below.

Table 2

[0078] In various aspects, each amino acid in the amino acid sequence is a D - amino acid, or each amino acid in the amino acid sequence is an L - amino acid. For example, in one aspect, each amino acid in the amino acid sequence is a D - amino acid. As will be understood by those skilled in the art, a polypeptide in which each amino acid is a D - amino acid can be useful, for example, for the preparation of a bispecific polypeptide in the D - configuration. In a further aspect, each amino acid in the amino acid sequence is an L - amino acid. A polypeptide in which each amino acid is an L - amino acid can be useful, for example, for the preparation of a bispecific polypeptide in the L - configuration.

[0079] Thus, in one aspect, a polypeptide is disclosed that comprises an amino acid sequence having 90% to 99% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 8), wherein each amino acid in the amino acid sequence is a D-amino acid.

[0080] In one aspect, the amino acid sequence KKKKKKX HH AEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS-N 2 H 4 A polypeptide is disclosed that comprises an amino acid sequence having at least 90% identity to the amino acid sequence (SEQ ID NO: 9), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid.

[0081] In one aspect, the amino acid sequence KKKKKKX HH A polypeptide is disclosed that comprises an amino acid sequence having at least 90% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 10), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid.

[0082] In various aspects, the polypeptide is D-trpavidin, D-streptactin, D-streptactin XT, or monovalent D-SA.

[0083] D. Method for Producing a Polypeptide In one aspect, a method for producing L-SA, D-SA, or a variant thereof, the method: (a) providing a first peptide having an amino acid sequence having at least 90% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYES-N 2 H 4 (SEQ ID NO: 2), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, (b) providing a second peptide having an amino acid sequence having at least 90% identity to the amino acid sequence AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS-N 2 H 4 (SEQ ID NO: 4), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, (c) providing a third peptide having an amino acid sequence having at least 90% identity to the amino acid sequence ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 5), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, and (d) ligating the first, second, and third peptides, is disclosed.

[0084] In one aspect, a method for producing L-SA, D-SA, or a variant thereof, the method being: (a) the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYES-N 2 H 4Coupling a first peptide having an amino acid sequence having at least 90% identity to (SEQ ID NO: 2) and a linker, wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; (b) functionalizing the linker with a positively charged amino acid residue, thereby providing a solubilizing residue; (c) the first peptide with the amino acid sequence AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS-N 2 H 4 Ligating to a second peptide having an amino acid sequence having at least 90% identity to (SEQ ID NO: 4), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; (d) ligating the second peptide to a third peptide having an amino acid sequence having at least 90% identity to the amino acid sequence ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 5), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; (e) cleaving the solubilizing residue from the polypeptide, is disclosed.

[0085] In one aspect, the amino acid sequence KKKKKKX HH A method for producing a polypeptide comprising an amino acid sequence having at least 90% identity to AEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 10), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, the method comprising: (a) the amino acid sequence KKKKKKX HHAEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS-N 2 H 4 To provide a first polypeptide having an amino acid sequence with at least 90% identity to (SEQ ID NO: 9), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; (b) to provide a third peptide having an amino acid sequence with at least 90% identity to the amino acid sequence ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 5), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid; and (c) to ligate the first polypeptide to the third peptide. A method is disclosed that includes these steps.

[0086] In one aspect, a method for producing L-SA, D-SA, or a variant thereof, the method comprising: (a) providing a polypeptide having an amino acid sequence with at least 90% identity to the amino acid sequence KKKKKKX HH AEAGITGTWYNQLGSTFIVTAGADGALTGTYES AVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS ATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 10), wherein each amino acid in the amino acid sequence is a D-amino acid or each amino acid in the amino acid sequence is an L-amino acid, and KKKKKK and X HH both contain solubilizing residues; and (b) cleaving the solubilizing residues from the polypeptide. A method is disclosed that includes these steps.

[0087] In various embodiments, the method further includes coupling a linker to the first peptide prior to the ligation step. As used herein, the term "linker" refers to a bifunctional, traceless linker that can be used, for example, to temporarily attach a solubilizing peptide sequence (e.g., a sequence of lysine residues) to a peptide (e.g., an insoluble peptide). See, e.g., Jacobsen et al. (2016) JACS 138: 11775-11782. When these solubilizing peptide sequences bind, the linker forms solubilizing residues in combination with the solubilizing peptide sequences, which can then be removed from the peptide.

[0088] Thus, in various embodiments, the linker has a structure represented by the following formula:

Chemical Formula

[0089] Thus, in various embodiments, the linker is C1-C12 alkyl and -(CH 2 CH 2 O) n- selected from, and n is selected from 1, 2, 3, and 4. In a further aspect, the linker is C1-C12 alkyl. In yet a further aspect, the linker is C1-C8 alkyl. In an even further aspect, the linker is C1-C4 alkyl (e.g., -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH(CH 3 )CH 2 - etc.). In an even more aspect, the linker is C4 alkyl.

[0090] In a further aspect, the linker is -(CH 2 CH 2 O) n -, and n is selected from 1, 2, 3, and 4 (e.g., -CH 2 CH 2 O-, -(CH 2 CH 2 O) 2 -, -(CH 2 CH 2 O) 3 - etc.). In yet a further aspect, n is selected from 1, 2, and 3. In an even further aspect, n is selected from 1 and 2. In an even more aspect, n is 1. In yet a further aspect, n is 2.

[0091] In various aspects, the amine protecting group is selected from 9-fluoroenylmethyl carbamate (Fmoc), tert-butyl carbamate (Boc), benzyl carbamate (Cbz), acetamide (Ac), trifluoroacetamide, and phthalimide. In a further aspect, the amine protecting group is Fmoc.

[0092] In a further aspect, the linker has the following structure:

Chemical formula

[0093] In a further aspect, the method further comprises deprotecting the linker prior to the ligation step. For example, as shown above, the deprotected linker may comprise:

Chemical formula

[0094] In a further aspect, the method further comprises functionalizing the linker with positively charged amino acid residues prior to the ligation step. In still a further aspect, the positively charged amino acid residue is lysine. In yet a further aspect, the free amine is functionalized with two or more positively charged amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8 positively charged amino acid residues), and the positively charged amino acid residues are the same or different. In an even further aspect, each positively charged amino acid residue is lysine. In still a further aspect, the free amine is functionalized with six lysine residues. For example, as shown above, the deprotected linker functionalized with positively charged amino acid residues (i.e., solubilizing residues) may comprise:

Chemical formula

[0095] In a further aspect, the first peptide is ligated to the second peptide before being ligated to the third peptide. Thus, in various aspects, the method includes preparing the polypeptide set forth in SEQ ID NO: 9, which can then subsequently be ligated to additional peptide fragments (e.g., fragment 3 (SEQ ID NO: 5)).

[0096] In a further aspect, the method further includes cleaving solubilizing residues. See, for example, SEQ ID NO: 8.

[0097] In a further aspect, ligation is performed via native chemical ligation or any other ligation chemistry. In yet a further aspect, ligation is performed via transesterification.

[0098] In a further aspect, the method produces L-SA or a variant thereof (e.g., L - streptavidin, monovalent L-SA).

[0099] In a further aspect, the method produces D-SA or a variant thereof (e.g., D - streptavidin, monovalent D-SA).

[0100] E. Bispecific polypeptide D-SA or a variant thereof In one aspect, a bispecific polypeptide is disclosed that includes a single-chain antibody (scFv) or F’ab fragment and the disclosed polypeptide. In a further aspect, the polypeptide includes an amino acid sequence having 90% to 99% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 8), wherein each amino acid in the amino acid sequence is a D - amino acid. In yet a further aspect, the scFv is an anti - CD30 antibody. In even a further aspect, the scFv is 5F11 scFv.

[0101] In one aspect, a bispecific polypeptide is disclosed that comprises an antibody or an antibody fragment thereof covalently bound to a D-SA or a variant thereof.

[0102] In various aspects, the antibody or antibody fragment thereof is an anti-IL-17 receptor antibody, an anti-IL-5 receptor antibody, an anti-PD-L1 antibody, an anti-FGF23 antibody, an anti-epidermal growth factor receptor antibody, an anti-GD2 antibody, an anti-HER-2 receptor antibody, an anti-RANKL antibody, an anti-C5 antibody, an anti-VEGF receptor antibody, an anti-VEGF-A antibody, an anti-VEGF receptor 2 antibody, an anti-IgE antibody, an anti-TNF-alpha antibody, an anti-IL-12 / 23 antibody, an anti-CTLA-4 antibody, an anti-CD30 antibody, an anti-CD4 antibody, an anti-CGRP receptor antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-CD25 antibody, or an anti-GP11b / 111a antibody. In a further aspect, the antibody or antibody fragment thereof is an anti-CD30 antibody.

[0103] In various aspects, the antibody or antibody fragment thereof specifically binds to a cell surface marker. In a further aspect, the cell surface marker is CD3, CD4, CD5, CD20, CD25, or a glycosphingolipid. In still a further aspect, the glycosphingolipid is GD 2 is.

[0104] In various aspects, the antibody or antibody fragment thereof is a single-chain antibody (scFv) or an F’ab fragment. In still a further aspect, the scFv is derived from blosumab, ibalizumab, enulumab, atezolizumab, reslizumab, pembrolizumab, nivolumab, ramucirumab, ipilimumab, brentuximab, ustekinumab, panitumumab, ranibizumab, necitumumab, dinutuximab, denosumab, exulizumab, bevacizumab, omalizumab, adalimumab, abelumab, durvalumab, brodalumab, muromonoab-CD3, abciximab, rituximab, daclizumab, infliximab, basiliximab, palivizumab, trastuzumab, gemtuzumab, or a biologically active variant thereof.

[0105] In various embodiments, the antibody or antibody fragment thereof is covalently bound to D-SA.

[0106] In various embodiments, the antibody or antibody fragment thereof is covalently bound to a variant of D-SA. In a further embodiment, the variant of D-SA is D-tryptavidin, D-streptactin, D-streptactin XT, or monovalent D-SA.

[0107] In various embodiments, D-SA or its variant specifically binds to D-biotin.

[0108] In various embodiments, the bispecific polypeptide further comprises a label or detection tag. In various further embodiments, the label or detection tag is a sortase tag.

[0109] 1. Label In various embodiments, the bispecific polypeptides and compositions described herein may further comprise one or more labels or detection tags (e.g., epitope or protein tags such as FLAG™ tag, myc tag, 6His, and fluorescent fusion proteins). In various embodiments, a label (e.g., FLAG™ tag) may be fused to a component of the disclosed bispecific polypeptide (e.g., a bispecific polypeptide comprising D-SA or its variant). In various embodiments, the disclosed methods and compositions further comprise a fusion protein, or a polynucleotide encoding the same. In various embodiments, the bispecific polypeptide or fusion protein comprises at least one epitope-providing amino acid sequence (e.g., an "epitope tag"), and the epitope tag is selected from i) an epitope tag added to the N-terminus and / or C-terminus of a protein (e.g., D-SA or its variant), or ii) an epitope tag inserted into a region of a protein (e.g., D-SA or its variant), and an epitope tag that replaces some amino acids in the protein (e.g., D-SA or its variant).

[0110] In various aspects, the bispecific polypeptides and compositions described herein may further comprise polyglycine and a sortase tag (i.e., Leu-Pro-Xxx-Thr-Gly-Xxx, where Xxx is any amino acid). Such tags can be useful, for example, in the implementation of sortase ligation (e.g., between D-SA and scFv).

[0111] As will be appreciated by those skilled in the art, tags can be placed at the N-terminus or C-terminus of the peptide, although smaller tags can be placed at almost any position within the synthetic protein (e.g., D-SA).

[0112] An epitope tag is a short stretch of amino acids against which a specific antibody can be generated and, in various aspects, enables the specific identification and tracking of tagged proteins added to a living body or cultured cells. Detection of tagged molecules can be achieved using several different techniques. Examples of such techniques include immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting ("western blotting"), and affinity chromatography. Epitope tags add a known epitope (e.g., an antibody-binding site) to the target protein, providing binding of a known, often high-affinity, antibody, thereby enabling the specific identification and tracking of tagged proteins added to a living body or cultured cells. Examples of epitope tags include, but are not limited to, myc, T7, GST, GFP, HA (hemagglutinin), V5, and FLAG tags. The first four examples are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, e.g., U.S. Pat. Nos. 4,703,004 and 4,851,341). Epitope tags can have one or more additional functions beyond recognition by an antibody.

[0113] In various aspects, the disclosed methods, bispecific polypeptides, and compositions include an epitope tag, which has a length of 6 to 15 amino acids. In alternative aspects, the epitope tag has a length of 9 to 11 amino acids. The disclosed methods and compositions may also include a bispecific polypeptide that contains two or more epitope tags, either spaced apart or directly tandem. Further, the disclosed methods and bispecific polypeptides or compositions may include 2, 3, 4, 5, or even more epitope tags as long as the bispecific polypeptide maintains its biological activity(ies) (e.g., “functional”).

[0114] In various aspects, the epitope tag can be a VSV-G tag, a CD tag, a calmodulin-binding peptide tag, an S tag, an Avi tag, an SF-TAP tag, a strep tag, a myc tag, a FLAG tag, a T7 tag, an HA (hemagglutinin) tag, a His tag, a GST tag, or a GFP tag. The sequences of these tags are described in the literature and are well known to those skilled in the art.

[0115] As used herein, the term “immunological binding” is a non-covalent form of binding between an epitope of an antigen (e.g., an epitope tag) and the antigen-specific portion of an antibody or a fragment thereof. The antibody is preferably monoclonal and needs to be specific for each epitope tag(s) used. Antibodies include mouse, human, and humanized antibodies. Antibody fragments are known to those skilled in the art and include, inter alia, single-chain Fv antibody fragments (scFv fragments) and Fab fragments. This antibody can be produced by conventional hybridomas and / or other recombinant techniques. Many antibodies are commercially available.

[0116] The construction of bispecific polypeptides from known protein domains, or from whole proteins or proteins and peptides, is well known. Generally, nucleic acid molecules encoding the desired protein and / or peptide moieties are ligated using genetic engineering techniques to create a single operably linked fusion oligonucleotide. Appropriate molecular biology techniques can be found in Sambrook et al. (Molecular Cloning: A laboratory manual Second Edition Cold Spring Harbor Laboratory Press, Cold spring harbor, NY, USA, 1989).Examples of genetically engineered multi-domain proteins that are linked by various linkers and contain peptide tags can be found in the following patent documents: U.S. Patent No. 5,994,104 ("Interleukin-12 fusion protein"), U.S. Patent No. 5,981,177 ("Protein fusion method and construction"), U.S. Patent No. 5,914,254 ("Expression of fusion polypeptides transported out of the cytoplasm without leader sequences"), U.S. Patent No. 5,856,456 ("Linker for linked fusion polypeptides"), U.S. Patent No. 5,767,260 ("Antigen-binding fusion proteins"), U.S. Patent No. 5,696,237 ("Recombinant antibody-toxin fusion protein"), U.S. Patent No. 5,587,455 ("Cytotoxic agent against specific virus infection"), U.S. Patent No. 4,851,341 ("Immunoaffinity purification system"), U.S. Patent No. 4,703,004 ("Synthesis of protein with an identification peptide"), and WO98 / 36087 ("Immunological tolerance to HIV epitopes").

[0117] The placement of the functionalized peptide moiety (epitope tag) within the target fusion protein can be influenced by the activity of the functionalized peptide moiety and the need to maintain at least substantially the biological activity of the fusion protein such as a TCR in the fusion. Two ways for the placement of the functionalized peptide are the N-terminus and the position within a protein moiety that shows adaptability to insertion. These are not the only positions where the functionalized peptide can be inserted, but they will serve as good examples and will be used for illustration. Other suitable insertion positions can be identified by inserting test peptide coding sequences (e.g., sequences encoding FLAG peptides) at various positions into the construct and then assaying the resulting fusions for appropriate biological activity and functionalized peptide activity using assays appropriate for the particular moiety used to construct the fusion. The activity of the target protein can be measured using any of a variety of known techniques, including those described herein.

[0118] Conjugates containing F.L-biotin In one aspect, conjugates are disclosed that include L-biotin covalently bound to a therapeutic or diagnostic agent.

[0119] In various aspects, the conjugate is covalently bound to a therapeutic agent. In a further aspect, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, doxorubicin, cisplatin, 5-fluorouracin (5-FU), etoposide, daunorubicin, camptothesin, methotrexate, carboplatin, or oxaliplatin.

[0120] In various aspects, the conjugate is covalently bound to a diagnostic agent. In a further aspect, the diagnostic agent is a cancer diagnostic agent.

[0121] G. Pharmaceutical compositions In one aspect, a pharmaceutical composition is disclosed that comprises an effective amount of the disclosed bispecific polypeptide and a pharmaceutically acceptable carrier. In a further aspect, the pharmaceutical composition comprises a composition comprising an effective amount of the disclosed bispecific polypeptide and a pharmaceutically acceptable carrier.

[0122] In various aspects, the bispecific polypeptide comprises a single-chain antibody (scFv) or F’ab fragment and a polypeptide comprising an amino acid sequence having 90% to 99% identity to the amino acid sequence AEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 8), wherein each amino acid in the amino acid sequence is a D-amino acid. In various further aspects, the bispecific polypeptide comprises an antibody or an antibody fragment covalently bound to D-SA or a variant thereof.

[0123] In various aspects, the bispecific polypeptide comprises L-biotin covalently bound to a therapeutic or diagnostic agent.

[0124] The composition can be formulated for administration by any of a variety of routes of administration and may contain one or more physiologically acceptable excipients, which may vary depending on the route of administration. As used herein, the term "excipient" means any compound or substance, including those that may also be referred to as "carriers" or "diluents". Preparing pharmaceutically and physiologically acceptable compositions is considered routine in the art, and thus, those skilled in the art can seek guidance from a number of authorities as needed.

[0125] The pharmaceutical compositions disclosed herein can be prepared for oral or parenteral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra - arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), or transdermal (e.g., topical) administration. Aerosol inhalation can also be used to deliver the nanoparticles. Thus, the compositions can be prepared for parenteral administration and contain nanoparticles dissolved or suspended in an acceptable carrier including, but not limited to, aqueous carriers such as water, buffered water, saline, buffered saline (e.g., PBS). One or more of the excipients included can help approximate physiological conditions such as pH adjusters and buffers, tonicity adjusters, wetting agents, detergents, etc. If the composition contains solid components (which can be for oral administration), one or more of the excipients can function as binders or fillers (e.g., for formulation into tablets, capsules, etc.). If the composition is formulated for application to the skin or mucosal surface, one or more of the excipients can be solvents or emulsifiers for formulation into creams, ointments, etc.

[0126] The pharmaceutical compositions may be sterilized, may be sterilized by conventional sterilization techniques, or may be sterile - filtered. Aqueous solutions may be packaged for use as such, or the lyophilized lyophilized preparations encompassed by this disclosure may be combined with a sterile aqueous carrier prior to administration. The pH of the pharmaceutical composition will typically be between 3 and 11 (e.g., about 5 to 9), or 6 to 8 (e.g., about 7 to 8). Compositions obtained in solid form may be packaged in multiple single - dose units, each containing a predetermined amount of one or more of the above - mentioned agents (e.g., sealed packages of tablets or capsules). Compositions in solid form may also be packaged in containers for flexible amounts, such as compressible tubes designed for topically applicable creams or ointments.

[0127] In various embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0128] H. Method of Treating a Disease or Condition In one aspect, a method of treating a disease or condition in a subject in need thereof, the method comprising: (a) administering to the subject an effective amount of the disclosed bispecific polypeptide; and (b) subsequently administering to the subject an effective amount of a composition comprising L-biotin covalently attached to a therapeutic agent, wherein the method is disclosed, wherein the D-SA or variant thereof specifically binds to L-biotin.

[0129] As will be appreciated by those skilled in the art, bispecific polypeptides comprising other proteins that bind biotin with high affinity (i.e., proteins other than streptavidin) can also be used in the disclosed methods. Thus, for example, in various aspects, avidin can be used in place of streptavidin (e.g., L-avidin, D-avidin).

[0130] Any of the compositions described herein can be administered as a "combination".

[0131] The pharmaceutical compositions described herein can be administered to a subject (e.g., a human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of a clinical disease. Thus, in various embodiments, the patient can be a human patient. In therapeutic use, the composition can be administered to a subject (e.g., a human patient) who already has cancer (or an autoimmune disease or disorder) or has been diagnosed with cancer (or an autoimmune disease or disorder) in an amount sufficient to at least partially improve the symptoms or signs or to inhibit (and preferably prevent) the progression of the symptoms of the condition, its complications, and the outcome. In various embodiments, the composition can be administered to a subject (e.g., a human patient) who already has cancer (or an autoimmune disease or disorder) or has been diagnosed with cancer (or an autoimmune disease or disorder). In various embodiments, the composition can be administered to a subject (e.g., a human patient) who already has cancer or has been diagnosed with cancer and also has an autoimmune disease or disorder or has been diagnosed with an autoimmune disease or disorder in an amount sufficient to at least partially improve the symptoms or signs or to inhibit (and preferably prevent) the progression of the symptoms of the condition, its complications, and the outcome. An amount sufficient to achieve this is defined as a "therapeutically effective amount". A therapeutically effective amount of a pharmaceutical composition can be an amount that achieves a cure, but the outcome is only one of several that can be achieved. As described above, a therapeutically effective amount includes an amount that provides a treatment in which the onset or progression of cancer (or an autoimmune disease or disorder) is delayed, impeded, or prevented or the symptoms of cancer (or an autoimmune disease or disorder) are improved. One or more of the symptoms may not be very severe. In the treated individual, recovery can be accelerated.

[0132] The effective amount for this use may depend on the severity of the cancer and on the weight, general condition, and health of the subject, but generally ranges from about 0.1 mg / kg body weight to about 10.0 mg / kg body weight per dose per subject. Suitable regimens for the initial and booster administrations are typified by an initial administration, followed by repeated administrations at intervals of once or more per hour, per day, per week, or per month by subsequent administrations. For example, a subject can receive nanoparticles in the range of about 0.1 mg / kg body weight to about 10 mg / kg body weight per dose one or more times per week (e.g., two, three, four, five, six, or seven or more times per week). For example, a subject can receive a dose of 0.1 mg / kg body weight to 10 mg / kg body weight per week (e.g., 0.3, 1.0, 3.0, 10.0 mg / kg body weight). A subject can also receive nanoparticles once every two or three weeks, in the range of 0.1 mg / kg body weight to 10 mg / Kg body weight per dose. The total effective amount of nanoparticles in the pharmaceutical compositions disclosed herein can be administered to a mammal as a single dose either as a bolus or as an infusion over a relatively short period of time, or multiple doses can be administered using a split treatment protocol (e.g., every 4 - 6, 8 - 12, 14 - 16, or 18 - 24 hours, or every 2 - 4 days, every 1 - 2 weeks, or once a month). Alternatively, continuous intravenous infusion sufficient to maintain a therapeutically effective concentration in the blood is also within the scope of the present disclosure.

[0133] One or more therapeutically effective amounts of the therapeutic agents present in the compositions described herein and used in the methods disclosed herein, when applied to a mammal (e.g., a human), can be determined by one of ordinary skill in the art, taking into account the individual differences in age, weight, and other general conditions (as described above).

[0134] The combination therapies disclosed herein can be administered as one or more pharmaceutical compositions and, in separate cases, can be administered simultaneously or sequentially in any order.

[0135] In various embodiments, the composition can comprise a mixture of two or more such compounds in equal or unequal amounts.

[0136] Specific combinations of agents can vary depending on a number of factors, such as the particular type of cancer, the severity of the cancer, any co-existing diseases, and the health of the patient.

[0137] When any combination of the compositions disclosed herein is administered to the same patient, they can be administered in a single formulation (e.g., a co-formulation), or in separate formulations (which may be the same or different) that are administered simultaneously or sequentially.

[0138] In various embodiments, the effective amount is a therapeutically effective amount. In various further embodiments, the effective amount is a prophylactically effective amount.

[0139] In various embodiments, the subject is a mammal. In various further embodiments, the subject is a human.

[0140] In various embodiments, the subject has been diagnosed as in need of treatment of a disease or condition prior to the administration step. In various further embodiments, the method further comprises a step of identifying a subject in need of treatment of a disease or condition.

[0141] In various embodiments, the method treats a disease. Exemplary diseases include, but are not limited to, cancer, non-Hodgkin lymphoma, multiple sclerosis, Crohn's disease, rheumatoid arthritis, asthma, macular degeneration, psoriasis, Hodgkin lymphoma, paroxysmal nocturnal hemoglobinuria, or X-linked hypophosphatemia. In a further embodiment, the disease is cancer. In still a further embodiment, the cancer is a primary cancer or a secondary cancer. In yet a further embodiment, the primary cancer or secondary cancer is a sarcoma, carcinoma, brain tumor, breast cancer, kidney cancer, pancreatic cancer, lung cancer, liver cancer, lymphoma, prostate cancer, colon cancer, ovarian cancer, gastrointestinal cancer, colorectal cancer, skin cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, glioma, leukemia, neuroblastoma, cervical cancer, chronic myeloproliferative disorders, myelodysplastic syndromes, blood cancers, myeloproliferative tumors, non-small cell lung cancer, gastroesophageal junction cancer, bladder cancer, Merkel cell cancer, urothelial skin cancer, or plasma cell tumor (myeloma). In an even further embodiment, the cancer is neuroblastoma.

[0142] In various embodiments, the method treats a condition. Exemplary conditions include, but are not limited to, prevention of thrombosis in angioplasty, kidney transplant rejection, prevention of migraine, HIV infection, or bone loss.

[0143] In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after the bispecific polypeptide. In further embodiments, the therapeutic agent is administered 24 hours after the bispecific polypeptide. In still further embodiments, the therapeutic agent is administered 48 hours after the bispecific polypeptide. In even further embodiments, the therapeutic agent is administered 1 week after the bispecific polypeptide. In yet further embodiments, the therapeutic agent is administered more than 1 week after the bispecific polypeptide.

[0144] In various embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, doxorubicin, cisplatin, 5-fluorouracin (5-FU), etoposide, daunorubicin, camptothesin, methotrexate, carboplatin, or oxaliplatin.

[0145] In various embodiments, D-SA or a variant thereof specifically binds to L-biotin.

[0146] I. Kit In one embodiment, a kit is disclosed that includes the disclosed bispecific polypeptide and one or more selected from (a) a therapeutic agent, (b) L-biotin, (c) instructions for administering the bispecific polypeptide in connection with the treatment of a disease or condition, and (d) instructions for treating a disease or condition.

[0147] In various embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and mTor inhibitors.

[0148] In various embodiments, the chemotherapeutic agent is an antitumor agent. In a further embodiment, the antitumor antibiotic is selected from doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or pharmaceutically acceptable salts thereof.

[0149] In various embodiments, the chemotherapeutic agent is an antimetabolite. In a further embodiment, the antimetabolite is selected from gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or pharmaceutically acceptable salts thereof.

[0150] In various embodiments, the chemotherapeutic agent is an alkylating agent. In a further embodiment, the alkylating agent is selected from carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or pharmaceutically acceptable salts thereof.

[0151] In various embodiments, the chemotherapeutic agent is a mitotic inhibitor. In an even further embodiment, the mitotic inhibitor is selected from irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, ixabepilone, vinorelbine, vinblastine, and teniposide, or pharmaceutically acceptable salts thereof.

[0152] In various embodiments, the chemotherapeutic agent is an mTor inhibitor. In still further embodiments, the mTor inhibitor is selected from everolimus, sirolimus, and temsirolimus, or pharmaceutically acceptable salts thereof.

[0153] In various embodiments, the therapeutic agent is covalently linked to L-biotin.

[0154] In various embodiments, the bispecific polypeptide and the therapeutic agent are co-packaged. In various further embodiments, the bispecific polypeptide and the therapeutic agent are co-formulated.

[0155] In various embodiments, the disease is cancer.

Examples

[0156] J. Examples The following examples are presented to provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are made and evaluated, and are intended purely as examples of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C or ambient temperature, and pressure is at or near atmospheric pressure.

[0157] The examples are provided herein to illustrate the invention and should not be construed as limiting the invention in any way. The examples are provided herein to illustrate the invention and should not be construed as limiting the invention in any manner.

[0158] 1. Proposed Strategy As detailed herein, mirror-image SA and biotin (D-SA / L-biotin) provide an elegant solution to avoid the limitations encountered in the L-SA / D-biotin system. For example, D-proteins are inert to L-proteases and thus cannot be digested for MHC presentation to the immune system. This property means that D-SA has significantly reduced immunogenicity and increased half-life compared to L-SA. Additionally, symmetry dictates that the mirror-image pair (D-SA / L-biotin) has exactly the same extraordinary affinity as the native pair (L-SA / D-biotin). Importantly, it has been found that the binding of D-biotin to D-SA is minimal. Thus, it is proposed that D-SA / L-biotin can be used as a biotin orthogonal streptavidin system (BOSS). Without wishing to be bound by theory, it is hypothesized that together with the low immunogenicity of D-SA, the orthogonality of BOSS overcomes the limitations of native SA / biotin.

[0159] BOSS can be created by using mirror-image SA and biotin (D-SA and L-biotin). Mirror images, or D-proteins (composed of mirror-image D-amino acids), are inert to L-proteases 21 and thus cannot be digested to present MHC to the immune system 22 . These properties mean that D-SA has significantly reduced immunogenicity and increased half-life compared to L-SA. Since native SA (L-SA) and native biotin (D-biotin) have extraordinary affinity, it is also true (by the law of mirror symmetry) that D-SA and L-biotin have exactly the same affinity. However, low immunogenicity, increased half-life, and strong binding interaction between D-SA and L-biotin are not sufficient to create BOSS. To be successful, it may be useful to demonstrate that native D-biotin shows minimal binding to D-SA (i.e., orthogonality). The usefulness of mirror-image SA was recently reported in a short preprint attempting to avoid biotin interference in immunoassay diagnostics 40 .

[0160] Since D-proteins cannot be recombinantly produced, D-SA is chemically synthesized via chemical protein synthesis (CPS) using D-amino acids. Proteins of this size (127 amino acids) require synthesis in multiple segments via solid-phase peptide synthesis (SPPS) (usually limited to about 60 amino acids due to aggregation on the resin). 41 Native chemical ligation (NCL) 42 is then used to ligate the segments together (Figure 3). NCL requires one peptide with an N-terminal Cys and the other peptide with a C-terminal thioester. The thioester peptide is made using C-terminal hydrazine on the resin during SPPS. Conversion after cleavage to azide makes the hydrazine a better leaving group, which can then be replaced by various thiols to create the thioester. NCL is then achieved by reacting the C-terminal thioester with the N-terminal Cys on a separate segment. This reaction forms a reversible thioester bond between the two segments, bringing the N-terminals close enough for a shift from S-acyl to N-acyl and forming an irreversible native amide bond (Figure 3). Since many proteins (including SA) lack Cys residues, the native N-terminal Ala residue can be replaced with a cysteine residue for use in NCL. After ligation, Cys can be converted to the native Ala in a simple desulfurization reaction. 43 This strategy significantly increases the number of available ligation junctions.

[0161] Proteins the size of SA have long been thought to be difficult to make by CPS. Recently, tools have been developed to make the synthesis of proteins of this size more feasible. One of these tools is the Automated Ligator (Aligator). 23It is a program developed to predict an optimal synthesis strategy based on parameters that minimize the number and length of peptide segments and optimize ligation junctions. This program significantly reduces the trial and error of CPS, which is one of the major rate-limiting steps for protein synthesis. In addition, peptide solubility can be a rate-limiting step. When a protein is divided into segments, regions with a high density of hydrophobic or negatively charged residues are often exposed, leading to a decrease in solubility and difficulty in purifying the segments and ligation. To counter insoluble peptides, a traceless linker called a helping hand (HH) was used. HH binds to a primary amine that can be functionalized with solubilizing residues (usually Lys or Arg). 44、45 (Figure 4). After the complete protein is assembled and solubility enhancement is no longer required, HH is removed under mild conditions to obtain natural amino acid side chains.

[0162] 2. Characterization of the properties of L-biotin and L-streptavidin binding Since tight cross-linking impairs the orthogonality of BOSS, it is necessary to characterize the binding of D-biotin to D-streptavidin. According to the law of mirror symmetry, the binding behavior of L-biotin to L-streptavidin is equivalent to that of D-biotin interaction. First, a control isothermal titration calorimetry (ITC) experiment was carried out. D-biotin was titrated into L-SA to confirm the folding and activity of recombinant SA (Novus Biologicals). This titration data was fitted using Origin software as shown in Figure 2A, and the binding stoichiometry (N = 0.97 ± 0.003 sites) and enthalpy (ΔH = -27.72 ± 0.18 kcal / mol) were calculated. These values were consistent with the literature values. 27、39 The extremely strong interaction between L-SA and D-biotin hinders the determination of the K D value from this experiment, but the binding constant was measured using a more sensitive technique and found to be 4.8x10 -14 M. 1

[0163] ​Next, the ITC experiment was repeated with L-biotin (Carbosynth) and L-SA. This interaction was expected to show a weaker binding to the hypothesized stereochemical mismatch between L-biotin and L-SA. In this case, the binding was weak enough that all parameters could be fit with good reliability (N = 0.96 ± 0.08 sites, ΔH = -10.09 ± 1.26 kcal / mol, and K D = 6.13 ± 2.16 μM, Figure 2B). This residual binding is approximately one billion times weaker than the native pair, suggesting that L-biotin does not bind to L-SA in the presence of D-biotin. Although not wishing to be constrained by theory, these data suggest that BOSS is highly orthogonal to D-biotin and that BOSS PTI is suitable for in vivo use without interference from endogenous biotin.

[0164] 3. Chemical synthesis of L-streptavidin The streptavidin synthesized is the 127-residue "core" SA, the most commonly used form, which has ideal biotin affinity and tetramer stability. 46 L-SA was synthesized prior to D-synthesis to save on initial troubleshooting costs and to evaluate the quality of synthesis and folding by comparing synthetic L-SA to recombinant L-SA. This verification was performed because synthetic L-SA has the same affinity for L-biotin as it does for D-biotin when D-SA is made using the same procedure. The three-segment synthetic strategy (SA1, SA2, and SA3 shown in Figure 5) guided by Aligator 23 was used. SA1 and SA2 were synthesized with hydrazide on the C-terminus. SA3 was made as a peptide acid.

[0165] All three peptides were synthesized via SPPS and purified via reverse-phase HPLC (RP-HPLC). SA1 and SA2 were synthesized with a C-terminal hydrazine for use in NCL after conversion to a thioester. SA1 was synthesized with a helping hand (HH) molecule added to the N-terminus because it has minimal solubility in ligation buffer. Then, six Lys residues were added to HH to create K 6 -HH-SA1, which dramatically improved solubility. SA2 and SA3 were sufficiently soluble in NCL without HH modification. K 6 -HH-SA1 was successfully ligated to SA2 to obtain K 6 -HH-SA1-SA2. Then, SA3 was ligated to K 6 -HH-SA1-SA2 to obtain K 6 -HH-SA1-SA2-SA3. After this final ligation, HH was cleaved using 1 M hydroxylamine, followed by desulfurization to obtain a small amount of SA1-SA2-SA3 (native L-SA). The full-length product was characterized via LC-MS (Figure 6) and has a small Ala deletion, likely due to incomplete Ala coupling during the SPPS of SA1.

[0166] The peptide was synthesized on a Prelude X instrument (Gyros Protein Technologies) using Fmoc solid-phase peptide synthesis (SPPS) on a 50 μmol scale. The deprotection cycle used treatment with 20% piperidine in 4 mL of DMF for 2 minutes twice, followed by three 30-second washes with 4 mL of DMF. The coupling cycle consisted of the addition of 200 mM amino acid in 1.3 mL of DMF, 195 mM HATU in 1.3 mL of DMF, and 600 mM NMM in 1 mL of DMF. Then, the resin and coupling reagents were mixed at room temperature for 25 minutes using nitrogen bubbling and then washed three times with 4 mL of DMF. In the case of peptide acid (SA3), 0.03 mmol of Fmoc-AA was dissolved in 2 mL of DMF / DCM, and then 0.3 mmol of DIPEA was added to the Fmoc-AA solution. This solution was added to 300 mg of polystyrene 2-chlorotrityl chloride resin and mixed on a rotisserie at room temperature for 1 hour. Unreacted groups were capped by rinsing the resin with 20 mL of 17:2:1 DCM:MeOH:DIPEA. 300 mg of 2-CTC resin was converted to 2-chlorotrityl Fmoc-hydrazine by reacting it with 61.2 μmol of Fmoc-hydrazine in 6 mL of 1:1 DCM and DMF and 0.532 mL of DIPEA for 2 hours. Unreacted residues were capped by adding 60 μL of MeOH and reacting for 10 minutes. The resin was rinsed thoroughly with DCM and DMF. After completion of the synthesis, the peptide resin was washed thoroughly with DCM and dried under vacuum.

[0167] Pseudoproline dipeptides were used to synthesize peptides of sufficient purity. GT, VT, DS, LT, KS, and DT were utilized in the synthesis of SA1-3. TMB-Gly was also used to increase the synthesis yield. Underlined Ala residues were synthesized as Cys residues to facilitate native chemical ligation and then desulfurized to obtain native alanine residues.

[0168] Cleavage of the peptide resin was achieved by stirring with 6 mL of TFA containing 2.5% water and 2.5% TIS per 50 μmol of peptide resin for 3 h. For peptides containing Cys, 2.5% EDT was added to the cleavage cocktail. The TFA solution was precipitated into 40 mL of ice-cold diethyl ether per 50 μmol of crude peptide and centrifuged at 4 °C and 4,000 g for 10 min. The supernatant was decanted while washing the pellet twice with ether, then dissolved in 50% acetonitrile in water and lyophilized overnight. Each peptide was then purified by preparative RP-HPLC using either a C4 (SA1) or C12 (SA2 and SA3) column. The purity of SA1, SA2, and SA3 by RP-HPLC and LC-MS is shown in Figure 7.

[0169] 4. Native Chemical Ligation, Helping Hand Removal, and Desulfurization SA1 was not sufficiently soluble for native chemical ligation (NCL), so two separate helping hand strategies were used. First, a glutamate helping hand was incorporated as an Fmoc-protected amino acid during SPPS at Glu2 in SA1 and then functionalized with six lysine residues. The resulting peptide had a greatly increased solubility and underwent NCL very efficiently, but there were complications in the removal of this helping hand. After this failed attempt, after Fmoc removal of the final residue, a Ddap helping hand was added to the N-terminus of SA1 and the resin was washed with DMF. The resin was stirred at 37 °C for 24 h in 200 mM Ddap in 4 mL of NMP. The resin was washed with DMF and six lysine residues were added to it via SPPS. This peptide had the same increased solubility and ligation efficiency, but the Ddap was easily removed.

[0170] Native chemical ligation reactions were carried out according to standard methods using the thiol additive MPAA and the TCEP reducing agent. All ligation reactions were performed using the peptide hydrazide method, by which the peptide was dissolved and activated in activation buffer (6 M GuHCl, 100 mM phosphate, pH 3) for 20 min at -20 °C by adding freshly prepared sodium nitrite solution (15 eq) (conversion of hydrazide to acyl azide). After activation, a solution containing freshly prepared MPAA at pH 7 in ligation buffer (6 M GuHCl, 100 mM phosphate, pH 7) was added and the final pH was adjusted to 7 to initiate the thiolysis and ligation reactions. Upon completion (based on analytical HPLC and LC / MS), the reaction was treated with freshly prepared 150 mM TCEP in 6 M GuHCl, diluted with 5% acetic acid in water, rotated at 5000 g, and the supernatant was purified by preparative HPLC. After the second ligation, 2 M hydroxylamine in ligation buffer (6 M GuHCl, 100 mM phosphate, pH 7) was added to give a final composition of 1:1 with the ligation reaction volume and reacted overnight at room temperature to remove the helping hand. The reaction mixture was dialyzed against ligation buffer (6 M GuHCl, 100 mM phosphate, pH 7) over several days and the buffer was changed once. Desulfurization was carried out using a metal-free radical-mediated procedure. Using desulfurization buffer (6 M GuHCl, 100 mM phosphate, pH 6.5), two stock solutions of solution A (120 mM VA-044 and 240 mM reduced GSH) and solution B (500 mM TCEP) were prepared. Equal volumes of solution A and B were added to the dialyzed ligation reaction. The final pH was adjusted to 6.5 and the reaction was stirred at room temperature until desulfurization was complete (about 3 h). The reaction was diluted with 5% AcOH, centrifuged at 5000 g, and the solution was purified by preparative HPLC on a C4 column. The purified ligation products for SA1 and SA2 ligations are shown in Figure 8. Desulfurized and purified full-length synthetic streptavidin with the helping hand removed is shown in Figure 6.

[0171] K. Predictive Examples 1. Chemical synthesis, property evaluation, and optimization of pharmacokinetics of L / D-SA a. Synthesis Before scaling up the synthesis, troubleshooting of SPPS of SA1 for removing Ala deletion is performed. The exposed amines are capped with acyl groups after Ala coupling to prevent peptides with incomplete Ala coupling from proceeding with the synthesis. Such capped products are cleaved and easily separated during RP-HPLC purification. Then, the synthesis of full-length L-SA can be scaled up using pre-synthesized SA2 and SA3 via the ligation procedure from small-scale synthesis.

[0172] Since NLC is carried out under denaturing conditions, L-SA needs to be folded. SA is a very stable protein, but recombinant SA may be completely denatured in 6 M guanidine thiocyanate. 47 This unfolded SA spontaneously refolds when dialyzed into neutral buffer (independent of chaperones). 47 Chemically synthesized SA has the same primary amino acid sequence as recombinant SA. Therefore, it folds under the same conditions as recombinant SA. When folded, it has the same activity as recombinant SA. This folding procedure is reproduced with recombinant SA before using it for L-SA. Using the optimized synthesis procedure, D-SA1, D-SA2, and D-SA3 are prepared using D-amino acids. HH is achiral and is added to the N-terminus of L- or D-peptides. N-terminal K 6 -HH is added to D-SA1 to increase solubility and facilitate NCL. Using the optimized NCL, HH removal, desulfurization, and folding procedures, D-SA is prepared (since D-proteins fold in the same way as L-proteins, the optimized procedures are expected to function to fold both enantiomers).

[0173] b. Property evaluation After synthesis and folding, the CD spectra of synthetic D- and L-SA are compared with the CD spectrum of the recombinant protein to confirm the folded state. SA binds to biotin with high affinity only when in the tetrameric state. Therefore, it is also important to analyze the oligomerization state of the protein using analytical ultracentrifugation (AUC) and size exclusion chromatography (SEC). SEC can also be used to purify the SA tetramer complex and remove aggregates and / or lower oligomerization states.

[0174] As mentioned above, the K of recombinant L-SA and L-biotin D was measured. Whether the binding affinity of the synthetic protein is the same as that of the recombinant is confirmed using ITC. The above ITC experiment is repeated with synthetic L-SA and then D-SA. The data are expected to be nearly identical to those observed with L-SA. This data supports that the correct protein has been made and is folded correctly.

[0175] The binding of L-biotin to recombinant SA is of particular interest. Since D-biotin has evolved to bind to SA with such high affinity, it is surprising that L-biotin does not bind at all. To clarify the mechanism behind the difference in binding between the two enantiomers, L-SA is crystallized with L-biotin and its structure is determined via X-ray crystallography. The native structure (D-biotin / L-SA) is overlaid with the mismatched structure (L-biotin / L-SA) to determine how SA discriminates between biotin enantiomers with a specificity of one billion-fold. Crystal growth conditions using native SA and biotin have been developed and high-resolution data have been collected. Droplets are set up to co-crystallize SA with L-biotin under the same conditions and millimolar concentrations of L-biotin are obtained. Preliminary crystals from these conditions diffract at a resolution of better than 2 Å.

[0176] c. Pharmacokinetics The biodistribution, half-life, and clearance pathways of D-SA are also of interest. There is increasing interest in using fluorescence-based whole-body imaging (FFI) to collect these data due to the cost reduction and safety increase compared to radiation studies. 48~50 This study is conducted using FFI in collaboration with the University of Utah’s Preclinical Research Resource (PRR). Since the hair of mice can interfere with fluorescence measurements, SKH1 hairless mice (Charles River) are selected for this study. Alexa Fluor 647 NHS ester (emitting magenta fluorescence, ThermoFisher) is added to amine-functionalized L-biotin to produce fluorescently labeled L-biotin (Mag-L-biotin). Fluorescently labeled D-biotin is also produced using the same method, but Alexa Fluor 568 NHS ester (emitting red fluorescence, ThermoFisher) is used to produce Red-D-biotin. Then, a 1:1 mixture of Mag-L-biotin to D-SA monomer in PBS and a 1:1 mixture of Red-D-biotin to L-SA in PBS are prepared. Then, as previously done, 200 μL of a 4 nmol mixture in PBS is intravenously injected into the mice. 48 In vivo fluorescence is measured at 0, 1, 2, 3, 4, 6, 8, 24, and 48 hours using an IVIS Spectrum In Vivo Imaging System (PerkinElmer) and compared to D-SA vs L-SA. At each time point, the concentration of the fluorophore in each organ is tracked. This will show how D-SA is removed from circulation (i.e., a fluorescent concentration in the liver would mean it is removed by the liver). The half-life is determined by quantifying the total level of fluorescence in the whole mouse at different time points. To ensure statistical significance, the time points and measurements in this study are performed on 8 mice (the group size is estimated by a power analysis, and 80% power is to detect a 50% difference at p < 0.05). An equal number of male and female mice are used to account for any differences due to gender as a biological variable.

[0177] In cooperation with PRR, the immunogenicity of D-SA in SKH1 hairless mice was also tested and compared with L-SA. A group of eight mice was intravenously injected with either 30 μg of L- or D-SA at days 0, 10, 20, and 40. Blood samples were collected before the injections at days 10, 20, and 40 and analyzed using an indirect ELISA assay as previously performed. 51 D-SA is immobilized on the wells of the plate. This is usually achieved by non-specific interactions between the hydrophobic residues on the well plastic and the protein. Since the interaction is not chiral-specific, this should function equally well using D-SA as using the L-protein. Blocking is achieved using bovine serum albumin, and then a goat anti-mouse IgG secondary antibody-horseradish peroxidase conjugate (ThermoFisher) is added to examine whether any antibody is bound to D-SA. Without wishing to be bound by theory, D-SA is expected to have minimal immunogenicity compared to L-SA. 51 。

[0178] 2. Demonstration of BOSS PTI a. BOSS-targeted construct For proof of concept, the SA PTI method previously described by Cheung et al. 7 was reproduced using BOSS (BOSS PTI). The single-chain variable fragment (scFv) used in this study, which is derived from the 5F11 antibody, was used. The scFv is a fusion protein of the variable regions of the heavy and light chains of the antibody. These small antibody derivatives can be cleared from circulation much faster than full-size antibodies, within less than 24 hours. 52、53 This property is essential for PTI so that unbound pre-targeting agents are rapidly cleared from circulation before the drug is administered. The 5F11 scFv binds to GD, a glycosphingolipid that is concentrated in the gray matter and synaptic junctions. GD 9、10、20、35 。 2 GD 2is upregulated in several types of tumors, including neuroblastoma (NB). 7、54、55 This upregulation was the reason for the NIH ranking GD 2 as one of the most promising tumor antigens. 56 Cheung et al. 7 expressed scFv fused to SA. They then used this fusion as a pretargeting tool. Their scFv-SA conjugates were all composed of L-amino acids and recombinantly expressed in E. coli. Since there is currently no method for recombinantly expressing D-proteins, conjugation chemistry is used to bind chemically synthesized D-SA to recombinantly expressed L-5F11scFv. The scFv is expressed according to published procedures, but a C-terminal sortase tag (Leu-Pro-Xxx-Thr-Gly-Xxx, where Xxx is any amino acid) is added 57 Sortase recognizes this tag and cleaves between Thr and Gly to form an enzyme-linked thioester on the C-terminus of the scFv. When bound to the scFv, sortase preferentially reacts with the N-terminal amine of the oligoglycine motif 57 Since glycine has no chirality, sortase recognizes the oligoglycine motif added to the N-terminus of D-SA. This conjugate (scFv-D-SA) is used for pretargeting experiments. The L version of this construct (scFv-L-SA) is also made by recombinant expression as a control.

[0179] b. BOSS PTI in NB cells The efficacy of scFv-D-SA in cells is first shown using fluorescence microscopy. Neuroblastoma (NB) cells (SK-N-SH from ATCC) and immortalized human cerebral cortical brain cells (HBEC-5i from ATCC) are cultured. Healthy cerebral cortical (CC) cells have a ganglioside density five times lower than NB cells 55、58~61, functions as a control cell line. Four separate experiments are conducted: NB or CC cells with scFv-D-SA, and NB or CC cells with scFv-L-SA. Previous studies have suggested that a 5 μM solution of the scFv construct during a 3-hour incubation period should be sufficient for it to bind to the cells. 48 . After 3 hours, the medium is exchanged, rinsed with PBS buffer to ensure that excess (unbound) scFvL / D-SA is thoroughly removed. Then, a 1:1 mixture of Red-D-biotin and Mag-L-biotin (5 μM each) is added. After allowing time for binding (3 hours), the medium is exchanged and rinsed with buffer. After these steps, scFv-D-SA and scFv-L-SA should bind to NB cells at a much higher concentration than to CC cells, and the biotin conjugates should bind to D- and L-SA. Whether there is any cross-binding of Red-D-biotin and Mag-L-biotin is determined using a fluorescence microscope. Red-D-biotin should bind exclusively to scFv-L-SA, and Mag-L-biotin should bind exclusively to scFv-D-SA. Additionally, due to the absence of competing biotin, a higher fluorescence signal is expected for scFv-D-SA than for scFv-L-SA.

[0180] c. BOSS PTI in mice Using the above procedure, FFI is used to determine the half-life and in vivo distribution of scFv-L-SA and scFv-D-SA in mice. This study determines how long it takes for unbound antibody to be removed from circulation for the BOSS PTI experiment. Then, collaboration with PRR is carried out for xenographing NB cells into BALB / c nude mice (Charles River). These mice are immunodeficient and hairless to accept the xenograft and facilitate FFI. 4 nmol of scFv-D-SA and scFv-L-SA are administered to xenographed and non-xenographed mice according to a previously published procedure. 7Allow an empirically determined time to elapse until the unbound scFv-D-SA and scFv-L-SA are removed. Then inject a 1:1 mixture of 5 nmol 48 of Red-D-biotin and Mag-L-biotin. Measure fluorescence at 0, 1, 2, 3, 4, 6, 8, 24, and 48 hours. Fluorescence is expected to localize around the tumor in xenograft mice and rapidly disappear in non-xenograft mice. There should be no cross-linking of Red-D-biotin and Mag-L-biotin with BOSS PTI or SA PTI. Additionally, due to the absence of interfering biotin, the fluorescence signal is expected to be brighter with BOSS PTI than with SA PTI.

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[0251] It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. Other embodiments of the present invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

1. A bispecific polypeptide comprising an antibody covalently bound to D-streptavidin (D-SA) or a variant thereof, or a fragment thereof.

2. The bispecific polypeptide according to claim 1, wherein the antibody or antibody fragment is an anti-IL-17 receptor antibody, an anti-IL-5 receptor antibody, an anti-PD-L1 antibody, an anti-FGF23 antibody, an anti-epidermal growth factor receptor antibody, an anti-GD2 antibody, an anti-HER-2 receptor antibody, an anti-RANKL antibody, an anti-C5 antibody, an anti-VEGF receptor antibody, an anti-VEGF-A antibody, an anti-VEGF receptor 2 antibody, an anti-IgE antibody, an anti-TNF-alpha antibody, an anti-IL-12 / 23 antibody, an anti-CTLA-4 antibody, an anti-CD30 antibody, an anti-CD4 antibody, an anti-CGRP receptor antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-CD25 antibody, or an anti-GP11b / 111a antibody.

3. The bispecific polypeptide according to claim 1, wherein the antibody or antibody fragment thereof is an anti-CD30 antibody.

4. The bispecific polypeptide according to claim 1, wherein the antibody or antibody fragment specifically binds to a cell surface marker.

5. The bispecific polypeptide according to claim 4, wherein the cell surface marker is CD3, CD4, CD5, CD20, CD25, or glycosphingolipid.

6. The aforementioned glycosphingolipide, GD 2 The bispecific polypeptide according to claim 5.

7. The bispecific polypeptide according to claim 1, wherein the antibody or antibody fragment is a single-chain antibody (scFv) or an F'ab fragment.

8. The aforementioned scFv is brosumab, ibalizumab, erenumab, atezolizumab, reslizumab, pembrolizumab, nivolumab, ramucirumab, ipilimumab, brentuximab, ustekinumab, panitumaumab, ranibizumab, necitumamab, dinutuximab, denosumab, exulizumab. The bispecific polypeptide according to claim 7, derived from bevacizumab, omalizumab, adalimumab, avelumab, durvalumab, brodalumab, muromonoab-CD3, absiximab, rituximab, daclizumab, infliximab, basiliximab, palivizumab, trastuzumab, gemtuzumab, or a biologically active variant thereof.

9. The bispecific polypeptide according to claim 1, wherein the antibody or an antibody fragment thereof is covalently bound to D-SA.

10. The bispecific polypeptide according to claim 1, wherein the antibody or an antibody fragment thereof is covalently bound to a variant of D-SA.

11. The bispecific polypeptide according to claim 10, wherein the variant of D-SA is D-traptoavidin, D-strept-tactin, D-strept-tactin XT, or monovalent D-SA.

12. The bispecific polypeptide according to claim 1, wherein the D-SA or a variant thereof specifically binds to L-biotin.

13. A pharmaceutical composition comprising an effective amount of a bispecific polypeptide according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is formulated for intravenous administration.

15. Use of a bispecific polypeptide according to any one of claims 1 to 12 in the manufacture of a pharmaceutical product for treating a disease or condition.

16. The use according to claim 15, wherein the disease is selected from cancer, non-Hodgkin lymphoma, multiple sclerosis, Crohn's disease, rheumatoid arthritis, asthma, macular degeneration, psoriasis, Hodgkin lymphoma, paroxysmal nocturnal hemoglobinuria, and X-linked hypophosphatemia.

17. The use according to claim 15, wherein the condition is selected from prevention of thrombosis in angioplasty, kidney transplant rejection, prevention of migraines, HIV infection, and bone loss.

18. A pharmaceutical composition for treating a disease or condition, comprising the bispecific polypeptide described in any one of claims 1 to 12.

19. The pharmaceutical composition according to claim 18, wherein the pharmaceutical composition is used in combination with an effective amount of a composition containing L-biotin covalently bonded to a therapeutic agent.

20. The pharmaceutical composition according to claim 19, wherein the therapeutic agent is a radiopharmaceutical or a chemotherapeutic agent.

21. The pharmaceutical composition according to claim 18, wherein the disease is selected from cancer, non-Hodgkin lymphoma, multiple sclerosis, Crohn's disease, rheumatoid arthritis, asthma, macular degeneration, psoriasis, Hodgkin lymphoma, paroxysmal nocturnal hemoglobinuria, and X-linked hypophosphatemia.

22. The pharmaceutical composition according to claim 18, wherein the condition is selected from prevention of thrombosis in angioplasty, kidney transplant rejection, prevention of migraines, HIV infection, and bone loss.

23. A conjugate containing L-biotin covalently bound to a therapeutic or diagnostic agent.

24. The conjugate according to claim 23, wherein the therapeutic agent is a chemotherapy agent, or the diagnostic agent is a cancer diagnostic agent.

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