Click-to-Release for Proteins and Peptides

Abstract

The present invention relates to the combination of a modified peptide or protein comprising one or more bioorthogonal functional groups, and a drug modified with one or more bioorthogonal functional groups complementary to the bioorthogonal groups of the peptide or protein.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION

[0001] The present invention relates to the combination of a modified drug with a peptide or protein containing one or more bioorthogonal functional groups for the bioorthogonal delivery of the drug to a subject in need thereof. [Background technology]

[0002]

[0002] Click chemistry has emerged as a versatile tool for in vivo chemical reactions: "Click chemistry refers to a group of reactions that are fast, simple to use, easy to purify, versatile, regiospecific, and produce high yields of products" [1]; therefore, these reactions are ideal tools for targeting and labeling biological targets in vivo. The inverse electron demand Diels-Alder (iEDDA) reaction between diene tetrazines and dienophiles exhibits exceptional kinetics (1-10 6 M -1 s -1 ), which are particularly suitable for such in vivo applications due to their speed and the metabolic stability of some of the developed reagents.

[0003]

[0003] Due to its speed and specificity, this reaction has been used for the targeted release of caged prodrugs. In this approach, biologically active molecules are caged (i.e., modified) with chemical groups that are active with iEDDA chemistry. This modification alters (reduces) the biological activity of the drug, thereby reducing systemic side effects. The biological target, i.e., the site where the drug should be active in vivo, is then targeted with the iEDDA reactive counterpart. Upon successful targeting, the caged prodrug is administered and reacts with the iEDDA reactive counterpart already present in the body, releasing its full biological activity only upon reaction upon removal of the caging group.

[0004]

[0004] The current literature describes various methods for biomarkers with iEDDA-reactive moieties, for example using local injection [2].

[0005] Here, an iEDDA-reactive polymer is injected directly into tumor tissue. After the polymer binds to the tumor, an iEDDA-reactive prodrug is administered, which is released upon contact with the tumor-bound polymer. This increases the local concentration of the active drug and reduces systemic toxicity (see Figure 2). The drawbacks of this approach are that the procedure is invasive and requires precise localization of the tumor before the polymer can be applied.

[0005]

[0006] Alternatively, endogenous chemical reactivity markers can be used [3]. In some diseases, oxidative stress can lead to the production of acrolein as a metabolic by-product. Acrolein is a compound active in 1,3-dipolar cycloaddition reactions, which can also result in the removal of a chemical group and subsequent release of the drug from the prodrug. This means that the prodrug is preferentially activated at the site of the lesion where oxidative stress occurs (see Figure 3). While this approach is advantageous, it suffers from the drawback of disease heterogeneity. For this potential treatment to be adequately effective, it must be administered to disease forms with very high local acrolein concentrations, which must be analytically determined in advance. The metabolic heterogeneity of disease forms inevitably leads to the existence of subpopulations of patients who do not respond.

[0006]

[0007] Additionally, protein conjugates can be used [4]. This approach involves the use of a protein modified with an iEDDA-reactive moiety. This protein has affinity for a surface marker of the biological target to be treated with the drug. The protein conjugate is administered and binds to the target. After a suitable time for proper target labeling, treatment is completed by tracking with an iEDDA-caged prodrug. Upon contact with the pre-labeled target, the prodrug reacts and releases the active drug (see Figure 4).

[0007]

[0008] The drawbacks of this approach are that the development of protein conjugates is often cumbersome and requires significant resources to develop appropriate supply chains and analytical methods to verify the quality of the drug. The conjugation of iEDDA-reactive moieties needs to be site-specific to obtain reproducible drug molecules, and access to such technologies is not always possible.

[0008]

[0009] International Publication No. 2014081301 discloses a combination of a masking moiety conjugated to a trigger moiety, which is further conjugated to a drug. The trigger moiety includes a dienophile and an activator includes a diene. The trigger moiety and activator undergo a rapid bioorthogonal reaction, resulting in release of the masking moiety and activation of the drug. International Publication No. 2017044983 describes a bioorthogonal composition for delivering a drug to a subject. The bioorthogonal composition includes a hydrogel-supported composition having different bioorthogonal functional groups. International Publication No. 2022032191 discloses trans-cyclooctene bioorthogonal agents and their use in cancer and immunotherapy.

[0009]

[0010] Fairhall JM et al. have disclosed the conjugation of functionalized trans-cyclooctene with cetuximab, providing a reagent for pretargeting and localization of bioorthogonal reagents [5].

[0010]

[0011] The drawbacks of state-of-the-art conjugates are that the development of protein conjugates is often cumbersome and requires significant resources to be dedicated to developing appropriate supply chains and analytical methods to verify pharmaceutical quality. The conjugation of iEDDA-reactive moieties needs to be site-specific to obtain reproducible drug molecules, and access to such technologies is not always possible. Summary of the Invention [Problem to be solved by the invention]

[0011]

[0012] Therefore, there remains a need for new approaches to treat diseases based on modifications with bioorthogonal functional groups. [Means for solving the problem]

[0012]

[0013] The object of the present invention is to provide a novel approach to the treatment of diseases based on modifications with bioorthogonal functional groups. This objective is achieved by the subject matter of the present invention. This novel approach is based on the combination of a modified peptide or protein containing one or more bioorthogonal functional groups with a drug modified with one or more bioorthogonal functional groups complementary to the bioorthogonal groups of the protein or peptide.

[0013]

[0014] The present invention relates to the combination of (i) a modified peptide or protein comprising one or more bioorthogonal functional groups, and (ii) a drug modified with one or more bioorthogonal functional groups complementary to the bioorthogonal groups of (i).

[0014]

[0015] The bioorthogonal functional group can be a dienophile or a diene. A dienophile is, for example, trans-cyclooctene. A diene is, for example, a tetrazine moiety.

[0016] According to one embodiment of the present invention, the modified peptide or protein is selected from the group consisting of antibodies, antibody fragments, diabodies, single chain variable fragment antibodies, single domain antibodies, nanobodies, small protein binders, carrier proteins, any peptide or protein with affinity for a human disease target.

[0015]

[0017] "Antibody fragments" include portions of an intact antibody, including the antigen-binding and / or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; linear antibodies; single-chain antibody molecules; multivalent single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0016]

[0018] According to one embodiment of the present invention, the modified peptide or protein has the general formula (I):

[0017] [ka]

[0018] (In the formula, X represents NH or O; R is halogen, -OR a , -C(O)R a , -COOR a , -NR a R a , -SR a , -C 1~6 selected from the group consisting of alkyl and phenyl; 1~6 The alkyl or phenyl moiety may optionally be halogen, -OR a , -C(O)R a , -COOR a , -NR a R a , -SR a and optionally substituted by R 2 is the amino acid residue that binds to the next residue towards the N-terminus and C-terminus of the protein or peptide, R a is hydrogen or C 1~6 The compound has at least one diene moiety of which the aryl group is alkyl.

[0019] According to one embodiment of the present invention, the drug may be conjugated to the dienophile moiety. The drug may be conjugated to the dienophile moiety via a carbamate moiety.

[0020] A further embodiment relates to the combination described herein wherein the dienophile moiety is a trans-cyclooctene moiety.

[0021] Further embodiments relate to the combinations described herein, wherein the agent is selected from the group consisting of cytotoxins, antiproliferative agents, antitumor agents, antiviral agents, antibiotics, anti-inflammatory agents, chemosensitizers, radiosensitizers, immunosuppressants, immunostimulators, immunomodulators, anti-angiogenic factors, DNA damaging agents, DNA crosslinking agents, DNA binders, DNA alkylating agents, DNA intercalators, DNA cleaving agents, microtubule stabilizing and destabilizing agents, and topoisomerase inhibitors.

[0021]

[0022] Drugs include colchicine, vinca alkaloids, anthracyclines, doxorubicin, epirubicin, idarubicin, daunorubicin, camptothecin, taxanes, taxol, vinblastine, vincristine, vindesine, calicheamicin, tublysin, tublysin M, cryptophycin, methotrexate, methopterin, aminopterin, dichloromethotrexate, irinotecan, enediyne, amanitin, dactinomycin, duocarmycin, maytansine, maytansinoids, and doxorubicin. The compound may be selected from the group consisting of rastatin, auristatin, pyrrolobenzodiazepines and dimers, indolinobenzodiazepines and dimers, pyridinobenzodiazepines and dimers, mitomycin, melphalan, leurosin, leurosidein, actinomycin, tallysomycin, lexitropsin, bleomycin, podophyllotoxin, etoposide, etoposide phosphate, staurosporine, esperamicin, pteridine drugs, platinum-based drugs, and cytotoxic nucleosides.

[0022]

[0023] One embodiment of the present invention relates to a combination described herein for use in the treatment of cancer, an infectious disease, or an autoimmune disease.

[0024] Further embodiments relate to a combination as described herein wherein the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, squamous cell carcinoma of the head / neck, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate cancer, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.

[0023]

[0025] Further embodiments relate to combinations described herein, wherein the modified peptide or protein and the agent are administered sequentially or simultaneously. [Brief explanation of the drawings]

[0024] [Figure 1]

[0026] iEDDA reaction of tetrazines with dienophiles. [Figure 2]

[0027] Pretargeted drug release using iEDDA-reactive polymers. [Figure 3]

[0028] Use of acrolein as a reaction partner for drug release in vivo. [Figure 4]

[0029] Use of protein conjugates for click-to-release chemistry of highly active toxins in tumor treatment. DETAILED DESCRIPTION OF THE INVENTION

[0025]

[0030] The present invention relates to the combination of (i) a modified peptide or protein comprising one or more bioorthogonal functional groups, and (ii) a drug modified with one or more bioorthogonal functional groups complementary to the bioorthogonal groups of (i).

[0026]

[0031] In chemistry, the Diels-Alder reaction is an important reaction in which a conjugated diene reacts with a dienophile (a substituted alkene) to produce a substituted cyclohexene derivative. This reaction has two partners that react with each other: the diene and the dienophile.

[0027]

[0032] Dienes are unsaturated hydrocarbons consisting of two double bonds between carbon atoms. Dienes are also known as diolefins or alkadienes. They are covalently bonded compounds containing two alkene units. Dienes usually occur as subunits of more complex organic molecules. Furthermore, dienes are found in naturally occurring compounds as well as synthetic chemicals. These chemicals are useful in organic synthesis reactions.

[0028]

[0033] Dienophiles are organic compounds that readily react with dienes. Dienophiles are commonly used in Diels-Alder reactions, which involve the reaction of a conjugated diene with a substituted alkene, with the substituted alkene acting as the dienophile.

[0029]

[0034] Suitable dienophiles typically have one or two of the following functional groups: CHO, COR, COOR, CN, C=C, Ph, or a halogen. Additionally, the diene must be highly electron-rich.

[0030]

[0035] Alkenes react readily with dienes, and are therefore commonly known as dienophiles. Heating is not usually required for the Diels-Alder reaction, but heating can sometimes improve the reaction yield.

[0031]

[0036] The term bioorthogonal chemistry refers to any chemical reaction that can occur within a living system without interfering with natural biochemical processes. The concept of bioorthogonal reactions has made it possible to study biomolecules, such as glycans, proteins, and lipids, in real time within living systems without cytotoxicity. Several chemical ligation strategies that meet the requirements of bioorthogonality have been developed, including the 1,3-dipolar cycloaddition reaction between azides and cyclooctynes ​​(also known as copper-free click chemistry) and tetrazine ligation.

[0032]

[0037] To be considered bioorthogonal, a reaction must meet several requirements: - Selectivity: the reaction must be selective between endogenous functional groups to avoid side reactions with biological compounds; - Biological inertness: the reaction partners and the resulting bonds must not have any reactivity capable of destroying the natural chemical functionality of the organism under study; - Chemical inertness: the covalent bond must be strong and inert to biological reactions; - Kinetics: Reactions must be rapid, so that covalent ligation is achieved prior to probe metabolism and clearance. Reactions must be rapid on the time scale of cellular processes (minutes) to prevent competing reactions that could diminish the weak signal of low-abundance species. Rapid reactions also provide the rapid response necessary to accurately track dynamic processes; - Reaction biocompatibility: reactions must be non-toxic and must function under biological conditions, taking into account pH, aqueous environment, and temperature. Pharmacokinetics is becoming an increasing concern as bioorthogonal chemistry expands into living animal models; - Accessible engineering: The chemical reporter must be capable of being incorporated into a biomolecule through some form of metabolic or protein engineering. Optimally, one of the functional groups must be small enough not to interfere with its native behavior.

[0033]

[0038] According to the present invention, peptides or proteins can be modified with one or more bioorthogonal functional groups.Peptides or proteins can have either dienes or dienophiles.For example, proteins can be modified by incorporating amino acids with diene moieties, such as tetrazine moieties.

[0034]

[0039] Such amino acid compounds having a tetrazine moiety are represented by the general formula (I):

[0035] [ka]

[0036] (In the formula, X represents N or O; R is halogen, -OR a , -C(O)R a , -COOR a , -NR a R a , -SR a , -C 1~6 selected from the group consisting of alkyl and phenyl; 1~6 The alkyl or phenyl moiety may optionally be halogen, -OR a , -C(O)R a , -COOR a , -NR a R a , -SR a is replaced by R 2 is the amino acid residue that binds to the next residue towards the N-terminus and C-terminus of the protein or peptide; R a is hydrogen or C 1~6 alkyl).

[0037]

[0040] Methods for producing such modified amino acids are described in International Patent Application No. PCT / EP2022 / 064273.

[0041] Preferably, the tetrazine moiety is incorporated into a peptide or protein at a predetermined site. The peptide or protein modified accordingly can contain a single amino acid or multiple amino acids bearing the tetrazine moiety. Having an amino acid bearing a tetrazine moiety at a predetermined site provides the ability to generate precisely defined peptides or proteins.

[0038]

[0042] Some embodiments of the present invention relate to methods for producing peptides or proteins containing single or multiple tetrazine moieties, comprising genetically incorporating synthetic amino acids containing tetrazine moieties into the peptide or protein. Genetic incorporation of tetrazine moieties allows for the precise construction of defined peptide or protein conjugates. The position of the tetrazine moiety can be precisely controlled. This advantage obviates the need to subject the entire peptide or protein to complex reaction steps that utilize chemical functional groups present in natural amino acids.

[0039]

[0043] The described method for producing a peptide or protein suitably comprises: (i) providing a nucleic acid encoding a peptide or protein, the nucleic acid comprising an orthogonal codon encoding an amino acid having a tetrazine moiety; (ii) translating the nucleic acid in the presence of an orthogonal tRNA synthetase / tRNA pair that can recognize the orthogonal codon and incorporate the amino acid having a tetrazine moiety into a peptide or protein chain. Preferably, the orthogonal codon comprises an amber codon (TAG), the tRNA comprises tRNAcuA, and the tRNA synthetase comprises PylRS derived from the organisms Methanosarcina mazei / Methanosarcina Bakeri / Methanomethylophilus alvus.

[0040]

[0044] In other embodiments, the peptide or protein comprises a dienophile moiety. The dienophile moiety may be selected from spirohexene, vinylboronic acid, norbornene, cyclopropene derivatives, cyclooctyne, and trans-cyclooctene (TCO). Preferably, trans-cyclooctene is used as the dienophile.

[0041]

[0045] Correspondingly modified proteins and peptides can be evolved to have affinity for any biological target indicative of disease.

[0046] According to one embodiment of the present invention, the drug is modified with one or more functional groups. The functional group is either a diene or a dienophile, and is complementary to the functional group used in the modified peptide or protein. Therefore, if the peptide or protein is modified with a diene, the drug is modified with a dienophile. If the peptide or protein is modified with a dienophile, the drug is modified with a diene.

[0042]

[0047] The term "drug" refers to an agent capable of treating and / or ameliorating a condition or disease, or one or more symptoms thereof, in a subject. Drugs according to the present disclosure also include prodrug forms of therapeutic agents.

[0043]

[0048] The agent may be selected from the group consisting of cytotoxins, antiproliferative agents, antitumor agents, antivirals, antibiotics, anti-inflammatory agents, chemosensitizers, radiosensitizers, immunosuppressants, immunostimulators, immunomodulators, anti-angiogenic factors, DNA damaging agents, DNA cross-linking agents, DNA binders, DNA alkylating agents, DNA intercalators, DNA cleaving agents, microtubule stabilizing and destabilizing agents, and topoisomerase inhibitors.

[0044]

[0049] Drugs include, for example, colchicine, vinca alkaloids, anthracyclines, doxorubicin, epirubicin, idarubicin, daunorubicin, camptothecin, taxanes, taxol, vinblastine, vincristine, vindesine, calicheamicin, tublysin, tublysin M, cryptophycin, methotrexate, methopterin, aminopterin, dichloromethotrexate, irinotecan, enediyne, amanitin, dactinomycin, duocarmycin, maytansine, maytansino amides, dolastatins, auristatins, pyrrolobenzodiazepines and dimers, indolinobenzodiazepines and dimers, pyridinobenzodiazepines and dimers, mitomycin, melphalan, leurocin, leurosidein, actinomycin, tallysomycin, lexitropsin, bleomycin, podophyllotoxin, etoposide, etoposide phosphate, staurosporine, esperamicin, pteridine drugs, platinum-based drugs, and cytotoxic nucleosides.

[0045]

[0050] Combinations of such modified peptides and proteins with drugs can be used to treat and / or diagnose in a subject a condition or disease suitable for treatment or diagnosis by administration of the modified drug.

[0046]

[0051] The combinations described herein can be used to treat cancer, infectious diseases, or autoimmune diseases.

[0052] In certain embodiments, the combination described herein can be used to treat cancer.Cancer can be melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, head / neck squamous cell carcinoma, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate cancer, nasopharyngeal carcinoma or cutaneous T-cell lymphoma.

[0047]

[0053] Incorporating one or more synthetic amino acids into proteins and peptides active in iEDDA reactions provides a way to address the aforementioned shortcomings of various approaches to targeted drug release for the treatment of various diseases, thereby enabling advances in treatment for a variety of indications. Bypassing conjugate development and directly using proteins or peptides in click-to-release reactions simplifies the development of such therapeutics, relying on established protein purification schemes without the added burden of protein conjugate development. The potency and release activity of targeted proteins or peptides can be easily tuned by incorporating multiple synthetic amino acids, resulting in increased drug release at the target site.

[0048] method: Incorporation of tetrazine-modified synthetic amino acids into proteins:

[0054] Mutant pyrrolysyl-tRNA synthetases derived from wild-type pyrrolysyl-tRNA synthetases, such as pyrrolysyl-tRNA synthetases from Methanosarcina, Methanocaldococcus, Methanomethylophilus, or others, and / or mutant pyrrolysyl-tRNA synthetases aminoacylate pyrrolysyl-tRNA and incorporate modified amino acids, as described herein, into proteins and peptides.

[0049] Incorporation of tetrazine amino acids into nanobody proteins and subsequent active drug release

[0055] The mutant pyrrolysyl-tRNA synthetase is derived from an archaeal pyrrolysyl-tRNA synthetase (e.g., Methanosarcina, Methanocaldococcus, Methanomethylophilus, or others), and is derived from a wild-type pyrrolysyl-tRNA synthetase. The mutant pyrrolysyl-tRNA synthetase aminoacylates pyrrolysyl-tRNA and incorporates an amino acid as described herein. The mutant pyrrolysyl-tRNA synthetase can be generated by cutting-edge protein engineering techniques such as structure-guided site-saturation mutagenesis or directed evolution, or a combination thereof. Other techniques, such as gene shuffling, are also possible.

[0050]

[0056] The mutant pyrrolysyl-tRNA synthetase and the corresponding amber suppressor pyrrolysine-tRNA were introduced into an expression vector carrying the pBR322 origin of replication, a nanobody protein with an in-frame amber stop codon at amino acid position 65, and a C-terminal hexahistidine tag and kanamycin resistance gene. The mutant pyrrolysyl-tRNA synthetase was expressed from an inducible promoter, and the suppressor pyrrolysine-tRNA was expressed from a constitutive promoter commonly used for this purpose.

[0051]

[0057] Escherichia coli (E. coli) cells harboring the above expression vectors were cultured in 250 mL flasks containing 50 mL of M9 minimal medium containing 1–2% glucose as a carbon source or standard 2xYT medium containing 50 μg / mL kanamycin (Roth). The cultures were incubated at 37 °C on an orbital shaker at 160–180 rpm. Expression of PylRS was induced by adding 0.2% (w / v) inducer (Roth) at a D600 of 0.8–1.0. Additionally, 0.1–10 mM tetrazine-lysine was dissolved in 0.1 M HCl, DMSO, HO, or a mixture thereof. Expression was carried out for 4–24 h (temperature was adjustable depending on the target protein; for nanobodies, 37 °C). Cells were harvested by centrifugation (5,000 g, 4 °C, 30 min). Nanobody variants were purified by Ni 2+ affinity chromatography using Ni-NTA agarose according to the manufacturer's instructions.

[0052]

[0058] Purified nanobody variants bearing tetrazine-lysine were contacted in solution with trans-cyclooctene-(TCO)-doxorubicin, a prodrug of doxorubicin, which led to the reaction of TCO-doxorubicin with the tetrazine-lysine in the nanobody, followed by the elimination and release of the active drug doxorubicin.

[0053]

[0059] The presence and release of doxorubicin was confirmed by high performance liquid chromatography coupled to mass spectrometry using appropriate standards. The direct release of doxorubicin on the nanobodies was confirmed by these measurements.

[0054] References [1] D. Hein, Christopher. Liu, Xin-Ming. Wang, D. Click Chemistry, a Powerful Tool for Pharmaceutical Sciences. Natl. Inst. Heal. J. 25, 1-7 (2008). [2] Oneto, J. M. M., Khan, I., Seebald, L. & Royzen, M. In vivo bioorthogonal chemistry enables local hydrogel and systemic pro-drug to treat soft tissue sarcoma. ACS Cent. Sci. 2, 476-482 (2016). [3] Pradipta, A. R. et al. Targeted 1,3-dipolar cycloaddition with acrolein for cancer prodrug activation. Chem. Sci. 12, 5438-5449 (2021). [4] Rossin, R. et al. Chemically triggered drug release from an antibody-drug conjugate leads to potent antitumour activity in mice. Nat. Commun. 9, 1-11 (2018). [5] Fairhal, J. M. et al., EGFR-targeted prodrug activation using bioorthogonal alkene-azide click-and-release chemistry. Bioorg. Med. Chem. 46, 1-11 (2021).

Claims

1. a. a modified peptide or protein comprising one or more bioorthogonal functional groups, and b. A drug modified with one or more bioorthogonal functional groups that are reactive with the bioorthogonal group of (a). A combination of A combination wherein the bioorthogonal functional group of (b) is removed upon contact with the bioorthogonal functional group of (a), releasing the unmodified drug.

2. 2. The combination of claim 1, wherein the bioorthogonal functional group is a dienophile or a diene.

3. 3. The combination of claim 2, wherein the dienophile is a trans-cyclooctene dienophile.

4. 4. The combination of claim 3, wherein the diene is a tetrazine moiety.

5. 2. The combination of claim 1, wherein the modified peptide or protein is selected from the group consisting of antibodies, antibody fragments, diabodies, single chain variable fragment antibodies, single domain antibodies, nanobodies, protein binders, carrier proteins, any peptide or protein with affinity for a human disease target.

6. The modified peptide or protein is of general formula (I) 【Chemical 1】 (In the formula, X represents NH or O; R １ is a halogen, -OR ａ , -C(O)R ａ , -COOR ａ , -NR ａ R ａ , -SR ａ , -C １～６ alkyl and phenyl, １～６ The alkyl or phenyl moiety may optionally be halogen, -OR ａ , -C(O)R ａ , -COOR ａ , -NR ａ R ａ , -SR ａ is replaced by; R ２ is the amino acid residue that binds the next residue towards the N-terminus and C-terminus of the protein or peptide; R ａ is hydrogen or C １～６ 10. The combination of claim 1, wherein the at least one diene moiety is alkyl.

7. 7. The combination of any one of claims 1 to 6, wherein the drug is conjugated to a dienophile moiety.

8. 8. The combination of claim 7, wherein the drug is conjugated to the dienophile moiety via a carbamate moiety.

9. 8. The combination of claim 7, wherein the dienophile moiety is a trans-cyclooctene moiety.

10. 10. The combination of any one of claims 1 to 9, wherein the agent is selected from the group consisting of cytotoxins, antiproliferative agents, antitumor agents, antivirals, antibiotics, anti-inflammatory agents, chemosensitizers, radiosensitizers, immunosuppressants, immunostimulators, immunomodulators, anti-angiogenic factors, DNA damaging agents, DNA cross-linking agents, DNA binders, DNA alkylating agents, DNA intercalators, DNA cleaving agents, microtubule stabilizing and destabilizing agents, and topoisomerase inhibitors.

11. Drugs include colchicine, vinca alkaloids, anthracyclines, doxorubicin, epirubicin, idarubicin, daunorubicin, camptothecin, taxanes, taxol, vinblastine, vincristine, vindesine, calicheamicin, tubulysin, tubulysin M, cryptophycin, methotrexate, methopterin, aminopterin, dichloromethotrexate, irinotecan, enediyne, amanitin, dactinomycin, duocarmycin, maytansine, maytansinoids, and dolastatins. , auristatins, pyrrolobenzodiazepines and dimers, indolinobenzodiazepines and dimers, pyridinobenzodiazepines and dimers, mitomycin, melphalan, leurosin, leurosidein, actinomycin, tallysomycin, lexitropsin, bleomycin, podophyllotoxin, etoposide, etoposide phosphate, staurosporine, esperamicin, pteridine drugs, platinum-based drugs, and cytotoxic nucleosides.

12. A combination according to any one of claims 1 to 11 for use in the treatment of cancer, an infectious disease or an autoimmune disease.

13. 13. The combination for use according to claim 12, wherein the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, squamous cell carcinoma of the head / neck, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate cancer, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.

14. 14. The combination for use according to claim 12 or 13, wherein the modified peptide or protein and the agent are administered sequentially or simultaneously.

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