Click release proteins and peptides

Abstract

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

Description

Technical Field

[0001] The present invention relates to combinations of modified drugs with peptides or proteins comprising one or more bioorthogonal functional groups for bioorthogonal delivery of drugs in a subject in need thereof. Background Art

[0002] Click chemistry has become a versatile tool for in vivo chemistry: "Click chemistry refers to a set of reactions that are fast, easy to use, easy to purify, versatile, regioselective, and high-yielding" [1]; thus, these reactions are ideal tools for in vivo targeting and labeling of biological targets. The inverse-electron demand Diels-Alder (IEDDA) reaction between a diene tetrazine and a dienophile exhibits excellent kinetics (1 - 10 6 M -1 s -1 ), and due to its speed and the metabolic stability of some of the developed reactants, it is particularly suitable for such in vivo applications.

[0003] Due to its speed and specificity, this reaction has been applied to the targeted release of caged prodrugs. In this approach, a bioactive molecule is surrounded (modified) by a chemical group with iEDDA chemical activity. This modification alters (reduces) the biological activity of the drug and thus also its systemic side effects. Then, the caged prodrug is targeted to the biological target where the drug should be active in a living organism. After successful targeting, the caged prodrug is administered and reacts with the iEDDA-active counterpart already present in the living organism, and its full biological activity is released only when reacting upon elimination of the caging group.

[0004] Current literature describes various methods for labeling biological targets with iEDDA-reactive moieties, such as using local injection [2].

[0005] This method involves directly injecting an iEDDA-reactive polymer into tumor tissue. After the polymer binds to the tumor, an iEDDA-reactive prodrug is administered, which is released upon contact with the polymer bound to the tumor. This achieves a high local concentration of the active drug while reducing systemic toxicity (see Figure 2 ). The drawback of this method is that the process is invasive and requires precise localization of the tumor before administering the polymer.

[0006] Another approach uses endogenous chemical reactivity markers [3]. Some diseases produce acrolein as a byproduct of their metabolism through oxidative stress. Acrolein is a compound with 1,3-dipolar cycloaddition reactivity, a reaction that can also eliminate chemical groups and subsequently release the drug from the prodrug. This means that the prodrug is preferentially activated at the disease site where oxidative stress occurs (see Figure 3 ). Although this approach is ingenious, it also has drawbacks due to disease diversity. For this potential treatment to be appropriately effective, it needs to be administered to disease types with very high local acrolein concentrations, which requires prior analysis and determination. Due to the metabolic diversity of disease types, there will inevitably be some patient subgroups that do not respond.

[0007] In addition, protein conjugates [4] can be used. This approach uses proteins modified with iEDDA reactive moieties. The protein has an affinity for surface markers of the biological target to be treated with the drug. The protein conjugate is administered and allowed to bind to the target. After an appropriate target labeling time, treatment is completed by tracking the IEDDa caged prodrug. When in contact with the pre-labeled target, the prodrug reacts and releases the active drug (see Figure 4 ).

[0008] The drawback of this method is that the development of protein conjugates is usually cumbersome and requires a large amount of resources to develop suitable supply chains and analytical methods to verify the quality of drug products. The binding of the iEDDA reactive moiety needs to be site-specific to produce reproducible drug molecules, and it is not always possible to obtain this technology.

[0009] WO2014081301A1 discloses a combination of a masking moiety linked to a triggering moiety, which is further linked to a drug. The triggering moiety includes a dienophile, and the activator includes a diene. The triggering moiety and the activator undergo a rapid bioorthogonal reaction to release and activate the masking moiety and the drug. WO2017044983A1 describes a bioorthogonal composition for delivering a pharmaceutical agent in a subject. The bioorthogonal composition includes a hydrogel carrier composition having different bioorthogonal functional groups. WO2022032191A1 discloses trans-cyclooctene bioorthogonal agents and their use in cancer and immunotherapy.

[0010] Fairhall J.M. et al. disclose the conjugation of a functionalized trans-cyclooctene with cetuximab, providing a reagent for pre-targeting and localization of bioorthogonal reagents [5].

[0011] Disadvantages of existing technology conjugates are that the development of protein conjugates is generally cumbersome and requires significant resources to develop suitable supply chains and analytical methods to verify the quality of drug products. The conjugation of iEDDA reactive moieties needs to be site-specific to produce reproducible drug molecules, and it is not always possible to obtain such technology.

[0012] Accordingly, there remains a need for new methods of treating diseases based on modified entities bearing bioorthogonal functional groups. Summary of the Invention

[0013] The object of the present invention is to provide a new method of treating diseases based on modified entities bearing bioorthogonal functional groups. This object is solved by the subject matter of the present invention. The new method is based on a 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 functional groups of the protein or peptide.

[0014] The present invention relates to a 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 functional groups of (i).

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

[0016] According to one embodiment of the present invention, the modified peptide or protein is selected from antibodies, antibody fragments, bispecific antibodies, single-chain variable fragment antibodies, single-domain antibodies, nanobodies, small protein conjugates, carrier proteins, any peptide or protein having an affinity for a human disease target.

[0017] "Antibody fragment" comprises a part of a complete antibody, including the antigen-binding region and / or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; linear antibodies; single-chain antibody molecules; multivalent single-domain antibodies; multispecific antibodies formed by antibody fragments.

[0018] According to one embodiment of the present invention, the modified peptide or protein bears at least one diene moiety of general formula (I),

[0019]

[0020] wherein,

[0021] X represents NH or O,

[0022] R is selected from halogen, -OR a , -C(O)R a , -COOR a , NRa R a 、 -SR a 、 -C 1-6 alkyl and phenyl, wherein -C 1-6 the alkyl or phenyl moiety is optionally substituted by halogen, -OR a 、 -C(O)R a 、 -COOR a 、 -NR a R a 、 -SR a substituted,

[0023] R 2 is an amino acid residue and is linked to the next residue at the N-terminus and C-terminus of a protein or peptide;

[0024] R a is hydrogen or C 1-6 alkyl.

[0025] According to one embodiment of the present invention, the drug can be conjugated to a dienophile moiety. The drug can be conjugated to the dienophile moiety through a carbamate moiety.

[0026] Another embodiment relates to a combination as described herein, wherein the dienophile moiety is a trans-cyclooctene moiety.

[0027] Another embodiment relates to a combination as described herein, wherein the drug is selected from cytotoxins, anti-proliferatives, anti-tumor agents, anti-viral agents, antibiotics, anti-inflammatory agents, chemosensitizers, radiosensitizers, immunosuppressants, immunostimulants, immunomodulators, anti-angiogenic factors, DNA damaging agents, DNA cross-linking agents, DNA binding agents, DNA alkylating agents, DNA intercalating agents, DNA cleaving agents, microtubule stabilizers and destabilizers, and topoisomerase inhibitors.

[0028] The drug can be selected from colchicine, vinca alkaloids, anthracyclines, doxorubicin, epirubicin, idarubicin, daunorubicin, camptothecins, taxanes, taxols, vinblastine, vincristine, vindesine, calicheamicins, tubulysins, tubulysin M, cryptophycins, methotrexate, amethopterin, aminopterin, dichloromethotrexate, irinotecans, enediynes, amanitins, dactinomycines, duocarmycins, maytansines, maytansinoids, dolastatins, auristatins, pyrrolobenzodiazepines and dimers, indolinobenzodiazepines and dimers, pyridinobenzodiazepines and dimers, mitomycins, melphalan, vinblastine oxide, vinorelbine, actinomycin, talipexole, lexitropsins, bleomycins, podophyllotoxins, etoposide, etoposide phosphate, staurosporine, esperamicin, pteridine drugs, platinum drugs, and cytotoxic nucleosides.

[0029] One embodiment of the present invention relates to the use of the combination as described herein for the treatment of cancer, infectious diseases or autoimmune diseases.

[0030] Another embodiment relates to the combination as described herein, wherein the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, head and neck squamous cell carcinoma, anal / vulvar cancer, esophageal cancer, pancreatic cancer, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Wilms tumor, neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal carcinoma or cutaneous T-cell lymphoma.

[0031] Another embodiment relates to the combination as described herein, wherein the modified peptide or protein and the drug are administered sequentially or concomitantly. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : The iEDDA reaction of a tetrazine with a dienophile.

[0033] Figure 2 : Drug release by pre-targeting with an iEDDA-reactive polymer.

[0034] Figure 3 : Using acrolein as a reaction partner for in vivo drug release.

[0035] Figure 4 : Using protein conjugates for click-release chemistry of highly active toxins in cancer therapy. Detailed implementation mode

[0036] The present invention relates to a 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 functional groups of (i).

[0037] In the field of chemistry, the Diels-Alder reaction is an important reaction in which a conjugated diene reacts with a dienophile (substituted alkene) to produce a substituted cyclohexene derivative. This reaction has two reacting partners: the diene and the dienophile.

[0038] A diene is an unsaturated hydrocarbon containing a double bond between two carbon atoms. A diene is also called a diolefin or an alkadiene. It is a covalent compound containing two alkene units. Dienes usually exist as subunits of more complex organic molecules. In addition, dienes are present in natural compounds and also in synthetic chemicals. These chemicals are useful in organic synthesis reactions.

[0039] A dienophile is an organic compound that readily reacts with a diene. Dienophiles are usually used in the Diels-Alder reaction, which involves the reaction between a conjugated diene and a substituted alkene, where the substituted alkene acts as the dienophile.

[0040] Suitable dienophiles usually carry one or two of the following functional groups: CHO, COR, COOR, CN, C═C, Ph, or halogen. In addition, the diene must be electron-rich.

[0041] Alkenes are usually referred to as dienophiles because they readily react with dienes. Generally, heating is not required in the Diels-Alder reaction, but heating can increase the yield of the reaction.

[0042] 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 enables the real-time study of biomolecules, such as glycans, proteins, and lipids, in living systems without cytotoxicity. Many chemical ligation strategies that meet the requirements of bioorthogonality have been developed, including the 1,3-dipolar cycloaddition between azides and cyclooctenes (also known as copper-free click chemistry) and tetrazine ligation.

[0043] A reaction must meet many requirements to be considered a bioorthogonal reaction:

[0044] - selective : The reaction must be selective between endogenous functional groups to avoid side reactions with biological compounds;

[0045] - biologically inert : The reactive partners and the resulting linkage should not have any reaction patterns that can disrupt the natural chemical functions of the biological being studied;

[0046] - chemically inert : The covalent bond should be strong and inert to biological reactions;

[0047] - kinetics : The reaction must be fast to achieve covalent linkage before probe metabolism and clearance. The reaction must be fast on the time scale of cellular processes (minutes) to prevent competition from reactions that may attenuate small signals from smaller amounts of material. A fast reaction also provides a rapid response, which is necessary for accurately tracking dynamic processes;

[0048] - reactive biocompatibility : The reaction must be non-toxic and must function under biological conditions considering pH, aqueous environment, and temperature. As bioorthogonal chemistry expands to in vivo animal models, pharmacokinetics is increasingly of concern;

[0049] - accessible engineering : The chemical reporter molecule must be able to be incorporated into biomolecules through some form of metabolism or protein engineering. Ideally, one of the functional groups is also very small and thus does not interfere with natural behavior.

[0050] According to the present invention, a peptide or protein can be modified with one or more bioorthogonal functional groups. The peptide or protein can carry a diene or a dienophile. For example, a protein can be modified by incorporating a diene moiety, such as an amino acid bearing a tetrazine moiety.

[0051] Such an amino acid compound bearing a tetrazine moiety has the general formula (I),

[0052]

[0053] Wherein,

[0054] X represents N or O,

[0055] R is selected from halogen, -OR a , -C(O)R a , -COOR a , NR a R a , -SR a , -C 1-6 alkyl and phenyl, wherein the -C 1-6 alkyl or phenyl moiety is optionally substituted by halogen, -OR a , -C(O)R a , -COOR a , -NR a R a , -SR a ;

[0056] R 2 is an amino acid residue and is linked to the next residue at the N-terminus and C-terminus of a protein or peptide;

[0057] R a is hydrogen or C 1-6 alkyl.

[0058] A method for producing such modified amino acids is described in International Patent Application PCT / EP2022 / 064273.

[0059] Preferably, the tetrazine moiety is attached to a pre-determined site on the peptide or protein. The correspondingly modified peptide or protein may contain a single or multiple amino acids bearing the tetrazine moiety. Having an amino acid bearing the tetrazine moiety at a pre-determined site provides the ability to produce a precisely defined peptide or protein.

[0060] Some embodiments of the present invention relate to methods for producing peptides or proteins comprising a single or multiple tetrazine moieties, said methods comprising genetically incorporating a synthetic amino acid comprising a tetrazine moiety into the peptide or protein. Genetically incorporating the tetrazine moiety allows for the precise construction of defined peptide or protein conjugates. The position of the tetrazine moiety can be precisely controlled. This advantageously avoids the need for complex reactions on the entire peptide or protein using chemical functional groups present in natural amino acids.

[0061] Suitably, the method for producing a peptide or protein comprises:

[0062] (i) providing a nucleic acid encoding a peptide or protein, said nucleic acid comprising an orthogonal codon encoding an amino acid having a tetrazine moiety;

[0063] (ii) Translate 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. Suitably, the orthogonal codon comprises the amber codon (TAG), the tRNA comprises tRNAcuA, and the tRNA synthetase comprises PylRS from Methanosarcina mazei / Methanosarcina Bakeri / Methanomethylophilus alvus.

[0064] In other embodiments, the peptide or protein comprises a dienophile moiety. The dienophile moiety can be selected from spihexene, vinyl boronic acid, norbornene, cyclopropene derivatives, cyclooctene, and trans-cyclooctene (TCO). Preferably, trans-cyclooctene is used as the dienophile.

[0065] The correspondingly modified proteins and peptides can be evolved to have an affinity for a biological target of any labeled disease.

[0066] According to one embodiment of the present invention, the drug is modified with one or more functional groups. The functional group is a diene or a dienophile and is complementary to the functional group used in modifying the peptide or protein. Thus, in the case where 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, then the drug is modified with a diene.

[0067] The term "drug" refers to an agent that can treat and / or ameliorate a disorder or disease or one or more symptoms thereof in a subject. The drugs of the present disclosure also include prodrug forms of therapeutic agents.

[0068] The drug can be selected from cytotoxins, anti-proliferatives, anti-tumor agents, antiviral agents, antibiotics, anti-inflammatory agents, chemosensitizers, radiosensitizers, immunosuppressants, immunostimulants, immunomodulators, anti-angiogenic factors, DNA-damaging agents, DNA cross-linking agents, DNA-binding agents, DNA alkylating agents, DNA intercalating agents, DNA cleaving agents, microtubule stabilizers and destabilizers, and topoisomerase inhibitors.

[0069] For example, the drug is selected from colchicine, vinca alkaloids, anthracyclines, doxorubicin, epirubicin, idarubicin, daunorubicin, camptothecins, taxanes, taxols, vinblastine, vincristine, vindesine, calicheamicins, tubulysins, tubulysin M, cryptophycins, methotrexate, amethopterin, aminopterin, dichloromethotrexate, irinotecans, enediynes, amanitins, dactinomycines, duocarmycins, maytansines, maytansinoids, dolastatins, auristatins, pyrrolobenzodiazepines and dimers, indolinobenzodiazepines and dimers, pyridinobenzodiazepines and dimers, mitomycins, melphalan, vinrosidine, vinorelbine, actinomycin, telithromycin, lexitropsins, bleomycins, podophyllotoxins, etoposide, etoposide phosphate, staurosporine, esperamicin, pteridine drugs, platinum drugs, and cytotoxic nucleosides.

[0070] The combination of a correspondingly modified peptide and protein with a drug can be used to treat and / or diagnose a disorder or disease in a subject, which can be treated or diagnosed by administering the modified drug.

[0071] The combinations described herein can be used to treat cancer, infectious diseases or autoimmune diseases.

[0072] In certain embodiments, the combinations described herein can be used to treat cancer. The cancer can be melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, head and neck squamous cell carcinoma, anal / vulvar cancer, esophageal cancer, pancreatic cancer, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, nephroblastoma, neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal carcinoma or cutaneous T-cell lymphoma.

[0073] By incorporating one or more synthetic amino acids into proteins and peptides with iEDDA reactivity, we provide a method that addresses the drawbacks of various methods for targeted drug release for treating various diseases. This enables us to develop therapeutic methods for various indications. By avoiding the development of conjugates and directly using proteins or peptides in click release reactions, we simplify the development of this therapy, relying on established protein purification protocols without the additional burden of developing protein conjugates. The efficacy and release activity of the targeted protein or peptide can be easily regulated by incorporating multiple synthetic amino acids, which in turn releases more drugs at the targeted site.

[0074] Method:

[0075] Incorporate tetrazine-modified synthetic amino acids into proteins:

[0076] A mutant pyrrolysyl-tRNA synthetase obtained from wild-type pyrrolysyl-tRNA synthetase, which is a pyrrolysyl-tRNA synthetase from Methanosarcina, Methanocaldococcus, Methanomethylophilus or other derived pyrrolysyl-tRNA synthetases, and / or the mutant pyrrolysyl-tRNA synthetase aminoacylates pyrrolysyl-tRNA to incorporate the modified amino acids described herein into proteins and peptides.

[0077] Incorporate tetrazine amino acids into nanobody proteins and subsequently release active drugs

[0078] A mutant pyrrolysyl-tRNA synthetase obtained from wild-type pyrrolysyl-tRNA synthetase, which is a pyrrolysyl-tRNA synthetase derived from archaea (such as Methanosarcina, or Methanocaldococcus, or Methanomethylophilus, or others), and / or the mutant pyrrolysyl-tRNA synthetase aminoacylates pyrrolysyl-tRNA to incorporate the amino acids described herein. The mutant pyrrolysyl-tRNA synthetase is generated by existing protein engineering techniques, such as structure-guided site saturation mutagenesis or directed evolution or a combination thereof. In addition, other techniques, such as gene shuffling, can also be employed.

[0079] Introduce the mutant pyrrolysine tRNA synthetase and the corresponding amber suppressor pyrrolysine tRNA into an expression vector that contains the pBR322 replication origin, a nanobody protein carrying an in-frame amber stop codon at the 65th amino acid, as well as a C-terminal hexahistidine tag and a kanamycin resistance gene. The mutant pyrrolysine tRNA synthetase is expressed by an inducible promoter, while the suppressor pyrrolysine tRNA is expressed by a constitutive promoter commonly used for this purpose.

[0080] Escherichia coli cells containing the above expression vector were cultured in 250 mL flasks, with each flask 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 a speed of 160 - 180 rpm. When D600 reached 0.8 - 1.0, the expression of PylRS was induced by adding 0.2% (w / v) inducer (Roth). In addition, 0.1 - 10 mM of tetrazine - lysine was dissolved in 0.1 M HCl or DMSO or H 2 2O or a mixture thereof. The expression was carried out for 4 - 24 hours (the temperature can be adjusted according to the target protein; for nanobodies, it was 37 °C). The cells were harvested by centrifugation (5000 g, 30 minutes at 4 °C). The nanobody variants were purified by Ni2+-affinity chromatography using Ni-NTA agarose according to the manufacturer's instructions.

[0081] The purified nanobody variant carrying tetrazine - lysine was contacted with trans - cyclooctene - (TCO)-doxorubicin (a prodrug of doxorubicin) in solution. This led to the reaction of TCO - doxorubicin with the tetrazine - lysine in the nanobody, followed by the release of the active drug doxorubicin due to elimination.

[0082] The presence and release of doxorubicin were confirmed by high - performance liquid chromatography coupled with a mass spectrometer using appropriate standards. These measurements confirmed the direct release of doxorubicin onto the nanobody.

[0083] References

[0084] [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).

[0085] [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).

[0086] [3]Pradipta, A. R. et al. Targeted 1,3-dipolar cycloaddition with acrolein for cancer prodrug activation. Chem. Sci. 12, 5438–5449 (2021).

[0087] [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).

[0088] [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. The combination of a and b: 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 react with the bioorthogonal functional groups of (a); wherein when the bioorthogonal functional groups of (b) come into contact with the bioorthogonal functional groups of (a), the bioorthogonal functional groups of (b) are eliminated, such that the unmodified drug is released.

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

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

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

5. The combination according to claim 1, wherein the modified peptide or protein is selected from antibodies, antibody fragments, bispecific antibodies, single-chain variable fragment antibodies, single-domain antibodies, nanobodies, protein conjugates, carrier proteins, any peptide or protein having an affinity for a human disease target.

6. The combination according to claim 1, wherein the modified peptide or protein bears at least one diene moiety of general formula (I), wherein, X represents NH or O, R 1 selected from halogen, -OR a , -C(O)R a , -COOR a , NR a R a , -SR a , -C 1-6 alkyl and phenyl, wherein the -C 1-6 alkyl or phenyl moiety is optionally substituted by halogen, -OR a , -C(O)R a , -COOR a , -NR a R a , -SR a ; R 2 is the amino acid residue linked to the next residue at the N-terminus and C-terminus of a protein or peptide; R a is hydrogen or C 1-6 alkyl group.

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

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

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

10. The combination according to any one of claims 1-9, wherein the drug is selected from cytotoxins, anti-proliferatives, anti-tumor agents, anti-viral agents, antibiotics, anti-inflammatory agents, chemosensitizers, radiosensitizers, immunosuppressants, immunostimulants, immunomodulators, anti-angiogenic factors, DNA-damaging agents, DNA cross-linking agents, DNA-binding agents, DNA-alkylating agents, DNA-intercalating agents, DNA-cleaving agents, microtubule stabilizers and destabilizers, and topoisomerase inhibitors.

11. The combination according to claim 10, wherein the drug is selected from colchicine, vinca alkaloids, anthracyclines, doxorubicin, epirubicin, idarubicin, daunorubicin, camptothecins, taxanes, paclitaxel, vinblastine, vincristine, vindesine, calicheamicins, tubulysins, tubulysin M, nostocarphins, methotrexate, amethopterin, aminopterin, dichloromethotrexate, irinotecans, enediynes, amanitins, dactinomycins, duocarmycins, maytansines, maytanols, dolostatins, auristatins, pyrrolobenzodiazepines and dimers, indolobenzodiazepines and dimers, pyridinobenzodiazepines and dimers, mitomycins, melphalan, vinrosidine, vinorelbine, actinomycin, talipexole, distamycins, bleomycins, podophyllotoxins, etoposide, etoposide phosphate, staurosporine, esperamicin, pteridine drugs, platinum drugs, and cytotoxic nucleosides.

12. The combination according to any one of claims 1-11 is used for treating cancer, infectious diseases or autoimmune diseases.

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

14. The combination according to claim 12 or 13, wherein the modified peptide or protein and the drug are administered sequentially or concomitantly.

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