Cuttable immune cell adapter

Abstract

The present invention relates to antibody-cytokine conjugates, wherein the cytokine is conjugated to the antibody via a cleavable linker. Conjugates according to the invention can be prepared by coupling a functionalized antibody Ab (F) x comprising x reactive moieties F, where x is an integer in the range of 1-10, with an immune cell-binding polypeptide comprising one or two reactive moieties Q, where the antibody is specific for tumor cells, and the immune cell-binding polypeptide is specific for immune cells, wherein the reaction forms a covalent bond between the functionalized antibody and the immune cell-binding polypeptide by reaction of Q with F. The invention further relates to antibody-cytokine conjugates obtainable by the method according to the invention and to medical uses thereof.

Description

Technical Field

[0001] This invention relates to immune cell conjugates generated from antibodies and other peptides. More particularly, this invention relates to conjugates, compositions, and methods suitable for attaching target immune cell-binding peptides to antibodies via cleavable linkers without requiring prior genetic engineering of the antibodies. The resulting antibody-immune cell conjugate conjugates, as compounds, compositions, and methods, can be used, for example, in immunotherapy for cancer patients. Background Technology

[0002] Antibody-drug conjugates (ADCs) are considered a panacea in therapy, consisting of an antibody and a drug formulation attached to it. Antibodies (also called ligands) can be in the form of small proteins (scFv, Fab fragments, DARPin, affinity molecules, etc.), but are typically monoclonal antibodies (mAbs). They are selected based on their high selectivity and affinity for a given antigen, their long circulating half-life, and minimal immunogenicity. Therefore, mAbs, as carefully selected protein ligands for biological receptors, provide an ideal delivery platform for the selective targeting of drugs. For example, monoclonal antibodies known to selectively bind to specific cancer-associated antigens can be used to deliver chemically conjugated cytotoxic agents to tumors via binding, internalization, intracellular processing, and ultimately the release of active catabolites. The cytotoxic agents can be small molecule toxins, protein toxins, or other forms (such as oligonucleotides). Thus, tumor cells can be selectively destroyed while normal cells untargeted by the antibody are preserved. Similarly, the chemical conjugation of antibacterial drugs (antibiotics) with antibodies can be used to treat bacterial infections, while conjugates of anti-inflammatory drugs are being investigated for the treatment of autoimmune diseases. For example, attaching oligonucleotides to antibodies is a promising approach for treating neuromuscular diseases. Therefore, the concept of targeted delivery of active drugs to selected specific cellular sites is a powerful approach to treating a variety of diseases, offering many advantages over systemic delivery of the same drug.

[0003] An alternative strategy for targeting and delivering specific protein agents using monoclonal antibodies is to fuse the latter protein with one or more genes at the end of the antibody, which can be an N-terminus or C-terminus on the light or heavy chain (or both). In this case, the target bioactive protein (e.g., a protein toxin such as Pseudomonas exotoxin A (PE38) or an anti-CD3 single-chain variable fragment (scFv)) is genetically encoded as a fusion with the antibody (possibly but not necessarily via a peptide spacer), and thus the antibody is expressed as the fusion protein. The peptide spacer may or may not contain a protease-sensitive cleavage site.

[0004] Monoclonal antibodies can also be genetically modified in their protein sequences to alter their structure, thereby introducing (or removing) specific properties. For example, mutations can be made in the Fc-fragment of an antibody to disable its binding to the Fc-γ receptor, binding to the FcRn receptor or to specific cancer targets can be modulated, or the antibody can be engineered to reduce pI and control clearance in circulation.

[0005] Emerging strategies in therapeutic treatment involve the use of antibodies that can bind to multiple antigens or epitopes simultaneously, namely so-called bispecific antibodies (targeting two different antigens or epitopes at the same time) or trispecific antibodies (targeting three different antigens or epitopes), such as those developed by Kontermann and Brinkmann. Drug Discov. Today [Today's Drug Discovery] 2015, 20 The findings summarized in 838–847 are incorporated herein by reference. Bispecific antibodies with 'dual-target' functionality can interfere with multiple surface receptors or ligands, for example, associated with cancer, proliferation, or inflammatory processes. Bispecific antibodies can also bring multiple targets close together to support the formation of protein complexes on a single cell or trigger cell-to-cell contact. Examples of 'forced-connection' functionality include bispecific antibodies that support protein complexation in coagulation cascades or tumor-targeting immune cell recruiters and / or activators. Depending on the method of production and structure, bispecific antibodies vary in the number of antigen-binding sites, their geometry, serum half-life, and effector functions.

[0006] Over the years, various forms of multispecific antibodies have been developed, which can be broadly categorized into IgG-like (with Fc fragments) and non-IgG-like (lacking Fc fragments) forms, such as those developed by Kontermann and Brinkmann. Drug Discov. Today [Today's Drug Discovery] 2015, 20 , 838–847 and Yu and Wang, J. Cancer Res. Clin. Oncol. [Journal of Cancer Research and Clinical Oncology] 2019, 145 The literature summarized in 941–956 is incorporated by reference. Most bispecific antibodies are generated through one of three methods: somatic cell fusion of two hybridoma lines (quadroma), genetic (protein / cell) engineering, or chemical conjugation with a cross-linking agent; there are now more than 60 different technology platforms in total.

[0007] IgG-like forms based on the complete IgG molecular architecture include, but are not limited to, IgG with dual variable domains (DVD-Ig), Duobody technology, kilobody-in-the-mouth (KIH) technology, common light chain technologies, and cross-linked mAb technologies, while truncated IgG forms include ADAPTIR, XmAb, and BEAT technologies. Non-IgG-like methods include, but are not limited to, BITE, DART, TandAb, and ImmTAC technologies. Bispecific antibodies can also be generated by fusing different antigen-binding moieties (e.g., scFv or Fab) with other protein domains, enabling the inclusion of additional functions. For example, two scFv fragments have been fused with albumin, which endows the antibody fragment with a long circulation time of serum albumin, as described by Müller et al. J. Biol. Chem. [Journal of Biochemistry] 2007, 282 As demonstrated in 12650–12660, this literature is incorporated by reference. Another example is the 'dock-and-lock' approach based on the heterodimerization of cAMP-dependent protein kinase A and protein A kinase-anchored proteins, as demonstrated by Rossi et al. Proc. Nat. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 2006, 103 The literature reported in 6841–6846 is incorporated herein by reference. These domains can be linked to Fab fragments and the entire antibody to form multivalent bispecific antibodies, as described by Rossi et al. Bioconj.Chem. [Chemistry of Bioconjugates] 2012, 23 As shown in 309–323, docking and locking strategies require the creation of fusion proteins between the targeting antibody and the peptide fragment to dock with protein A kinase-anchored proteins. Therapeutic Ab fragments (scFv, biantibodies) can also be fused with albumin or albumin-binding proteins, which increases the drug's half-life in the bloodstream by up to five to six times. The construction of such molecules produces unpredictable results, thus limiting the application of bispecific antibodies generated from different Ab-fragment fusions or Ab binding to other proteins in the research and development of new therapeutic molecules.

[0008] Brennan et al. Science [Science] 1985, 229 Articles 81–83 first used chemical conjugation to generate non-IgG bispecific antibodies, which are cited in this paper: two Fab2 fragments obtained by dissolving rabbit IgG with pepsin were reduced and then oxidized to produce bispecific Fab2. Similarly, Glennie et al. (1987)... 139Articles 2367–2375 report isobifunctional and heterobifunctional reagents that interact with cysteine ​​residues; this literature is incorporated by reference. T cells with bispecific antibody-coated surfaces are obtained using chemical conjugation of Abs targeting CD3 and CD20 (rituximab), as described by Gall et al. Exp. Hematol. [Experimental Hematology] 2005, 33 As shown in 452–459, this literature is incorporated by reference. The bispecific CD20 × CD3 generation was ensured by treating OKT3 (anti-CD3) with Traut's reagent, followed by mixing with maleimide-functionalized rituximab (obtained by pretreatment of rituximab with 4-(N-maleimide-methyl)cyclohexane-1-carboxylic acid sulfosuccinimide (sulfon-SMCC)). Through random chemical conjugation of the two antibodies, followed by random heterodimerization, the bispecific antibody was inevitably obtained as a highly heterogeneous mixture (also containing multimers). The only reported, and site-specific, chemical method to date is the CovX-Body technique, as described by Doppalapudi et al. Bioorg. Med. Chem. Lett. [Letters in Bioorganic and Medicinal Chemistry] 2007, 17 The technique reported in 501–506 (incorporated by reference) is based on the insertion of an alcohol catalyzed antibody site into a targeting antibody, followed by treatment with a peptide fragment chemically modified by an azacyclic butane ketone motif, resulting in spontaneous linking. By adding two short peptides that inhibit VEGF or angiopoietin 2, along with a branched linker, and then adding an antibody, bispecific antibodies were generated, as described by Doppalapudi et al. Proc. Nat. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 2010, 107 The references 22611–22616 reported are incorporated by reference.

[0009] Bispecific antibody generation based on chemically conjugated Abs or Ab fragments is currently not used, primarily due to low yields of products (low purity) and high cargo costs. Furthermore, advances in recombinant DNA technology have enabled the efficient generation of fusion proteins, leading to positive clinical outcomes. In any case, non-genetically modified chemical methods can significantly accelerate time to clinical application, provided appropriate control over stoichiometry and site specificity can be ensured.

[0010] Examples of bispecific antibodies that are already in or currently in clinical development include catumaxomab (EpCAM × CD3), blinatumomab (CD19 × CD3), GBR1302 (Her2 × CD3), MEDI-565 (CEA × CD3), BAY2010112 (PSMA × CD3), RG7221 (angiopoietin × VEGF), RG6013 (FIX × FX), RG7597 (Her1 × Her3), MCLA128 (Her2 × Her3), MM111 (Her2 × Her3), MM141 (IGF1R × Her3), ABT122 (TNFα × IL17), ABT981 (IL1a × IL1b), ALX0761 (IL17A × IL17F), SAR156597 (IL4 × IL13), and AFM13 (CD30 × IL17). CD16) and LY3164530 (Her1 × cMET).

[0011] A popular strategy in cancer therapy employs bispecific antibodies that bind to both upregulated tumor-associated antigens (TAAs, or simply targets) and receptors present on immune cells that destroy cancer (e.g., T cells or NK cells). Such bispecific antibodies are also known as T-cell or NK-cell redirected antibodies, respectively. Although immune cell redirection methods have been around for over 30 years, new technologies continue to overcome the limitations of first-generation immune cell redirected antibodies, particularly by extending the half-life to allow for intermittent dosing, reducing immunogenicity, and improving the safety profile. Currently, one approved drug (bonatumab or Blincyto)... ® It is available in over 30 other bispecific forms at various stages of clinical development. The basis for bonatetumab's approval (2014) stemmed from a single-arm trial that achieved a 32% complete response rate and a minimal residual disease (MRD) response (31%) in all treated patients. Currently, 51 clinical trials of bonatetumab are underway for ALL (39 trials), NHL (10 trials), multiple myeloma (1 trial), and lymphoma with Richter transformation (1 trial). However, bonatetumab has a major drawback due to its short serum half-life (2.11 h, due to its relatively small molecule and simple structure) and the need for continuous intravenous infusion.

[0012] As with other treatments for serious diseases, therapeutic bispecific antibodies (BsAbs) cause a variety of side effects, the most common of which are nausea, vomiting, abdominal pain, fatigue, leukopenia, neutropenia, and thrombocytopenia. In many patients, the Ab against the therapeutic bispecific antibody appears in the bloodstream during treatment. Most adverse events occur during the start of therapy, and in most cases, side effects normalize with continued treatment. Most data on the adverse effects of therapeutic BsAbs are available for bonnetumab and caputozumab, as these drugs have undergone numerous clinical trials. Common side effects of bonnetumab and caputozumab therapy are “cytokine storm,” elevated cytokine levels, and several neurological events. Cytokine release-related symptoms are a common side effect of many therapeutic mAbs and occur due to a specific mechanism of action: the use of cytotoxic T cells as effectors. Using a low initial dose of the drug in combination with subsequent high doses, and pre-treatment with corticosteroids (dexamethasone) and antihistamines, may minimize cytokine release syndrome.

[0013] One approach to mitigating adverse events associated with immune cell conjugation therapy (particularly cytokine release syndrome) and avoiding the use of escalating dosing regimens was proposed by Bacac et al. Clin. Cancer Res. [Clinical Cancer Research] 2018, 24 Reports 4785–4797, which are incorporated by reference, demonstrate that significantly higher potency and safer administration can be achieved by generating a CD20 × CD3 T-cell conjugate in a 2:1 molecular form (i.e., bivalently binding to CD20 and monovalently binding to CD3), achieved by inserting an anti-CD3 fragment into one of the Fab arms of a fully IgG anti-CD20 antibody. The resulting bispecific antibody is associated with a long half-life and high potency, achieved through high-affinity bivalent binding to CD20 and head-to-tail orientation of the B-cell and T-cell binding domains in a 2:1 molecular form. A long circulating half-life is achieved by incorporating the Fc region of a heterodimer human IgG1 carrying the “PG LALA” mutation to eliminate binding to the Fcg receptor and complement component C1q while maintaining neonatal Fc receptor (FcRn) binding. The bispecific CD20-T cell binder has demonstrated significantly higher potency than other CD20-TCB antibodies in clinical development and is effective against tumor cells expressing low levels of CD20. CD20-TCB also exhibits potent activity in primary tumor samples with low effector:target ratios.

[0014] To date, the most studied receptors for T-cell conjugation purposes include the CD3 receptor on activated T cells. T-cell retargeting bispecific antibodies are the most commonly used approach in cancer treatment, and the first report was published 30 years ago. These bispecific antibodies specifically bind to CD3 on T cells on one side and specifically bind to an antigen on the cancer cell independent of its T-cell receptor (TCR) on the other side. In the past decade, T-cell retargeting antibodies have made considerable progress in the treatment of hematologic malignancies and solid tumors. Caputuximab was the first bispecific antibody of its kind targeting both epithelial cell adhesion molecule (EpCAM) and CD3, and it was approved in Europe (2009) for the treatment of malignant ascites (but withdrawn in 2017 for commercial reasons). Following this discovery, a bispecific targeting of CD19 and CD3 (bonatumab) was successfully developed, and the FDA approved it in 2014 for the treatment of relapsed or refractory precursor B-cell acute lymphoblastic leukemia (ALL). Currently, although many patients have benefited from bonatetumab, there are many T-cell redirection antibodies with different forms and characteristics that have shown potential anti-tumor efficacy in clinical studies.

[0015] The concept of redirecting T cells to tumors is now being extended to other receptors that also have co-stimulatory effects, such as CD137 (4-1BB), CD134 (OX40), CD27, or ICOS.

[0016] In the CD137-targeting field, agonistic monoclonal antibodies (and therefore not bispecific antibodies) have shown great preclinical promise, but their clinical development has been slow due to poor therapeutic index, particularly hepatotoxicity. CD137 is expressed on T cells that have been activated to recognize tumor antigens via MHC / TCR interactions. It is a member of the TNFRSF (tumor necrosis factor receptor superfamily), which requires clustering to deliver activation signals to T cells. Monospecific monoclonal antibodies that can activate CD137 are in clinical use and are known to be potent T cell activators, but have therapeutically limiting hepatotoxicity issues due to Fc receptors and clustering driven by multivalent forms. Bispecific tumor-targeting antibodies that are monovalent for CD137 do not induce CD137 clustering in normal tissues. Only when the bispecific antibody binds to tumor-associated antigens on tumor cells does it induce co-conjugated CD137 clustering on tumor-associated T cells. This drives highly potent but tumor-specific T cell activation. Tumor-targeting crosslinks of Cd137 / 4-1BB can provide a safe and effective way to co-stimulate T cells for cancer immunotherapy, and their combination with T-cell bispecific antibodies can provide a convenient "off-the-shelf" approach to systemic cancer immunotherapy for many tumor types. Examples of anti-CD137-based bispecific antibodies in clinical development include MP0310 (FAP × CD137), RG7827 (FAP × CD137), ALG.APV-527 (5T4 × CD137), MCLA145 (PD-1 × CD137), PRS342 (phosphatidylinositol proteoglycan-3 × CD137), PRS-343 (Her2 × CD137), and CB307 (PSMA × CD137). The various bispecific antibodies described above were intentionally selected to be monovalent for CD137 and therefore do not induce CD137 clustering in normal tissues. For example, only after the bispecific CB307 binds to PSMA on tumor cells does it induce the clustering of co-conjugated CD137 on tumor-associated T cells, thereby driving highly potent but tumor-specific T cell activation.

[0017] Antibodies that bind to T cells are known in the art, developed by Martin et al. Clin. Immunol. [Clinical Immunology] 2013, 148 , 136–147 and Rossi et al., Int. Immunol. [International Journal of Immunology] 2008, 20 ,1247–1258 highlight (both incorporated by reference), for example, OKT3, UCHT3, BMA031 and their humanized forms. Antibodies known to bind to V□9V□2 T cells are also known; see, for example, de Bruin et al. J. Immunol. [Journal of Immunology] 2017, 198 , 308–317, this document is incorporated by reference.

[0018] Similar to T-cell conjugation, the recruitment of NK cells into the tumor microenvironment is under extensive investigation. NK cell conjugation is typically based on binding to CD16, CD56, NKp46, or other NK cell-specific receptors, as described in Konjevic et al., 2017. http: / / dx.doi.org / 10.5772 / intechopen.69729 The literature summarized herein is incorporated by reference. NK cell binders can be generated by fusing or inserting NK-binding antibody (fragments) into complete IgG bound to tumor-associated antigens. Alternatively, specific cytokines can be used, given that NK cell antitumor activity is regulated by a number of activating and inhibitory NK cell receptors, and alterations in NK cell receptor expression and signaling constitute the basis for the attenuation of cytotoxic NK cell function. Based on this and on predicted in vitro findings, cytokines (including IFNα, IL-2, IL-12, IL-15, and IL-18) have been used systemically or for the in vitro activation and expansion of NK cells, and have improved NK cell antitumor activity by increasing the expression of NK cell activation receptors and by inducing cytotoxic effector molecules. Furthermore, this cytokine-based therapy enhances NK cell proliferation and regulatory function, and has been shown to induce NK cells exhibiting cytokine-induced memory-like properties, representing a newly defined NK cell subset with improved NK cell activity and lifespan. Several cytokine payloads have been developed for cancer therapy and for the treatment of chronic inflammation and have been tested in preclinical trials. Pro-inflammatory cytokines such as IL-2, TNF, and IL-12 have been investigated for use in cancer therapy because they have been found to increase and activate local leukocyte infiltration at tumor sites. For example, IL-2 monotherapy has been approved as proleukin. ® And it is currently undergoing a phase III clinical trial in combination with nivolumab (NKTR-214). Similarly, various recombinant forms of IL-15 (rhIL-15 or ALT-803) are being clinically evaluated. Specific mutants of IL-15 have been reported, for example by Behár et al. Prot. Engin. Des. Sel. [Protein Engineering and Design Selection] 2011, 24 , 283–290 and Silva et al., Nature [Nature] 2019, 565, Reports 186–191 (both incorporated by reference), and the complex of IL-15 with its receptor (IL-15R), such as those by Rubinstein et al., Proc. Nat. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 2006,103 Reports 9166–9171 (incorporated by reference) also evaluated the antitumor activity of fusion constructs of IL-15 and IL-15R (Sushi domain), see, for example, Bessard et al. Mol. Canc.Ther. [Molecular Cancer Therapy Agents] 2009, 8 , 2736–2745, which is incorporated by reference. In contrast, immunosuppressive cytokines (such as IL-10) can be considered as a payload for treating chronic inflammatory conditions or other diseases (e.g., endometriosis).

[0019] Systemic administration of pro-inflammatory cytokines can lead to serious off-target-related adverse effects, which may limit dosage and hinder escalation to therapeutically active regimens. Some cytokine products (e.g., IL-2, TNF, IL-12) exhibit adverse effects at or below the recommended dose in the order of a few milligrams per person. Adverse effects associated with intravenous administration of pro-inflammatory cytokines may include hypotension, fever, nausea, or flu-like symptoms, and occasionally serious hematologic, endocrine, autoimmune, or neurological events. Given these considerations, there is a clear biomedical need to develop 'next-generation' cytokine products that are better tolerated and exhibit preferential action at disease sites, helping to preserve normal tissue, such as those developed by Murer and Neri. New Biotechnol. [New Biotechnology] 2019, 52 The findings summarized in , 42–53 are incorporated herein by reference. Therefore, targeted delivery of cytokines to tumors aims to induce a local pro-inflammatory environment, which can activate and recruit immune cells. A list of antibody-cytokine fusions described in the literature has been compiled by Hutmacher and Neri, Adv. Drug Deliv. Rev. [Advanced Drug Delivery Review] 2018, 141 Reports 67–91 are cited and incorporated herein by reference. A list of clinical cytokine fusions is provided by Murer and Neri. New Biotechnol. [New Biotechnology] 2019, 52In , 42–53, this literature is incorporated by reference. Preclinical evaluations of various IL-15 fusion proteins are underway, such as those summarized in “T-cell & NK-Cell Engaging Bispecific Antibodies 2019: A Business, Stakeholder, Technology and Pipeline Analysis”, published by La Merie Publishing, 2019, and incorporated by reference, such as OXS-3550 (CD33-IL-15-CD16 fusion) prepared using Trike technology, which is close to Phase I evaluation.

[0020] A common strategy in the field of immune cell conjugation involves the inactivation or removal of antibody binding to Fc-γ receptors, which has multiple pharmacological implications. The first consequence of removing binding to Fc-γ receptors is reduced Fc-γ receptor-mediated antibody uptake, such as by macrophages or megakaryocytes, which can lead to dose-limiting toxicities, as seen, for example, against Kadcyla. ® (Trastuzumab-DM1) and LOP628 have been reported. Selective deglycosylation of antibodies in vivo offers an opportunity to treat patients with antibody-mediated autoimmunity. Removal of the high-mannose glycoform from recombinant therapeutic glycoproteins may be beneficial, as the high-mannose glycoform is known to impair therapeutic efficacy through specific uptake by endogenous mannose receptors and resulting in rapid clearance, as exemplified by, for example, those by Gorovits and Krinos-Fiorotti. Cancer Immunol. Immunother. [Cancer Immunology and Immunotherapy] 2013, 62 , 217-223 and Goetze et al., Glycobiology [Glycobiology] 2011, 21 The descriptions in 949-959 (both incorporated by reference). Additionally, Van de Bovenkamp et al., J. Immunol. [Journal of Immunology] 2016, 196 , 1435-1441 (incorporated by reference), describes how high levels of mannose can affect immunity. Reusch and Tejada, Glycobiology [Glycobiology] 2015, 25 , 1325-1334 (incorporated by reference) describes how inappropriate glycosylation in monoclonal antibodies can lead to invalid generation from expressed Ig genes.

[0021] In the field of immunotherapy, the binding of glycosylated antibodies to Fc-γ receptors on immune cells can induce systemic activation of the immune system before the antibody binds to tumor-associated antigens, leading to a cytokine storm (cytokine release syndrome, CRS). Therefore, to reduce the risk of CRS, the vast majority of immune cell conjugates used clinically are based on Fc-silencing antibodies, lacking the ability to bind to Fc-γ receptors. Additionally, companies in the bispecific antibody field are customizing molecular architectures with defined ratios of target binding to immune cell-binding antibody domains. For example, Roche is developing T-cell conjugates based on asymmetric monoclonal antibodies that retain bivalent binding to TAAs (e.g., CD20 or CEA) through two CDRs, but an additional anti-CD3 fragment is engineered into only one of the two heavy chains (target binding:CD3 binding ratio of 2:1). Similar strategies can be used for the binding / activation of T cells with anti-CD137 (4-1BB), anti-OX40, or anti-CD27, or for the binding / activation of NK cells with anti-CD16, CD56, NKp46, or other NK cell-specific receptors.

[0022] Eliminating binding to the Fc-γ receptor can be achieved in various ways, such as through specific mutations in antibodies (especially Fc fragments) or by removing naturally occurring glycans (C14) from the Fc fragment. H 2. Fc domain (around N297). Glycan removal can be achieved through genetic modifications in the Fc domain (e.g., N297Q mutation or T299A mutation) or by enzymatic removal of the glycan using, for example, PNG enzyme F or glycosidases after antibody recombinant expression. For example, glycosidase H is known to prune high-mannose and heterozygous glycoforms, while glycosidase S can prune complex glycans and to some extent heterozygous glycans. Glycosidase S2 can prune both complex, heterozygous, and high-mannose glycoforms. Glycosidase F2 can prune complex glycans (but not heterozygous glycans), while glycosidase F3 can only prune complex glycans that are also 1,6-fucosylated. Another glycosidase, glycosidase D, can only hydrolyze Man5 (M5) glycans. A review of the specific activities of different glycosidases was disclosed in Freeze et al. Curr.Prot. Mol. Biol [Current Molecular Biology Protocols], 2010, 89 Reference 17.13A.1-17 is incorporated herein by reference. Another advantage of protein deglycosylation for therapeutic purposes is the improved batch-to-batch consistency and significantly enhanced uniformity.

[0023] Inspiration can be drawn from the field of ADC technology to prepare antibody-protein conjugates for generating bispecific antibodies or antibody-cytokine fusions.

[0024] Many techniques for bioconjugate are known, such as those summarized in G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition, 2013, which are incorporated herein by reference. Two main techniques for preparing ADCs via random conjugation (based on acylation of lysine side chains or alkylation of cysteine ​​side chains) can be recognized. Acylation of the □-amino group in the lysine side chain is typically achieved by subjecting the protein to treatment with reagents based on activated ester or activated carbonate derivatives, for example, the application of SMCC in the manufacture of Kadcyla. ® The main chemical process for alkylation of thiol groups in the cysteine ​​side chain is based on the use of maleimide reagents, such as, for example, Adcetris. ® In addition to standard maleimide derivatives, a range of maleimide variants have also been used for more stable cysteine ​​coupling, such as those developed by James Christie et al. J. Contr.Rel. [Sustained-Release Journal] 2015, 220 , 660–670 and Lyon et al., Nat. Biotechnol. [Nature Biotechnology] 2014, 32 As demonstrated in 1059–1062, both are incorporated by reference. Another important technique for coupling with cysteine ​​side chains is via disulfide bonds, a bioactivated linker that has been used to reversibly link protein toxins, chemotherapeutic agents, and probes to carrier molecules (see, for example, Pillow et al.). Chem. Sci. [Chemical Sciences] 2017, 8 (366–370). Other methods for cysteine ​​alkylation involve nucleophilic substitution, for example, with haloacetamides (typically bromoacetamide or iodoacetamide), see, for example, Alley et al. Bioconj.Chem. [Bio-coupled Chemistry] 2008, 19 , 759–765 (incorporated by reference), or various methods based on Michael addition over unsaturated bonds, such as reactions with acrylate reagents, see, for example, Bernardim et al. Nat. Commun. [Nature Communications] 2016, 7 , DOI: 10.1038 / ncomms13128 and Ariyasu et al., Bioconj.Chem. [Bio-coupled Chemistry] 2017, 28 , 897–902 (both incorporated by reference), react with phosphonamide esters, see, for example, Kasper et al., Angew.Chem. Int. Ed. [Angewandte Chemie International Edition] 2019, 58, 11625–11630 (incorporated by reference), reacting with allenamides, see, for example, Abbas et al. Angew.Chem. Int. Ed. [Angewandte Chemie International Edition] 2014, 53 , 7491–7494 (incorporated by reference), react with cyanoacetylene reagents, see, for example, Kolodych et al., Bioconj.Chem. [Bio-coupled Chemistry] 2015, 26 , 197–200 (incorporated by reference), reaction with vinyl sulfone, see, for example, Gil de Montes et al., Chem. Sci. [Chemical Sciences] 2019, 10 , 4515–4522 (incorporated by reference), or reactions with vinylpyridine, see, for example, https: / / iksuda.com / science / permalink / (accessible January 7, 2020). Reactions with methanesulfonylphenyloxadiazole have also been reported for cysteine ​​coupling, by Toda et al. Angew.Chem. Int. Ed. [Angewandte Chemie International Edition] 2013, 52 Reports 12592–12596 are incorporated by reference.

[0025] Numerous methods have been developed that enable the generation of antibody-drug conjugates with defined drug-antibody ratios (DARs) by site-specific coupling to one or more predetermined sites in an antibody. Site-specific coupling is typically achieved by engineering specific amino acids (or sequences) into the antibody to act as anchors for payload attachment; see, for example, Aggerwal and Bertozzi. Bioconj.Chem. [Bio-coupled Chemistry] 2014, 53 , 176–192 (incorporated by reference), most typically achieved through the engineering of cysteine. In addition, a range of other site-specific conjugation techniques have been explored over the past decade, primarily targeting the genetically encoded non-natural amino acids, such as acetylphenylalanine suitable for oxime linkage, or p-azidomethylphenylalanine suitable for click chemical conjugation. Most antibody-based gene reengineering methods yield ADCs with a DAR of approximately 2. Alternative methods of antibody conjugation without reengineering the antibody involve the reduction of interchain disulfide bridges followed by the addition of a payload attached to a cysteine ​​crosslinking agent such as a disulfone reagent, see, for example, Balan et al. Bioconj.Chem. [Bio-coupled Chemistry] 2007, 18 , 61–76 and Bryant et al., Mol. Pharmaceutics [Molecular Pharmaceutics] 2015, 12, 1872–1879 (both incorporated by reference), monobromomaleimide or dibromomaleimide, see, for example, Smith et al. J. Am. Chem. Soc. [Journal of the American Chemical Society] 2010, 132 , 1960–1965 and Schumacher et al., Org. Biomol. Chem. [Organic and Biomolecular Chemistry] 2014, 37 , 7261–7269 (both incorporated by reference), bismaleimide reagents, see, for example, WO 2014114207, bis(phenylthio)maleimide, see, for example, Schumacher et al. Org.Biomol.Chem. [Organic and Biomolecular Chemistry] 2014, 37 , 7261–7269 and Aubrey et al., Bioconj.Chem. [Bio-coupled Chemistry] 2018, 29 , 3516–3521 (both incorporated by reference), dibromopyridazine dione, see, for example, Robinson et al. RSC Advances [Progress of the Royal Society of Chemistry] 2017, 7 9073–9077 (incorporated by reference), bis(halomethyl)benzene, see, for example, Ramos-Tomillero et al. Bioconj.Chem. [Bio-coupled Chemistry] 2018, 29 ,1199–1208 (incorporated by reference) or other bis(halomethyl) aromatic compounds, see, for example, WO 2013173391. Typically, ADCs prepared by cysteine ​​crosslinking have a drug antibody loading of about 4 (DAR4).

[0026] Ruddle et al. ChemMedChem [Chemistry and Pharmaceutical Chemistry] 2019, 14 Recent studies (1185–1195) have shown that DAR1 conjugates can be selectively reduced by C H 1 and C L Interchain disulfide chains were subsequently rebridged from antibody Fab fragments (prepared via papain digestion of complete antibody or recombinant expression) by treatment with symmetrical PDB dimers containing two maleimide units. The resulting DAR1-type Fab fragments demonstrated high homogeneity, stability in serum, and excellent cytotoxicity. In a subsequent publication, White et al., MAbs [Monoclonal Antibody] 2019, 11In , 500–515, and in WO 2019034764 (incorporated by reference), it has been demonstrated that DAR1 conjugates can also be prepared from whole IgG antibodies after the antibody has been pre-engineered: using antibodies having only one intrachain disulfide bridge in the hinge region (Flexmab technology, in Dimasi et al.), J. Mol. Biol. [Journal of Molecular Biology] 2009, 393 As reported in 672–692 (this literature is cited and incorporated) or by using antibodies with additional free cysteine, which can be achieved through mutation of the native amino acid (e.g., HC-S239C) or by insertion into the sequence (e.g., HC-i239C, by Dimasi et al.), Mol. Pharmaceut. [Molecular Pharmaceutics] 2017, 14 (Reported 1501–1516). It was shown that any engineered antibody could generate a DAR1 ADC by reacting the resulting cysteine-engineered ADC with a bismaleimide-derived PBD dimer. The Flexmab-derived DAR1 ADC demonstrated high resistance to loss of payload in serum and exhibited potent antitumor activity in a HER2-positive gastric cancer xenograft model. Furthermore, this ADC showed twice the tolerable dose in rats compared to a site-specific DAR2 ADC prepared using a single maleimide-containing PBD dimer. However, no improvement in the therapeutic window was noted, as the minimum effective dose (MED) of the DAR1 ADC increased by the same multiple of 2 relative to the DAR2 ADC.

[0027] In WO 2014065661 and van Geel et al., Bioconj.Chem. [Bio-coupled Chemistry] 2015, 26 In 2233–2242 (both incorporated by reference), it was demonstrated that site-specific conjugation of antibodies can be achieved based on the enzymatic remodeling of natural antibody glycans at N297 (by glycosidase pruning and the introduction of an azide-modified GalNAc derivative via glycosyltransferase), followed by click chemistry attachment of cytotoxic payloads. Verkade et al. Antibodies [Antibody] 2018, 712 demonstrated that the introduction of acylated sulfonamides further improved the glycan remodeling technique in terms of therapeutic index, and that the resulting antibody-drug conjugates (ADCs) could be customized for DAR2 or DAR4 by selecting specific linkers. It was also demonstrated that glycan pruning prior to conjugation resulted in ineffective binding (Fc silencing) of the resulting ADCs to the Fc-γ receptor. ADCs prepared using this technique were found to exhibit a significantly expanded therapeutic index compared to a range of other conjugation techniques currently used clinically, such as ADCT-601 (ADC Therapeutics).

[0028] A similar enzymatic method for converting antibodies into azide-modified antibodies with Fc silencing, developed by Lhospice et al. Mol. Pharmaceut. [Molecular Pharmaceutics] 2015, 12 A report from 1863–1871 (incorporated via citation) describes the use of bacterial transglutaminase (BTG or TG enzyme). It was demonstrated that deglycosylation of the native glycosylation site N297 with PNG enzyme F releases the adjacent N295 as a substrate for TG enzyme-mediated introduction. This substrate, in the presence of TG enzyme, is treated with a molecule carrying an azide to convert the deglycosylated antibody into a bisazido antibody. Subsequently, the bisazido antibody is reacted with a DBCO-modified cytotoxin to produce an ADC with DAR2. (Cheng et al.) Mol. Cancer Therap. [Molecular Cancer Therapy] 2018, 17 , 2665–2675 (incorporated by reference) reported a genetic approach based on the introduction of azide mediated by C-terminal TG enzyme, followed by conversion to ADC via metal-free click chemistry.

[0029] Besides the attachment of small molecules, various click chemistry methods have been well demonstrated to be suitable for the generation of protein-protein conjugates. For example, Witte et al., Proc. Nat. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 2012, 109 ,1993–1998 (incorporated by reference) have shown that non-natural N-N and C-C protein dimers can be obtained by introducing two different proteins into a sorting enzyme mediated by a combination of two complementary click probes (azidide and DBCO), followed by seamless ligation based on metal-free click chemistry (tension-promoted azidide-alkyne cycloaddition or SPAAC). Wagner et al., Proc. Nat. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 2014, 11116820–16825 (incorporated by reference) have applied this method to prepare bispecific antibodies based on C-terminal sorting labeling with anti-influenza scFv. This method has been further extended to metal-free click chemistry based on the anti-electron-demanded Diels-Alder cycloaddition with tetrazines, as described by Bartels et al. Methods [Methods] 2019, 154 , 93–101 (incorporated by reference). Tetraazine linkages were also previously, for example, by Devaraj et al. Angew.Chem. Int. Ed. [Angewandte Chemie International Edition] 2009, 48 , 7013–7016 and Robillard et al., Angew.Chem. Ed. Engl. [Angewandte Chemie International Edition] 2010, 49 Applications 3375–3378 (both incorporated by reference) are used for antibody modification by first (random) chemically attaching trans-cyclooctene (TCO) to the antibody. In contrast, site-specific introduction of TCO (or tetrazine or other click moieties for tetrazine linkage) to the antibody can be achieved through a variety of methods based on pre-genetic modification of the antibody as described above, and, for example, by Lang et al. J. Am. Chem. Soc. [Journal of the American Chemical Society] 2012, 134 , 10317–10320, Seitchik et al., J. Am. Chem. Soc. [Journal of the American Chemical Society] 2012, 134 , 2898–2901 and Oller-Salvia, Angew.Chem.Int.Ed. [Angewandte Chemie International Edition] 2018, 57 Reports 2831–2834, all of which were incorporated by reference.

[0030] Sorting enzymes are suitable enzymes used for site-specific modification of proteins after a sorting enzyme recognition sequence has been pre-introduced, such as those first developed by Popp et al. Nat. Chem. Biol. [Nature Chemistry and Biology] 2007, 3 Reports 707–708. Many other enzyme-enzyme recognition sequence combinations are also known for site-specific protein modifications, such as those described by Milczek. Chem. Rev. [Chemical Review] 2018, 118 The summary, 119–141 (incorporated by reference), and particularly applicable to antibodies, as by Falck and Müller, Antibodies [Antibody] 2018, 7, 4 (doi:10.3390 / antib7010004) and van Berkel and van Delft, Drug Discov. Today: Technol.[Today's Drug Discovery: Technology] 2018, 30 The points summarized in 3–10 are both incorporated by reference. Furthermore, various methods can be used for the non-genetic modification of natural proteins, such as those proposed by Koniev and Wagner. Chem. Soc. Rev. [Chemical Society Review] 2015, 44 The summary, 5495–5551 (by reference incorporated), and used for N-terminal modifications, as by Rosen and Francis, Nat. Chem. Biol. [Nature Chemistry and Biology] 2017, 13 , 697–705 and Chen et al., Chem. Sci. [Chemical Sciences] 2017, 8 The above methods can be used to mount appropriate click probes into peptides / proteins, as summarized by, for example, Chen et al. Acc. Chem. Res. [Review of Chemical Research] 2011, 44 , 762–773 and Jung and Kwon, Polymer Chem. [Polymer Chemistry] 2016, 7 The methods summarized in ,4585–4598 (both incorporated by reference) are applied to immune cell conjugates or cytokines. Immune cell conjugates can be readily generated by attaching complementary click probes to antibodies targeting tumor-associated antigens, and the stoichiometry of the tumor-binding antibody with the immune cell conjugate can be customized through appropriate technical selection.

[0031] Chemical methods have also been developed for site-specific modification of antibodies without prior genetic modification, such as those developed by Yamada and Ito. ChemBioChem. [Chemistry & Biochemistry] 2019, 20 , 2729–2737 are highlighted.

[0032] Chemical coupling via affinity peptides (CCAP) for site-specific modification has been developed by Kishimoto et al. Bioconj.Chem. [Bioconjugated Chemistry] 2019, describes a method that utilizes peptides that bind with high affinity to human IgG-Fc, enabling the selective modification of single lysine residues in the Fc fragment with either a biotinylated portion or a cytotoxic payload. Similarly, Yamada et al., Angew.Chem.Int.Ed. [Angewandte Chemie International Edition] 2019, 58 , 5592–5597 and Matsuda et al., ACS Omega [ACS Omega] 2019, 4, 20564–20570 (both incorporated by reference) have demonstrated that a similar approach (AJICAP™ technology) can be applied to the site-specific introduction of thiol groups on single lysine residues in antibody heavy chains. CCAP or AJICAP™ technology can also be used for the site-specific introduction of azide groups or other functional groups.

[0033] Chemical conjugation of immune cell conjugates with antibodies has been applied, but this typically produces heterogeneous mixtures. WO2021144315 reports the preparation of homogeneous bispecific antibodies or antibody-cytokine fusions that do not require prior reengineering of full-length IgG and / or enable customization of the number of immune cell conjugate peptides and the length and structure of the spacers between IgG and peptides.

[0034] Fusion proteins containing cleavable linkers have been described, for example, in WO 2021198490, WO 2021188835, and WO 2019 / 173832. As described in WO 2021188835 or WO 2019 / 173832, the cleavable linker can link a masking agent to the remainder of the fusion protein. To date, no method has been reported for preparing homogeneous antibody-cytokine conjugates linked via chemical linkers, wherein the cytokine is released upon cleavage by a protease. Summary of the Invention

[0035] This invention relates to an antibody-cytokine conjugate in which a cytokine is linked to an antibody via a chemical linker, the antibody-cytokine conjugate being capable of targeted delivery of the cytokine by releasing it at a target site. The antibody-cytokine conjugate can be produced without the need for genetic modification of IgG. This invention enables the molecular form of the antibody-cytokine conjugate to be defined in a 2:1 or 2:2 ratio, i.e., the ratio of the complement-dependent region (2) in a complete IgG CDR to the immune cell binding peptide (1 or 2).

[0036] The inventors have surprisingly discovered that the conjugates according to the invention have higher efficacy and therapeutic window compared to gene fusions of antibodies and immune cell conjugates.

[0037] Unbound by theory, the inventors believe that the immune cell-binding peptide of the fusion protein is glycosylated, which produces a heterogeneous product with variable efficacy, the average efficacy of which is considered to be lower than that of the homogeneous deglycosylated product.

[0038] Therefore, a first aspect of the present invention relates to a cleavable antibody-cytokine conjugate. A second aspect relates to a cleavable antibody-cytokine conjugate for medical treatment. A third aspect relates to a method for preparing an antibody-cytokine conjugate. Detailed Implementation

[0039] definition The verb "to comprise" and its inflections, as used in this specification and claims, are used in their non-limiting sense to mean including the item following the word, but not excluding items not specifically mentioned. Furthermore, the reference to an element by the indefinite article "a" or "an" does not preclude the possibility of more than one element, unless the context explicitly requires the presence of one and only one element. Therefore, the indefinite article "a" or "an" generally means "at least one".

[0040] The compounds disclosed in this specification and claims may contain one or more asymmetric centers, and these compounds may exist in different diastereomers and / or enantiomers. Unless otherwise stated, the description of any compound in this specification and claims is intended to include all diastereomers and mixtures thereof. Additionally, unless otherwise stated, the description of any compound in this specification and claims is intended to include individual enantiomers, as well as any racemic mixtures or other mixtures of enantiomers. When the structure of a compound is described as a particular enantiomer, it should be understood that the invention of this application is not limited to that particular enantiomer.

[0041] Compounds may occur in different tautomer forms. Unless otherwise stated, compounds according to the present invention are intended to include all tautomer forms. When the structure of a compound is described as a particular tautomer, it should be understood that the invention of this application is not limited to that particular tautomer.

[0042] The compounds disclosed in this specification and claims may further exist as exo- and endo-diastereomers. Unless otherwise stated, the description of any compound in this specification and claims is intended to include individual exo- and endo-diastereomers of the compound, and mixtures thereof. When the structure of a compound is described as a particular endo- or exo-diastereomer, it should be understood that the invention of this application is not limited to that particular endo- or exo-diastereomer.

[0043] Furthermore, the compounds disclosed in this specification and claims may exist as cis and trans isomers. Unless otherwise stated, the description of any compound in this specification and claims is intended to include a single cis and a single trans isomer of the compound, and mixtures thereof. By way of example, when the structure of a compound is described as a cis isomer, it should be understood that the corresponding trans isomer or mixture of cis and trans isomers is not excluded from the invention of this application. When the structure of a compound is described as a specific cis or trans isomer, it should be understood that the invention of this application is not limited to that specific cis or trans isomer.

[0044] The compounds of this invention can exist in the form of salts, which are also covered in this invention. Salts are generally pharmaceutically acceptable salts containing pharmaceutically acceptable anions. The term "its salt" means a compound formed when an acidic proton (typically the proton of an acid) is replaced by a cation (such as a metal cation or an organic cation). Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts not intended for administration to patients. For example, in a salt of a compound, the compound may be protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of the inorganic or organic acid serves as the anionic component of the salt.

[0045] The term "pharmaceutically acceptable" salt means a salt that is acceptable for administration to patients (such as mammals) (a salt containing a counterion that has acceptable mammalian safety for a given dosage regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases, as well as pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound derived from various organic and inorganic counterions known in the art, and includes, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium salts, etc.; and, when the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochlorides, hydrobromides, formates, tartrates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, etc.

[0046] The term "protein" is used in its normal scientific sense in this article. In this article, a polypeptide containing approximately 10 or more amino acids is considered a protein. Proteins may contain natural amino acids, but they may also contain non-natural amino acids.

[0047] The term "monosaccharide" is used in its normal scientific sense in this article, referring to an oxygen-containing heterocycle formed by the intramolecular formation of a hemiacetal during the cyclization of a chain of 5–9 (hydroxylated) carbon atoms (most commonly containing five carbon atoms (pentoses), six carbon atoms (hexoses), or nine carbon atoms (sialic acid)). Typical monosaccharides are ribose (Rib), xylose (Xyl), arabinose (Ara), glucose (Glu), galactose (Gal), mannose (Man), glucuronic acid (GlcA), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylneuraminic acid (NeuAc).

[0048] The term "cytokine" is used in its normal scientific sense in this article and refers to small protein molecules (5–20 kDa) that regulate the activity of immune cells by binding to their homologous receptors and triggering subsequent cell signaling. Cytokines include chemokines, interferons (IFNs), interleukins, monokines, lymphokines, colony-stimulating factors (CSFs), and tumor necrosis factor (TNF). Examples of cytokines are IL-1α (IL1a), IL-1β (IL1b), IL-2 (IL2), IL-4 (IL4), IL-5 (IL5), IL-6 (IL6), IL-8 (IL-8), IL-10 (IL10), IL-12 (IL12), IL-15 (IL15), IFN-α (IFNA), IFN-γ (IFN-G), and TNF-α (TNFA).

[0049] The term "antibody" is used herein in its normal scientific sense. An antibody is a protein produced by the immune system that recognizes and binds to a specific antigen. An antibody is an example of a glycoprotein. The term "antibody" is used herein in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" is also intended to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" is intended to include complete immunoglobulins, including antigen-binding fragments of antibodies. Furthermore, the term also includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art. Typical examples of antibodies include abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, efalizumab, alemtuzumab, adalimumab, tositumomab-I131, cetuximab, ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, and certolizumab. pegol, golimumab, canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab, ipilimumab, and brentuximab.

[0050] This document defines an "antibody fragment" as a portion of a complete antibody, containing its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2 and Fv fragments, biantibodies, microantibodies, triantibodies, tetraantibodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from one or more antibody fragments, one or more fragments generated from a Fab expression library, or epitope-binding fragments of any of the above that bind immunospecifically to a target antigen (e.g., cancer cell antigen, viral antigen, or microbial antigen).

[0051] This article defines "antigen" as an entity that an antibody specifically binds to.

[0052] This document defines the term "substantial" or "substantially" as the majority of a total, mixture, or sample, i.e., >50%, preferably more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total.

[0053] This document defines a linker as a portion that connects two or more elements of a compound. For example, in an antibody-conjugate, the antibody and the payload are covalently linked to each other via a linker. A linker may include one or more linkers and spacer portions connecting the various portions within the linker.

[0054] This article defines a "polar linker" as a linker containing structural elements whose specific purpose is to increase the polarity of the linker, thereby improving water solubility. A polar linker may, for example, contain one or more units or combinations thereof selected from ethylene glycol, carboxylic acid moiety, sulfonate moiety, sulfone moiety, acylated sulfonamide moiety, phosphate moiety, hypophosphite moiety, amino group or ammonium group.

[0055] This article defines a "spacer" or "spacer portion" as a portion that separates (i.e. provides distance between) two (or more) parts of a connector and covalently connects them. A connector can be, for example, part of a connector-construction, connector-coupler, or bioconjugate, as defined below.

[0056] The term "click probe" refers to a functional part capable of undergoing a click reaction, i.e., two compatible click probes click each other, such that they are covalently linked in the product. Compatible probes for click reactions are known in the art and preferably include (cyclic) alkynes and azides. In the context of this invention, a click probe Q in a compound according to the invention is capable of reacting with a click probe F on a (modified) protein, such that a conjugate is formed upon a click reaction, wherein the protein is conjugated to a compound according to the invention. Hereinafter, F and Q are compatible click probes.

[0057] The term "(hetero)alkyl" refers to both alkyl groups and heteroalkyl groups. A heteroalkyl group is one in which one or more carbon units (e.g., CH2, CH, or C) in an alkyl chain are surrounded by heteroatoms (such as O, S, S(O), S(O)2, or NR). 4 The alkyl group is replaced by another alkyl group. In other words, the alkyl chain is selected from O, S, S(O), S(O)2, and NR. 4 One or more elements are interrupted. Such interruptions differ from substituents because they occur within the chain of an alkyl group, while substituents are side-chain groups attached in a monovalent form to, for example, a carbon atom of an alkyl chain. In preferred embodiments, the (hetero)alkyl group is an alkyl group, such as ethyl (Et), isopropyl (i-Pr), n-propyl (n-Pr), tert-butyl (t-Bu), isobutyl (i-Bu), n-butyl (n-Bu), or n-pentyl.

[0058] Similarly, the term "(hetero)aryl" refers to both aryl groups and heteroaryl groups. A heteroaryl group is one in which one or more carbon units (e.g., CH) in a ring are surrounded by heteroatoms (such as O, S, N, or NR). 4 ) replaced aryl groups.

[0059] In this document, "acylsulfonamide moiety" is defined as a sulfonamide moiety (H2NSO2NH2) that is N-acylated or N-carbamylated at one end of the molecule and N-alkylated (mono- or di-) at the other end of the molecule. In the context of this invention, and especially in the examples, this group is also referred to as "HS".

[0060] A "domain" can be any region of a protein, is typically defined based on sequence homology, and is usually associated with a specific structural or functional entity. Members of the CEACAM family are known to consist of Ig-like domains. In this document, the term "domain" is used to refer to individual Ig-like domains such as "N-domain," or continuous groups of domains such as "A3-B3 domain."

[0061] A “coding sequence” or sequence that “encodes” an expression product (such as RNA, polypeptide, protein, or enzyme) is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme; that is, the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. The coding sequence of a protein may include a start codon (usually ATG) and a stop codon.

[0062] The term "gene" refers to a DNA sequence that encodes or corresponds to a specific amino acid sequence, containing all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences such as promoter sequences that determine, for example, the conditions for gene expression. Some genes are not structural genes and may be transcribed from DNA into RNA but not translated into amino acid sequences. Other genes may function as regulators of structural genes or regulators of DNA transcription. In particular, the term "gene" can refer to a genomic sequence that encodes a protein, i.e., a sequence containing regulators, promoters, introns, and exons.

[0063] This article defines "biomolecule" as any molecule that can be isolated from nature or any molecule composed of smaller molecular structural units that are components of macromolecular structures derived from nature, particularly nucleic acids, proteins, glycans, and lipids. Examples of biomolecules include enzymes, (non-catalytic) proteins, polypeptides, peptides, amino acids, oligonucleotides, monosaccharides, oligosaccharides, polysaccharides, glycans, lipids, and hormones.

[0064] The term "payload" refers to a portion covalently attached to a target site (such as an antibody). Therefore, a payload is a monovalent portion having an open end that is covalently attached to the target site via a linker. The payload can be a small molecule or a biomolecule. In this invention, the payload is an immune cell binder polymer.

[0065] The term "complement-dependent region" or "CDR" refers to a variable segment of an antibody that enables it to bind to a specific receptor or antigen.

[0066] This invention This invention provides an antibody-cytokine conjugate with an extended in vivo half-life and increased in vitro and in vivo activity, capable of activating an immune response in the tumor microenvironment, thereby conferring protective antitumor immunity. The antibody-cytokine conjugate according to the invention can target and deliver cytokines by releasing them at the target site. The conjugate according to the invention exhibits higher efficacy and therapeutic window than gene fusions of antibodies and immune cell conjugates, and compared to antibody-cytokine conjugates without cleavable sites.

[0067] This antibody-cytokine conjugate can be produced without the need for genetic modification of IgG. This invention enables the molecular form of the antibody-cytokine conjugate to be customized to a 2:1 or 2:2 ratio, i.e., the ratio of the complement-dependent region (2) in the complete IgG CDR to the immune cell binding peptide (1 or 2).

[0068] Therefore, a first aspect of the present invention relates to a cleavable antibody-cytokine conjugate. A second aspect relates to a cleavable antibody-cytokine conjugate for medical treatment. A third aspect relates to a method for preparing an antibody-cytokine conjugate. The present invention also covers pharmaceutical compositions comprising the conjugate according to the present invention.

[0069] Couplers having a general structure (1) or structure (2) This invention relates to antibody-cytokine conjugates having a universal structure (1) or (2): Antibody-cytokine conjugates having structure (1) or structure (2) in: Preferably, L exists 6 And β is 1.

[0070] Antibody AB AB is an antibody. Antibodies are known in the art and include IgA, IgD, IgE, IgG, IgM, Fab, VHH, scFv, biantibodies, microantibodies, affinity molecules, affylin, affimilin, atrimer, fynomer, cysteine ​​knot, DARPin, adnectin / centryin, knottin, anticalin, FN3, Kunitz domain, OBody, bicyclic peptides, and tricyclic peptides. Preferably, the antibody is a monoclonal antibody, more preferably selected from the group consisting of IgA, IgD, IgE, IgG, and IgM antibodies. Even more preferably, AB is an IgG antibody. The IgG antibody can be any IgG isotype. The antibody can be any IgG isotype, such as IgG1, IgG2, IgI3, or IgG4. Preferably, AB is a full-length antibody, but AB can also be an Fc fragment.

[0071] Antibody Abs typically exhibit specificity for extracellular receptors on tumor cells, preferably selected from the group consisting of: 5T4 (TPBG), ADAM9, ALPP, ALPPL2, AMHRII, ASCT2 (SLC1A5), ASLG659, ASPHD1, av-integrin, avb3-integrin / ITGAV / CD51, Axl, B7-H3 (CD276), B7-H4 (VTCN1), BAFF-R, BCMA (CD269), BMPR1B, short proteoglycans, c-KIT (CD117), c-Met, C4.4a (LYPD3), CA-IX (CA9) / MN, cadherin-6, CanAg, CCR7, CD117 (c-KIT), CD123 (IL-3Rα), CD 13. CD133, CD138 / cohesin-1, CD166 (ALCAM), CD19, CD20, CD203C, CD205, Ly75, CD21, CD22, CD228 (P79, SEMF), CD25 (IL-2R-α), CD30 (TNFRSF8), CD324 (CDH1 / E-cadherin), CD33, CD352 (SLAMF6, NTB-A), CD37, CD38, CD44v6, CD45, CD46, CD47, CD48a (SLAMF2), CD56 (NCAM), CD70, CD71 (TF-R), CD72, CD74, CD79a, CD79b, CDH6, cadherin-6, CEACAM5 (CD66e), sealing protein, CLDN18.2. CLDN6 (Skullin), CLEC12A, CLL-1 / CLEC12A, Cripto, CS1 (SLAMF7, CD319), CXCR5, DKL-1, DLL3 (δ-like 3), DPEP3, E16, EGFR, EGFRvIII, ENPP3, CD203c (AGS-16), EpCAM, EphA2, EphB2R, liver glycoside A4 (EFNA4), ETBR, FAP, FGFR2, FGFR3, fibronectin EDB, FLT3, FOLR1 (FR-a), Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, GPC3 (phosphatidylinositol proteoglycan-3), gpNMB, GPR172A, GPR19, GPR54, GRP20, guanylate cyclase C (GCC), HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Ra, integrin avb6, KAAG-1, LAMP-1 (CD107a), Lewis Y (CD174), LGR5, LIV-1 (SLC39A6, ZIP6), LRRC15, LY64, Ly6E (lymphocyte antigen 6), Ly6G6D, LY6K, mesothelin ( MSLN), MFI2 (TAA), MICA / B, MOSPD2, MPF, MUC1 (CA6), MUC16 / CA-125, MUC1c, NaPi2b (SLC34A2), NCA, conjugin-4 (PVRL4), Notch3, p-cadherin (pCAD, CDH3), P2X5, PD-L1 (CD274, B7-H1), PMEL17, PRLR (prolactin), PSCA, PSMA, PTK7 (CCK4), RET, RNF43, RON, ROR1, ROR2, Serna 5b, SEZ6, SLITRK6 (SLC44A4), STEAP1, STEAP2, STn, TAG72, TENB2, TF (CD142, prothrombin kinase), TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, TNF-α, TROP-2 (TACSTD2), TWEAKR, receptor tyrosine kinase (RTK), tendinin, or antibodies specific to extracellular proteins and / or tumor-associated carbohydrate antigens (TACA) induced by viral infection. In preferred embodiments, the extracellular receptor is selected from PD-L1, HER2, and conjugation-4, more preferably from PD-L1 and HER2.

[0072] Antibodies can also be specific for extracellular proteins caused by viral infections (such as human poliovirus (HPV), human cytomegalovirus (HCMV), or human papillomavirus (HPV)). Antibodies can also be specific for tumor-associated carbohydrate antigens (TACAs) selected from the following group: Tn, STn, T antigen, LDN, Lewis antigen. c (Le) c ), sialic acid-Lewis c (SLe) c ), 6-sialic acid-Lewis c (6SLe) c ), LN, α-Gal, 3SLN, 6SLN, H antigen, A antigen, B antigen, Lewis antigen a (Le) a ), sialic acid-Lewis a (SLe) a ), 6-sialic acid-Lewis a (6SLe) a Louis b (Le) b ), sialic acid-Lewis b (SLe) b ), 6-sialic acid-Lewis b (6SLe) b Louis x (Le) x ), sialic acid-Lewis x (SLe) x ), 6-sialic acid-Lewis x (6SLe) x Louis y (Le) y ), sialic acid-Lewis y (SLe) y ), 6-sialic acid-Lewis y (6SLe) y Antibodies can be specific to both extracellular proteins and TACA.

[0073] In this art, the number of payloads D attached to a single antibody is referred to as DAR (drug-antibody ratio). In this invention, the drug is an immune cell binder polypeptide that is specific to immune cell receptors. In this invention, the DAR value is an integer in the range of 1-10, preferably a DAR value of 1, 2, or 4, and more preferably a DAR value of 1 or 2.

[0074] Part of the antibody can be the reaction part F 1 Or linking group Z 1 Linker L, which connects to the peptide portion of the cell binder6 Preferably, the linking group Z 1 It is linked to the antibody via glycan.

[0075] Connector L 6 Preferably, there is a connector L 6 This allows for the introduction of reactive groups F at specific sites on the antibody. 1 For example, via artificially introduced reactive groups F. 1 This is the case with coupling, for example, using transglutaminase, sorting enzymes, or through enzymatic modification of the polysaccharide (e.g., glycosyltransferases or α-1,3-mannosyl-glycoprotein-2-bN-acetylglucosamine transferase). For example, a modified sugar residue S(F) can be introduced at the polysaccharide. 1 )2, thereby extending the glycan with a single monosaccharide residue S, which introduces two reactive groups F onto the antibody glycan. 1 In the most preferred embodiment, coupling occurs via the antibody's glycans. The coupling site is preferably located on the antibody's heavy chain.

[0076] All recombinant antibodies generated in mammalian host systems contain a conserved N-glycosylation site at or near the asparagine residue at position 297 of the heavy chain (Kabat number), which is modified by a complex type of glycan. This naturally occurring glycosylation site of the antibody is preferred; however, other glycosylation sites, including artificially introduced ones, can also be used for the linker L. 6 The connection. Therefore, in a preferred embodiment, L 6 The antibody is linked to an amino acid located at position 250–350 of the heavy chain, preferably at position 280–310, more preferably at position 295–300, and most preferably at position 297. Using this conserved glycosylation position of the antibody, the resulting conjugate forms a symmetrical dimer, wherein each half of the antibody contains one F... 1 Some antibodies may have a second glycosylation site in each half, which is preferably not used as a conjugation site. Technicians can perform enzymatic conversion in such a way that conjugation is performed using only the major glycosylation site. Alternatively, technicians can perform enzymatic conversion in such a way that conjugation is also performed using the second glycosylation site, thereby doubling the DAR of the antibody-drug conjugate.

[0077] L 6 It connects AB to F. 1 or Z 1 The connector, and by –(H) v –S–(L 7 ) w’– indicates that H is a monosaccharide, v is an integer in the range 0–10, S is a sugar or sugar derivative, w' is 0 or 1, and L 7 It is –N(H)C(O)CH2–, –N(H)C(O)CF2–, or –CH2–. (H) v It can be linear or branched, and S can be related to (H). v Any monosaccharide linkage. Typically, L 6 It is formed at least in part by the glycans of antibodies.

[0078] L 6 -(H) v – Parts are glycans or portions thereof. Therefore, the –(H) of glycans… v – Typically derived from the original antibody. (H) v Preferably selected from structures (H1), (H2), and (H3). In this article, w is 0 or 1, j is an integer in the range 0–9, and GlcNAc is... N - The acetylglucosamine moiety, Fuc is the fucose moiety, Gal is the galactose moiety, the tilde * indicates the linkage with the antibody peptide, and the tilde ** indicates the linkage with S. Fuc typically binds to GlcNAc via an α-1,6-glycosidic bond. Normally, antibodies may or may not be fucosylated (w = 1) or unfucosylated (w = 0). In the context of this invention, the presence of the fucosylated moiety is irrelevant; similar effects can be obtained using fucosylated (w = 1) and unfucosylated (w = 0) antibody conjugates. The GlcNAc residue can also be referred to as the core-GlcNAc residue and is a monosaccharide directly attached to the antibody peptide moiety.

[0079] S can be directly connected to the core - GlcNAc(Fuc). w The part, i.e., j = 0, means that the rest of the glycan is attached to the core-GlcNAc(Fuc) before S attachment. w The glycan is partially removed. Such pruning of the glycan is well known in the art and can be achieved by the action of glycosidases. (H3) can be obtained from the pruned glycan by introducing galactose with galactosyltransferase and S-fucose with fucosyltransferase, as described in WO 2022037665. Alternatively, the core-GlcNAc(Fuc) can be used. w There are one or more monosaccharide residues between the part and S, i.e., j is an integer in the range of 1–9, preferably j = 1–5. In a preferred embodiment, (G) jIt is an oligosaccharide fraction containing j monosaccharide residues G, where j is an integer in the range of 2–5. In another preferred embodiment, (G) j It is a monosaccharide fraction containing j monosaccharide residues G, where j is 1. (G) j Typically linked to GlcNAc(Fuc) via α-1,4 bonds. w The GlcNAc portion. In a preferred embodiment, j is 0, 1, 3, 4, or 5; more preferably, j is 0 or 1; and most preferably, j is 0.

[0080] Although any monosaccharide present in the polysaccharide can be used as G, each G is preferably individually selected from the group consisting of: galactose, glucose, N -acetylgalactosamine, N - Acetylglucosamine, mannose and N - Acetylneuraminic acid. A more preferred option for G is galactose, N - Acetylglucosamine, mannose. When j = 1, G = galactose and S = N - Acetylneuraminic acid.

[0081] When j is 3 - 10, (G) j It can be linear or branched. Branched oligosaccharides (G) j Preferred examples are shown in (a), (b), (c), (d), (e), (f), and (h). In this article, the wavy line represents the core GlcNac(Fuc). w The connection. In (G) jIn the presence of a moiety where j > 2, it is preferred that its terminal portion is GlcNAc or Gal, preferably GlcNAc. In other words, the monosaccharide residue directly linked to S is preferably GlcNAc or Gal. The presence of the GlcNAc moiety is beneficial for the synthesis of functionalized antibodies because the monosaccharide derivative S can be readily introduced to the terminal GlcNAc residue via glycosyltransferase. The presence of the Gal moiety is beneficial for the synthesis of functionalized antibodies because the monosaccharide derivative S sialic acid can be readily introduced to the terminal Gal residue via sialyltransferase. Alternatively, the presence of the Gal moiety allows for the introduction of the monosaccharide derivative S fucose to the adjacent GlcNac via fucosyltransferase, as described in WO 2022037665. (G) comprising structures (a) – (h) j In the preferred embodiments described above, a portion of S can be linked to any terminal GlcNAc residue (i.e., not a residue with a tilde bond) that is linked to the core GlcNAc residue on the antibody.

[0082] Antibodies and antibody-drug conjugates with j = 0 or 1 show no binding or significantly reduced binding to the Fc-γ receptor, while antibodies and antibody-drug conjugates with j in the range of 4–10 do bind to the Fc-γ receptor. Therefore, by selecting a certain value of j, the desired degree of binding to the Fc-γ receptor can be obtained. Thus, j = 0, 1, 4, 5, 6, 7, 8, 9, or 10 is preferred, j = 0, 1, 4, or 5 is more preferred, and most preferably the antibody is pruned and j = 0.

[0083] S is a sugar or a sugar derivative. The term "sugar derivative" is used herein to refer to a derivative of a monosaccharide (i.e., a monosaccharide containing substituents and / or functional groups). Suitable examples of S include glucose (Glc), galactose (Gal), mannose (Man), fucose, amino sugars, and sugar acids such as glucosamine (GlcNH2), galactosamine (GalNH2), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) (also known as N-acetylneuraminic acid (NeuNAc)), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Preferably, S is selected from Gal, GalNAc, and NeuNAc. In a particularly preferred embodiment, S is GalNAc.

[0084] Linking group Z 1 or reactive group F 1 It can be directly attached to S, or between S and Z. 1 or F 1 Connectors L can exist between them. 7 Therefore, L 7It can exist (w' = 1 or 2) or not exist (w' = 0). Typically, each Z part can be connected via connector L. 7 Connected to S, therefore in one embodiment, w' = 0 for x. Preferably, L 7 It does not exist and each connection part Z is directly attached to S. If it exists, L 7 It can be selected from -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-. In a preferred embodiment, x = 1 and w' = 0 or 1, and most preferably x = 1 and w' = 0.

[0085] Cuttable site The conjugates of this invention contain a cleavable site to enable the release of the immune cell binder peptide at the target site. The cleavable site can be present as part of the payload D (i.e., D is present). 1 And α = 1) or exists in the connector L (preferably as L) 2 and L 3 That is, o = p = 1). In a preferred embodiment, α = 1 and o = p = 0.

[0086] Immune cell binder peptide D The payload of the conjugate of the present invention is an immune cell binder peptide D, also known as a cytokine. The payload D is linked to an antibody via a cleavable linker. The cleavable unit may be present in the linker L (i.e., in one embodiment, L is cleavable) or within the peptide chain of D (i.e., α = 1 and the cleavable peptide linker D is present). 1 Typically, if α = 1, then L is not cut.

[0087] Polypeptide D or its precursor is preferably produced via microbial recombinant expression. Particularly preferred is that the recombinant expression occurs in prokaryotes, in a cell-free protein expression system, and is carried out by synthetic methods or a combination of synthetic and protein-linking methods. Proteins obtained through prokaryotic microbial expression, cell-free methods, or synthetic methods are not glycosylated; therefore, when D or its precursor is obtained through prokaryotic microbial expression, cell-free expression, or synthetic methods, it is more homogeneous than when obtained from eukaryotes. Most preferably, D is obtained through microbial recombinant expression of *Escherichia coli*. In other words, it is preferred that the peptide chain of D is not glycosylated, i.e., D is an unglycosylated polypeptide.

[0088] Mode of action D is based on –(D) 1 ) α –D 2 The polypeptide, of which D1 It is a cleavable peptide linker; D 2 It is an immune cell binder polypeptide specific to immune cell receptors, and α is 0 or 1. If L is not cleavable, then D exists. 1 Preferably, α is 1 and D exists. 1 More preferably, D via D 1 The N-terminus or C-terminus, most preferably the N-terminus is connected to the Z-terminus. 2 .

[0089] D 2 It is specific to immune cell receptors, and by activating the receptors, it triggers subsequent cell signaling. D 2 The ability to bind to at least one of its associated receptors is completely or reduced through its conjugation with an antibody. Therefore, D 2 By cutting D 1 Or it may be activated by the linker L. Because the probability of cleavage at a specific target site is increased due to the cleavable site, cell signaling primarily occurs at the target site. Because antibodies spatially hinder D... 2 The ability to target at least one of the relevant receptors, therefore preferably, D 2 The distance between D and AB should be as short as possible. More preferably, D 2 The shortest atomic chain between AB and B is 10-100 atoms, more preferably, the atoms are selected from B, C, O, N, S and P. In a preferred embodiment, the atomic chain consists of 15-50 atoms selected from C, O, N, S and P, more preferably selected from C, O, N and S.

[0090] Cuttable connector D 1 D 1 It is a cleavable peptide linker as known in the art. D 1 Containing a cleavable site of a protease, preferably a mammalian protease. In therapeutic applications, the protease cleavage site can be cleaved by a protease present (typically overexpressed) in or near the target cell (e.g., cancer cells, infected cells, or pathogens). These proteases can be extracellular enzymes produced by the target cell or intracellular enzymes that have leaked to the outside of the target cell. Preferably, the target is a cancer cell, and the enzyme is present in the tumor microenvironment. Typically, the cleavable peptide linker is specifically cleaved by a protease present in the microenvironment of the target cell (typically the tumor microenvironment). Such proteases are often overexpressed in the target microenvironment.

[0091] In a preferred embodiment, D 1It contains a cleavable site that is recognized by one of the following: serine protease, cysteine ​​protease, aspartic protease, threonine protease, glutamate protease, metalloproteinase, gelatinase, and asparagine peptide lyase.

[0092] In another preferred embodiment, D 1 It contains a cleavable site that is recognized and cleaved by one of the following: cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, kallikrein, hKl, hKlO, hKT5, plasmin, collagenase, type IV collagenase, matrix lysozyme, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspase, caspase-3, miR1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymotrypsin, renin, pepsin, matrix protease, membrane serine protease 1 (MT-SP1), and podase. Legumain, plasmepsin, nepenthesin, extrametalopeptidase, endopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM12, urokinase plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1b convertase, thrombin, FAP (FAP-α), type II transmembrane serine protease (TTSP), neutrophil elastase, protease 3, neutrophil serine protease 4, mast cell chymotrypsin, mast cell trypsin, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26). Preferably, the cleavable site is recognized and cleaved by one of MMP, podase, matrix protease, urokinase and thrombin, more preferably by at least one of podase, matrix protease and urokinase.

[0093] Any protease-specific cleavable peptide linker known in the art can be used as D 1Such peptide linkers are known, for example, from US 2016 / 0194399, which is incorporated herein by reference. Suitable cleavable peptide linkers comprise peptide sequences selected from the following: RASRAN (SEQ. ID NO: 43), LSGRSDNH (SEQ. ID NO: 44), TGRGPSWV (SEQ. ID NO: 45), SARGPSRW (SEQ. ID NO: 46), TARGPSFK (SEQ. ID NO: 47), GGWHTGRN (SEQ. ID NO: 48), HTGRSGAL (SEQ. ID NO: 49), PLTGRSGG (SEQ. ID NO: 50), AARGPAIH (SEQ. ID NO: 51), RGPAFNPM (SEQ. ID NO: 52), SSRGPAYL (SEQ. ID NO: 53), RGPATPIM (SEQ. ID NO: 54); RGPA (SEQ. ID NO: 55), GGQPSGMWGW (SEQ. ID NO: 56), FPRPLGITGL (SEQ. ID NO: 57), VHMPLGFLGP (SEQ. ID NO: 58). 58), SPLTGRSG (SEQ. ID NO: 59), SAGFSLPA (SEQ. ID NO: 60), LAPLGLQRR (SEQ. ID NO: 61), SGGPLGVR (SEQ. ID NO: 62), PLGL (SEQ. ID NO: 63), ISSGLLSS (SEQ. ID NO: 64), QNQALRMA (SEQ. ID NO: 65), AQNLLGMV (SEQ. ID NO: 66), STFPFGMF (SEQ. ID NO: 67), PVGYTSSL (SEQ. ID NO: 68), DWLYWPGI (SEQ. ID NO: 69), MIAPVAYR (SEQ. ID NO: 70), RPSPMWAY (SEQ. ID NO: 71), WATPRPMR (SEQ. ID NO:72), FRLLDWQW (SEQ. ID NO: 73), LKAAPRWA (SEQ. ID NO: 74), GPSHLVLT (SEQ. ID NO: 75), LPGGLSPW (SEQ. ID NO: 76), MGLFSEAG (SEQ. ID NO: 77), SPLPLRVP (SEQ. ID NO: 78), RMHLRSLG (SEQ. ID NO: 79), LAAPLGLL (SEQ.ID NO: 80), AVGLLAPP (SEQ. ID NO: 81), LLAPSHRA (SEQ. ID NO: 82), PAGLWLDP (SEQ. ID NO: 83), ISSGLSS (SEQ. ID NO: 84), GPRSFGL (SEQ. ID NO: 85) and GPRSFG (SEQ. ID NO: 86). .

[0094] Peptide sequences having SEQ ID NO: 43–63 can be cleaved by at least one of the proteases stigmoproteinase, matrix protease, and urokinase. Peptide sequences having SEQ ID NO: 64–84 can be cleaved by at least one of the proteases MMP. Peptide sequences having SEQ ID NO: 85 and 86 are cleaved by thrombin. Preferred cleavable peptide linkers comprise peptide sequences selected from SEQ ID NO: 43–63, more preferably peptide sequences SEQ ID NO: 43 or SEQ ID NO: 44, and most preferably peptide sequence SEQ ID NO: 43.

[0095] Peptide sequences at cleavable sites can be incorporated into D. 1 In longer peptide sequences, the cleavable sites typically contain 3–12 amino acids, preferably 4–10 amino acids, and more preferably 6–8 amino acids. Peptide D 1 The overall length can be, for example, up to 50 amino acids long at the cleavable site as defined herein, preferably 3–30 amino acids long, and more preferably 8–25 amino acids long. Furthermore, peptide D... 1 The overall length can be, for example, 4–60 amino acids, preferably 10–50 amino acids, more preferably 15–40 amino acids. Preferred cleavable peptide linkers are cleavable peptide linkers 1 (SEQ. ID NO: 1) and 2 (SEQ. ID NO: 2).

[0096] Particularly preferred cleavable peptide linkers comprise the peptide sequence RASRAN (SEQ. ID NO: 43), and most preferably comprise cleavable peptide linker 2 (SEQ. ID NO: 2). These peptide linkers are recognized by at least three different tumor-specific proteases (lecithin, matrix protease, and urokinase), which facilitates site-specific cleavage of immune cell binder peptides at target sites.

[0097] Immune cell binding peptide D 2 Immune cell binding peptide D 2Cytokines are specific to immune cell receptors, typically to cytokine receptors on the surface of immune cells. It should be understood that references to cytokines include mutated variants and cytokines with attached cofactors. For example, unless otherwise specified, IL-15 refers to normal IL-15, but also to IL15α, sushi-IL15, IL15 with point mutations, etc. Preferably, cytokines do not have cofactors. In D 2 In the case of a mutant immune cell conjugating peptide, preferably, the mutation enhances or reduces the binding affinity to the receptor. Preferably, the immune cell conjugating peptide is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26, IL-28, IL-29, IL-33, IL-36, IL-37, IL-38, IFN. -α (including IFN-α1 / 13, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α14, IFN-α16, IFN-α17, and IFN-α21), IFN-β, IFN-γ, IFN-A, TFN-α, TNF-β, TGF-β1, M-CSF, G-CSF, GM-CSF, and CXL10, including mutated variants. More preferably, the immune cell conjugating peptide is IL-2 or IL-15. In the most preferred embodiment, the immune cell conjugating peptide is IL-15. In alternative embodiments, the immune cell conjugating peptide is not IL-15.

[0098] Therefore, in one embodiment, D 2 The IL-15 is preferred, and preferably IL-15 is mutated to enhance or decrease its binding affinity to the receptor, more preferably IL-15 is mutated to decrease its binding affinity to IL15Ra. IL15Ra binding leads to rapid clearance, therefore mutants with reduced IL15Ra binding affinity produce better PK and higher potency. More preferably, the IL15 according to sequence SEQ. ID NO: 3 contains amino acid substitutions at E46 and V49, even more preferably, these amino acid substitutions are E46G, V49R, and most preferably no other substitutions are present. Advantageously, for IL15Rβ and γ... c The binding affinity is reduced by the steric hindrance of the antibody. However, after cleavage, the binding affinity with IL15Rβ and γ is reduced. c The binding site becomes available, and the immune response is activated.

[0099] Linking group Z 1 and Z 2 Z 1 and Z 2 It is a linker group that covalently links the antibody to the payload of the conjugate according to the invention. The term "linker group" as used herein refers to a structural element generated by the reaction between Q and F, used to link one part of the conjugate to another part of the same conjugate. 1 Through Q 1 With F 1 Cycloaddition or nucleophilic substitution between them. Preferably, Z 1 Formed via cycloaddition. Similarly, Z 2 Preferably via Q 2 With F 2 Cycloaddition or nucleophilic substitution between them. Preferably, Z 2 Formed via cycloaddition. In this paper, Z refers to Z0. 1 and Z 2 Q refers to Q 1 and Q 2 And F refers to F 1 and F 2 .

[0100] As those skilled in the art will understand, the exact nature of the linking groups depends on the exact structures of the reactive moieties Q and F. Those skilled in the art know that they are reactive with each other and form suitable reaction partners Q. 1 / F 1 and Q 2 / F 2 The complementary click probes. For example, when F contains an alkynyl group, the complementary group Q contains an azide group. For example, when F contains an alkynyl group, the complementary group Q contains an alkynyl group. For example, when F contains a cyclopropenyl group, a trans-cyclooctene group, a cycloheptyne group, or a cyclooctyne group, the complementary group Q contains a tetrazinyl group. In these special cases, Z is only an intermediate structure and will expend N2 to generate dihydropyridazine (reacting with alkenes to form) or pyridazine (reacting with alkynes to form), as shown in Figure 1.

[0101] In a preferred embodiment, the linking group Z is obtained via a cycloaddition reaction, preferably wherein the cycloaddition is a [4+2] cycloaddition or a 1,3-dipolar cycloaddition. Coupling reactions via cycloaddition are known to those skilled in the art, and those skilled in the art can select appropriate reaction partners F and Q and will understand the properties of the resulting linking group Z. Preferred cycloadditions are [4+2]-cycloadditions (e.g., Diels-Alder reactions) or [3+2]-cycloadditions (e.g., 1,3-dipolar cycloadditions). Preferably, the cycloaddition is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. Preferred Diels-Alder reactions are electron-demanding Diels-Alder cycloadditions. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition. Cycloadditions (such as Diels-Alder reactions and 1,3-dipolar cycloadditions) are known in the art, and those skilled in the art know how to perform them.

[0102] Preferably, Z contains a moiety selected from the group consisting of: triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazoline, pyrazoline, piperazine, thioether, amide, or imide groups. The triazole moiety is particularly preferred to be present in Z. In one embodiment, Z comprises a (hetero)cyclic alkene moiety, i.e., formed from Q which comprises a (hetero)cyclic alkyne moiety. In alternative embodiments, Z comprises a (hetero)cyclic alkane moiety, i.e., formed from Q which comprises a (hetero)cyclic alkene moiety. In this document, aromatic rings (such as triazole rings) are considered heterocyclic alkane rings because they are formed by the reaction of alkyne and azide moieties.

[0103] In a preferred embodiment, Z has a structure (Z1): (Z1) In this article, it is described as – – – The key is either a single key or a double key. Additionally: - Ring Z is obtained by cycloaddition, preferably selected from (Za) – (Zm) as defined below, where the carbon atom marked with ** corresponds to the one fused with ring Z in (Z1) as described below. – – – The two carbon atoms in the bond; -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24(Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups; -Y 2 It is C(R) 31 )2、O、S、S (+) R 31 S(O)R 31 S(O)=NR 31 or NR 31 S (+) Is with B (-) Mutually balanced cation sulfur atoms, of which B (-) It is an anion, and each R in it is an anion. 31 R alone 15 Or a connection to D (via L); -u is 0, 1, 2, 3, 4 or 5; -u' is 0, 1, 2, 3, 4 or 5, where u + u' = 0, 1, 2, 3, 4, 5, 6, 7 or 8; -v = an integer in the range 8–16; The ring Z is formed by cycloaddition and is preferably selected from (Za) – (Zm). In a preferred embodiment, u + u' = 0, 4, 5, 6, 7, or 8, more preferably 0, 4, or 5. If described as – – – If the bond is a double bond, then u + u' = 4, 5, 6, 7, or 8 is preferred, and u + u' = 4 or 5 is more preferred. If described as – – – If the bond is a single bond, then u + u' = 0 or 5 is preferred. Preferably, the tilde marked with * connects to AB, optionally via L. 6 The tilde marked with ** is connected to L.

[0104] Particularly preferred is Z comprising a (hetero)cyclic olefin moiety, i.e., described as – – –The bond is a double bond. In a preferred embodiment, Z is selected from the structure (Z2) – (Z20c) described below: In this article, the connection with L is depicted using a tilde. B (–) It is an anion, preferably a pharmaceutically acceptable anion. B (+) It is a cation, preferably a pharmaceutically acceptable cation. R 36 It is a halogen selected from fluorine, chlorine, bromine and iodine, preferably R. 36 It's fluorine. Y 4 It is a heteroatom, preferably Y. 4 It is O or NH. R 35 Choose from the following groups: hydrogen, C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups, these C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups are optionally substituted and optionally selected from O, Si, S and NR. 14 One or more heteroatoms are interrupted, where R 14 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups, preferably, R 35 Selected from H, C5H 11 CH3, CH2CH3, CH2OH or CH2OTBS.

[0105] Ring Z is formed via a cycloaddition reaction and is preferably a triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazoline, oxazolidinone, pyrazoline, or piperazine. Most preferably, ring Z is a triazole ring. Ring Z can have a structure selected from (Za)–(Zm) as described below, wherein the carbon atom marked with ** corresponds to two carbon atoms of the (hetero)cycloalkane ring fused with ring Z in (Z2)–(Z20). Since, in the context of this embodiment, the linking group Z is formed by reacting with a (hetero)cycloalkyne, it is described above as – – – The key is a double bond. In this article, R 29 Selected from hydrogen, C1-6 Alkyl, aryl, C(O)-C 1-6 Alkyl, C(O)-aryl, C(O)-OC 1-6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1-6 Alkyl and C(O)-NR 33 -Aryl, where R 33 Is it H or C? 1-4 Alkyl group. Preferably, R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl, and pyrimidinyl. It has been found that when R... 29 When the hydrogen is present, the reactivity in the cycloaddition reaction, especially in the case of the formation of ring (Zl), is optimal. Therefore, in a preferred embodiment, ring Z is (Zl), wherein R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl and pyrimidinyl, more preferably R 29 It is hydrogen.

[0106] For isoxazoline (Zh) and rearranged products (Zh'), the following applies: -R 1 It is L 10 XR 4 ; -L 10 It has a structure (C(R) 3 )2) z The connector, where z is 2 or 3, and each R 3 Selected individually from H and C 1-4 Alkyl groups, where R appears twice 3 They can be connected together to form C 3-6 (Hetero)cycloalkyl groups; -R 4 Selected from H and C 1-4 alkyl; -X is S, O, or NH. -R 2 Selected from H and C 1-4 alkyl.

[0107] Preferred embodiments are defined below for the nitrone reactive group (F3a), which are also applicable to isoxazoline (Zh) and rearrangement products (Zh').

[0108] The inventors discovered that the substituent R 1 The stability of isoxazoline-based conjugates has been improved. Typically, isoxazolines can be rearranged according to the following scheme: The 4-isoxazoline ring may undergo disintegration via this rearrangement. The isoxazoline ring (a) is rearranged into an aziridine ring (b), with ring strain released from the aziridine ring in a zwitterionic species (c). Addition of water yields hydroxylamine (e), from which the aldehyde (f) is cleaved to yield a ketamine (g). This not only disrupts the isoxazoline ring but also breaks the covalent bond between the substituent at R* and the substituent at R**. Therefore, in cases where the isoxazoline ring is part of a bioconjugate, the payload is removed from the biomolecule, effectively eliminating its use and potentially causing serious side effects.

[0109] The solutions to the aforementioned problems developed by the inventors involve specific substituents R on the nitrogen atom of nitroketones. 1 The heteroatom X can capture the rearranged imine intermediate (d) by reacting with the imine carbon atom. The inventors discovered that this does not lead to the splitting of the aldehyde (f), but rather to a rearrangement in the following manner (R). 1 = (CH2)2OH (an exemplary scheme), in which an oxazoline ring (h) is formed. This structure is stable and does not disintegrate. Therefore, the covalent connection between the substituent at R* and the substituent at R** remains intact. Therefore, the bioconjugate remains intact and effective, wherein the payload is attached to the biomolecule. Therefore, even if the rearrangement of the 4-isooxazoline ring formed by the SPANC reaction is disrupted, the covalent connection is not broken, and thus the bioconjugate according to the invention has a significant improvement in the attachment stability of the payload to the biomolecule. When Z contains a (hetero)cyclic olefin moiety, it is preferred that the ring Z is selected from (Za), (Zh), (Zj), (Zk) or (Zl), and more preferably the ring Z is based on the structure (Za) or (Zl).

[0110] In another preferred embodiment, Z is selected from the structures (Z21) – (Z38d) described below: In this document, the connection with L is depicted using a tilde. Structure (Z29) can be an internal or external configuration, preferably an internal configuration. In structure (Z38), B (–) It is an anion, preferably a pharmaceutically acceptable anion. B (+) It is a cation, preferably a pharmaceutically acceptable cation. The ring Z is selected from the structure (Za) – (Zm) as defined above.

[0111] In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety or a (hetero)cycloheptene moiety, preferably according to structures (Z8), (Z26), (Z27), (Z28), (Z37), or (Z38a), which may optionally be substituted. Each of these preferred options for Z is further defined below.

[0112] Therefore, in a preferred embodiment, Z comprises a heterocyclic heptene moiety according to structure (Z37) or (Z38a), which is optionally substituted. Preferably, the heterocyclic heptene moiety according to structure (Z37) or (Z38a) is not substituted.

[0113] In a preferred embodiment, Z comprises a heterocyclooctene moiety according to structure (Z8), more preferably according to (Z29), which is optionally substituted. Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is not substituted. In the context of this embodiment, Z preferably comprises a heterocyclooctene moiety according to structure (Z39) as shown below, wherein V is (CH2). l And l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, even more preferably l is 0, 1 or 2, and most preferably l is 0 or 1. In the context of group (Z39), l is most preferably 1. Most preferably, Z is further defined below according to structure (Z42).

[0114] In alternative preferred embodiments, Z comprises a (hetero)cyclooctene moiety according to structure (Z26), (Z27), or (Z28), which is optionally substituted. In the context of this embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z40) or (Z41) as shown below, wherein Y 1 Is it O or NR? 11 , where R 11 Independently selected from the following groups: hydrogen, straight-chain or branched C1–C 12 alkyl groups or C4–C 12 (Hetero)aryl groups. The aromatic ring in (Z40) is optionally O-sulfated at one or more positions, while the ring in (Z41) may be halogenated at one or more positions. Preferably, the (hetero)cyclic octene moiety according to structure (Z40) or (Z41) is not further substituted. Most preferably, Z is according to structure (Z43), as further defined below.

[0115] In an alternative preferred embodiment, Z comprises a heterocyclic heptenyl group and is according to structure (Z37). In a particularly preferred embodiment, Z comprises a cyclooctenyl group and is according to structure (Z42): (Z42) In this article: - The key marked with * connects to AB, while the tilde key marked with ** connects to L; -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2 -CN、-S(O)2R 16 -S(O)3 (-) C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups; -R 18 Independently select from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups; -R 19 Choose from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups, which are optionally interrupted by one or more heteroatoms selected from the group consisting of: O, N, and S, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups, and (hetero)arylalkyl groups are independently and optionally substituted, or R 19 It is the second occurrence of Z (or Q) or D connected via a gap; and -l is an integer in the range of 0 to 10. In a preferred embodiment of the group according to structure (Z42), R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 C1–C6 alkyl groups, C5–C6 (hetero)aryl groups, wherein R 16 It is hydrogen or C1–C6 alkyl, more preferably R 15 Independently selected from the group consisting of hydrogen and C1–C6 alkyl groups, most preferably all R 15 All are H. In a preferred embodiment of the group according to structure (Z42), R 18 Independently selected from the group consisting of hydrogen and C1–C6 alkyl groups, most preferably two R groups. 18 All are H. In a preferred embodiment of the group according to structure (Z42), R 19 It is H. In a preferred embodiment of the group according to structure (Z42), l is 0 or 1, more preferably l is 1. In a particularly preferred embodiment, Z comprises a (hetero)cyclooctenyl group and is according to structure (Z43): (Z43) In this article: - The key marked with * connects to AB, while the tilde key marked with ** connects to L; -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups; -Y is N or CR15 ; - The carbon atom in the fused aromatic ring can be replaced by a nitrogen atom as shown in (Z6a)–(Z6d), preferably where Y is a nitrogen atom. 15 .

[0116] In a preferred embodiment of the group according to structure (Z43), R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -S(O)3 (-) C1–C6 alkyl groups, C5–C6 (hetero)aryl groups, wherein R 16 It is hydrogen or C1–C6 alkyl, more preferably R 15 Independently select from the following groups: hydrogen and -S(O)3 (-) In a preferred embodiment of the group according to structure (Z43), Y is N or CH, more preferably Y = N.

[0117] In a particularly preferred embodiment, Z comprises a heterocyclic heptenyl group and is according to structure (Z37) or (Z38a), wherein ring Z is a triazole. In an alternative preferred embodiment, the linking group Z comprises a (hetero)cycloalkane moiety, i.e., described as – – – The bond is a single bond. The (hetero)cycloalkane group can also be referred to as a heterocycloalkyl group or cycloalkyl group, preferably a cycloalkyl group, wherein the (hetero)cycloalkyl group is optionally substituted. Preferably, the (hetero)cycloalkyl group is a (hetero)cyclopropyl group, a (hetero)cyclobutyl group, a norbornyl group, a norbornenyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group, all of which may be optionally substituted. Particularly preferred are the (hetero)cyclopropyl group, the (hetero)cycloheptyl group, or the (hetero)cyclooctyl group, wherein the (hetero)cyclopropyl group, the (hetero)cycloheptyl group, or the (hetero)cyclooctyl group is optionally substituted. Preferably, Z comprises a cyclopropyl moiety according to structure (Z44), a heterocyclic butane moiety according to structure (Z45), a norbornene or norbornene group according to structure (Z46), a (hetero)cyclic heptyl moiety according to structure (Z47), or a (hetero)cyclic octyl moiety according to structure (Z48). In this document, Y... 3 Selected from C(R) 23 2. NR 23 Or O, where each R 23 It is hydrogen alone, C1–C6 alkyl, or linked to L (optionally via a spacer), and is labeled with – – –The bond is a single or double bond. In another preferred embodiment, the cyclopropyl group is according to structure (Z49). In yet another preferred embodiment, the (hetero)cycloheptyl group is according to structure (Z50) or (Z51). In yet another preferred embodiment, the (hetero)cyclooctyl group is according to structure (Z52), (Z53), (Z54), (Z55), or (Z56). In this document, one or more R groups on Si in (Z50) and (Z51) are typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is formed during a cycloaddition reaction and is typically selected from the structure (Zn)–(Zu), where the carbon atom marked with ** corresponds to the two carbon atoms of the (hetero)cycloalkane ring fused with ring Z in (Z44)–(Z56), and the carbon marked with * is connected to AB. Since, in the context of this embodiment, the linking group Z is formed by reaction with a (hetero)cycloalkene, it is described above as... – – – The key is a single key. In this article, R 29 Selected from hydrogen, C 1-6 Alkyl, aryl, C(O)-C 1-6 Alkyl, C(O)-aryl, C(O)-OC 1-6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1-6 Alkyl and C(O)-NR 33 -Aryl, where R 33 Is it H or C? 1-4 Alkyl group. Preferably, R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl, and pyrimidinyl. It has been found that when R... 29 When the hydrogen is present, the reactivity in the cycloaddition reaction, especially in the case of forming the (Zu) ring, is optimal. Therefore, in a preferred embodiment, ring Z is (Zu), wherein R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl and pyrimidinyl, more preferably R 29 It is hydrogen.

[0118] When Z contains a (hetero)cycloalkane moiety, it is preferred that ring Z is selected from (Zn), (Zs), (Zt) or (Zu), and most preferably ring Z is according to the structure (Zu).

[0119] In a preferred embodiment, the linking group Z comprises a portion selected from (Z1) – (Z56), wherein the ring Z is selected from (Za) – (Zu).

[0120] In this invention, Z1 and Z 2 The exact structure is typically different because Z 1 Through Q 1 and F 1 The reaction formed by Z, and 2 Through Q 2 and F 2 The reaction forms. Preferably, in this paper, F 2 Q 2 It is reactive, but to Q 1 It is not reactive, therefore Q 1 and Q 2 They should be different.

[0121] In a preferred embodiment, F 1 It is an azide and Q 1 It is a benzo-cyclic or tetramethylated (hetero)cyclic alkyne, while F 2 It is tetrazine, and Q 2 It is a bicyclic nonyne. In this paper, F 1 and Q 1 The reaction will preferably form a linking group Z according to the structure (Z5), (Z6), (Z7), (Z11), (Z17), (Z18), (Z19), or (Z19a). 1 Wherein ring Z is based on structure (Za), preferably on structure (Z26), (Z27), (Z28), (Z32), (Z37), (Z38) or (Z38a), more preferably on structure (Z40), (Z41) or (Z43). In this document, F 2 and Q 2 The reaction will preferably form a linking group Z according to the structure (Z8). 2 The ring Z is based on structure (Z1), preferably on structure (Z29), and more preferably on structure (Z42).

[0122] In yet another preferred embodiment, F 1 It is an azide and Q 1 It is a cyclic alkyne, and Q 2 It is a nitroketone, an ortho-quinone, or a tetrazine, and F 2 It is a cyclic olefin, preferably trans-cyclooctene. In this paper, F 1 and Q 1 The reaction will preferably form a linking group Z according to the structure (Z2) – (Z20). 1 Where ring Z is based on structure Za, preferably based on (Z8). In this paper, F 2 and Q 2 The reaction will preferably form a linking group Z according to the structure (Z44) – (Z56).2 The ring Z is based on the structure (Zo), (Zp), (Zq), (Zr), (Zq), preferably based on the structure (Zo) or (Zu), and more preferably based on the structure (Zo).

[0123] In another preferred embodiment, Z 1 It can be obtained through a click reaction between cycloalkynes and azides, while Z 2 It can be obtained through a click reaction between cycloalkynes and nitrones.

[0124] In the second preferred embodiment, Z, preferably Z 1 Formed via a nucleophilic reaction, preferably via nucleophilic substitution or Michael addition, more preferably via Michael addition. A preferred Michael reaction is a thiol-maleimide linkage, most preferably where Q is a maleimide and F is a thiol group, wherein the thiol may be part of a disulfide bridge. Preferably, the thiol is present in the side chain of a cysteine ​​residue. Such a coupling reaction with a thiol can also be referred to as thiol alkylation or thiol arylation. In a preferred embodiment, the linking group Z... 1 Containing a succinimide ring or its open-ring succinic amide derivative, which can be formed by hydrolysis of the succinimide ring. In cases where nucleophilic reactions are used for coupling, the preferred structural moiety is Q-(L... 1 ) a -BM-(L 2 bQ is selected from bromomaleimide, bisbromomaleimide, bis(benzenethiol)maleimide, bisbromopyridazine dione, bis(halomethyl)benzene, bis(halomethyl)pyridazine, bis(halomethyl)pyridine, or bis(halomethyl)triazole.

[0125] Alternatively, Z 1 Formed via a nucleophilic reaction at the amino group in the side chain of a lysine residue (F), this amino group can react with an amino reactive group Q. Such coupling reactions with thiols can also be referred to as amide bond formation or urethane bond formation. Typical amino reactive groups Q include N-hydroxysuccinimide (NHS) esters, p-nitrophenyl carbonates, and pentafluorophenyl carbonates. 、 Isocyanates, isothiocyanates, and benzoyl halides.

[0126] Linking group Z 1 The preferred options include the portions selected from (Z57) – (Z71) described below. In this document, one or more tilde keys marked with * in (Z57)-(Z66) are connected to AB via connector L, and unmarked tilde keys are connected to the payload via connector L. Additionally, R... 29It is C 1-12 Alkyl, preferably C 1-4 Alkyl, most preferably ethyl, and X 1 It is O or S, preferably X 1 = O. The nitrogen atoms marked with ** in (Z67)-(Z71) correspond to the nitrogen atoms of the side chains of the lysine residues of the antibody, and the unmarked wavy bonds are linked to the payload via linker L. The carbon atoms of the phenyl groups in (Z69) and (Z70) are optionally substituted, preferably optionally fluorinated.

[0127] Connector L Linker L via linker group Z 2 The payload D and the linking group Z 1 The linker can be connected (in the conjugate according to the invention), or the payload D can be linked to the reactive group Q (in the linker-cytokine construct), or the antibody can be linked to the reactive group (in the linker-antibody construct). Linkers are known in the art and can be cleavable or non-cleavable. In the case where the linker contains a cleavable site, the linker L is referred to as cleavable. Typically, the cleavable site is represented by L. 2 and L 3 Formation. Therefore, in the preferred embodiment, D 1 There is no (i.e., α = 0) and L 2 and L 3 It exists (i.e., o = p = 1), or D 1 There exists (i.e., α = 1) and L 2 and L 3 It does not exist (i.e., o = p = 0). Most preferably, D 1 Existence and L 2 and L 3 It does not exist.

[0128] In a preferred embodiment, the connector L is represented by the following structure. –(L 1 )–(L 2 ) o –(L 3 ) p –(L 4 ) q – in: -L 1 With Z 1 Connect, and L 4 (If it exists) with Z 2 connect; -L 1 L 2 L3 and L 4 Each individually is Z 1 With Z 2 Connectors that are linked together; -o, p, and q are each individually 0 or 1, preferably o = p = q = 0.

[0129] Connector L 1 L 1 For example, you can choose from groups consisting of straight chains or branches C1-C. 200 alkylene groups, C2-C 200 imide groups, C2-C 200 Imynyl group, C3-C 200 Cycloalkyl groups, C5-C 200 Cycloalkylene groups, C8-C 200 cycloalkynyl group, C7-C 200 alkylarylene groups, C7-C 200 arylalkylene groups, C8-C 200 aryl imidene group, C9-C 200 Arylynylene group. Optionally, the alkylene group, alkenylene group, ynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylynylene group can be substituted, and optionally, the group can be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, said heteroatoms preferably selected from the group consisting of: O, S(O). y’ and NR 21 , where y' is 0, 1 or 2, preferably y' = 2, and R 21 Independently select from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups. In one embodiment, optional substituents may be selected from polar groups, such as oxo groups, (poly)ethylene glycol diamine, (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, carboxylic acid groups, carbonate groups, carbamate groups, cyclodextrins, crown ethers, sugars (e.g., monosaccharides, oligosaccharides), phosphoric acid or its esters, phosphonic acid or esters, hypophosphonic acid or esters, sulfoxides, sulfones, sulfonic acids or esters, sulfinic acids or hyposulfonic acids.

[0130] In a preferred embodiment, connector L 1 It contains a polar group, which can also be present in L1 The polar group can be selected from (poly)ethylene glycol diamine (e.g., 1,8-diamino-3,6-dioxane or equivalents containing a longer ethylene glycol chain), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, and 1,x'-diaminoalkanes (where x' is the number of carbon atoms in the alkane, preferably x' = 1–10), –(O). a –C(O)–NH–S(O)2–NR 13 – (as further defined below, see structure (23)), – C(S(O)3 (–) –、–C(C(O)2 (–) –S(O)2–, –P(O)2 (–) –、–O(CH2CH2O) t –、–NR 30 (CH2CH2NR 30 ) t – and the following two structures: For the polar group defined above, which end is attached to Z? 1 Which end is connected to (L) 2 ) o It's irrelevant.

[0131] The polar group may also contain an amino acid, including non-natural amino acids, preferably selected from Arg, Glu, Asp, Ser, Thr, or sulfoalanine. In this document, R is further defined below for structure (23). 13 t is an integer in the range of 0–15, preferably 1–10, more preferably 2–5, and most preferably t = 2 or 4. Each R 30 H and C alone 1-12 Alkyl, C 1-12 Aryl, C 1-12 alkylaryl or C 1-12 Aryl group. Linker L 1 It may contain more than one such polar group, such as at least two polar groups. Polar groups may also be present in the linker L. 1 In the branch, this branch originates from a branch portion defined elsewhere. In L 1 In this context, nitrogen or carbon atoms are preferably used as branching parts. Particularly preferred is the presence of –O(CH2CH2O) within the branch. t - Polar groups.

[0132] In a preferred embodiment, connector L 1It is or contains a sulfonamide group, preferably a sulfonamide group according to structure (23): (twenty three) The wavy line indicates a connection to the rest of the compound, typically associated with Q. 2 or Z 2 and with L 2 L 3 L 4 Or a connection of D. Preferably, (O) a C(O) part is connected to Q 2 or Z 2 And NR 13 Partially connected to L 2 L 3 L 4 Or D, preferably connected to L 2 .

[0133] In structure (23), a = 0 or 1, preferably a = 1, and R 13 Choose from the following groups: hydrogen, C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups, these C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups are optionally substituted and optionally selected from O, S and NR. 14 One or more heteroatoms are interrupted, where R 14 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups. Alternatively, R 13 It is optional via the interval portion, preferably via Sp as defined below. 2 In one embodiment, D connected to N is via –(K) 2 ) e –(K 1 ) f –(K 2 ) g –C(O)- is connected to N. Alternatively, R 13 Optionally connected to other parts of the connector via spacers to form a ring structure. For example, R 13It can be connected to the linker via the CH2CH2 spacer to form a piperazine ring, wherein the connection with D is via the second nitrogen of the piperazine ring.

[0134] In a preferred embodiment, R 13 It is hydrogen, C1–C 20 Alkyl groups, preferably C1–C 16 Alkyl groups, more preferably C1–C 10 An alkyl group, or optionally linked via a spacer portion to another D or to other locations on the linker. In this document, the alkyl group is optionally substituted and optionally selected from O, S, and NR. 14 Preferably, one or more heteroatoms of O are interrupted, wherein R 14 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups. In another preferred embodiment, R 13 It is C1–C 20 Alkyl groups, more preferably C1–C 16 Alkyl groups, or even more preferably C1–C 10 An alkyl group, wherein the alkyl group is optionally interrupted by one or more O atoms, and wherein the alkyl group is optionally substituted with a -OH group, preferably a terminal -OH group. In this embodiment, R is further preferred. 13 It is a (poly)ethylene glycol chain containing terminal -OH groups. In another preferred embodiment, R 13 The group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, and tert-butyl is selected, or optionally linked to other parts of the linker via a spacer portion. More preferably, the group consisting of hydrogen, methyl, ethyl, n-propyl, and isopropyl is selected, or optionally linked to another D or to other parts of the linker via a spacer portion. Even more preferably, the group consisting of hydrogen, methyl, and ethyl is selected, or optionally linked to another D or to other parts of the linker via a spacer portion. Even more preferably, R... 13 It is hydrogen or optionally connected via a spacer portion to another D or to other locations connected to the linker, and most preferably R 13 It is hydrogen.

[0135] In a preferred embodiment, L 1 It is based on structure (24): (twenty four) In this paper, a and R 13 As defined above, Sp 1 and Sp 2Each is an independent spacer portion, and b and c are independently 0 or 1. Preferably, b = 0 or 1 and c = 1, more preferably b = 0 and c = 1. In one embodiment, the spacer Sp 1 and Sp 2 Independently select from the following groups: straight chain or branched chain C1-C 200 alkylene groups, C2-C 200 imide groups, C2-C 200 Imynyl group, C3-C 200 Cycloalkyl groups, C5-C 200 Cycloalkylene groups, C8-C 200 cycloalkynyl group, C7-C 200 alkylarylene groups, C7-C 200 arylalkylene groups, C8-C 200 aryl imide groups and C9-C 200 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynylene group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1–C 24 alkyl groups, C2–C 24 alkenyl groups, C2–C 24 alkynyl groups and C3–C 24 Cycloalkyl groups, including alkyl groups, alkenyl groups, ynyl groups, and cycloalkyl groups, are optionally substituted. When the alkylene group, alkenyl group, ynylene group, cycloalkylene group, cycloalkylene group, cycloalkylene group, cycloalkylene group, alkylarylene group, arylalkylene group, arylalene group, and arylynylene group are interrupted by one or more heteroatoms as defined above, it is preferred that the group is interrupted by one or more O atoms and / or by one or more SS groups. More preferably, the interval portion Sp 1 and Sp 2 (If present) Independently select from the following groups: straight chains or branches C1-C 100 alkylene groups, C2-C 100 imide groups, C2-C 100 Imynyl group, C3-C 100 Cycloalkyl groups, C5-C 100 Cycloalkylene groups, C8-C 100 cycloalkynyl group, C7-C 100 alkylarylene groups, C7-C100 arylalkylene groups, C8-C 100 aryl imide groups and C9-C 100 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynylene group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1–C 24 alkyl groups, C2–C 24 alkenyl groups, C2–C 24 alkynyl groups and C3–C 24 Cycloalkyl groups, of which alkyl groups, alkenyl groups, ynyl groups and cycloalkyl groups may optionally be substituted.

[0136] Even more preferably, the interval portion Sp 1 and Sp 2 (If present) Independently select from the following groups: straight chains or branches C1-C 50 alkylene groups, C2-C 50 imide groups, C2-C 50 Imynyl group, C3-C 50 Cycloalkyl groups, C5-C 50 Cycloalkylene groups, C8-C 50 cycloalkynyl group, C7-C 50 alkylarylene groups, C7-C 50 arylalkylene groups, C8-C 50 aryl imide groups and C9-C 50 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynylene group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1–C 24 alkyl groups, C2–C 24 alkenyl groups, C2–C 24 alkynyl groups and C3–C 24 Cycloalkyl groups, of which alkyl groups, alkenyl groups, ynyl groups and cycloalkyl groups may optionally be substituted.

[0137] Even more preferably, the interval portion Sp 1 and Sp2 (If present) Independently select from the following groups: straight chains or branches C1-C 20 alkylene groups, C2-C 20 imide groups, C2-C 20 Imynyl group, C3-C 20 Cycloalkyl groups, C5-C 20 Cycloalkylene groups, C8-C 20 cycloalkynyl group, C7-C 20 alkylarylene groups, C7-C 20 arylalkylene groups, C8-C 20 aryl imide groups and C9-C 20 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynylene group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1–C 24 alkyl groups, C2–C 24 alkenyl groups, C2–C 24 alkynyl groups and C3–C 24 Cycloalkyl groups, of which alkyl groups, alkenyl groups, ynyl groups and cycloalkyl groups may optionally be substituted.

[0138] In these preferred embodiments, it is further preferred that the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalene group, and arylalkynylene group are unsubstituted and optionally selected from O, S, and NR. 16 One or more heteroatoms of the group, preferably O interrupted, wherein R 16 It is independently selected from the group consisting of hydrogen and C1–C4 alkyl groups, preferably hydrogen or methyl.

[0139] Most preferably, the interval portion Sp 1 and Sp 2 (If present) Independently select from the following groups: straight chains or branches C1-C 20 Alkylene groups, which are optionally substituted and optionally selected from O, S, and NR. 16 One or more heteroatoms in the group are interrupted, where R 16 Independently select from the following groups: hydrogen, C1–C 24 alkyl groups, C2–C 24 alkenyl groups, C2–C24 alkynyl groups and C3–C 24 Cycloalkyl groups, including alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups, are optionally substituted. In this embodiment, it is further preferred that the alkylene groups are unsubstituted and optionally selected from O, S, and NR. 16 One or more heteroatoms of the group, preferably O and / or SS interrupted, wherein R 16 It is independently selected from the group consisting of hydrogen and C1–C4 alkyl groups, preferably hydrogen or methyl.

[0140] Therefore, the preferred interval portion Sp 1 and Sp 2 Includes -(CH2) r -、-(CH2CH2) r -、-(CH2CH2O) r -、-(OCH2CH2) r -、-(CH2CH2O) r CH2CH2-、-CH2CH2(OCH2CH2) r -、-(CH2CH2CH2O) r -、-(OCH2CH2CH2) r -、-(CH2CH2CH2O) r CH2CH2CH2- and -CH2CH2CH2(OCH2CH2CH2) r - where r is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, and even more preferably in the range of 1 to 15. More preferably n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably 1, 2, 3, 4, 5 or 6, and even more preferably 1, 2, 3 or 4.

[0141] Alternatively, the preferred connector L 1 It can be represented by the following: –(K 3 ) k –(K 1 ) d –(K 2 ) e –(K 1 ) f –(C(O)) g –or[–(K) 3 ) k –(K 1 ) d –(K 2 ) e –(K1 ) f –]2BM–(C(O)) g – (K 1 ) d’ –(K 2 ) e’ –(K 1 ) f’ –(C(O)) g’ -,in -d and d' are either 0 or 1 individually. -e and e' are individually integers in the range of 1–10. -f and f' are either 0 or 1 individually. -g and g' are integers in the range of 0–10, -k = 0 or 1, provided that if k = 1, then d = 0. -K 1 It is based on the sulfonamide group in structure (23) (twenty three), -K 2 It is the –CH2–CH2–O– or –O–CH2–CH2– part, or (K 2 ) e It is –(CH2–CH2–O) e1 –CH2–CH2– or –(CH2–CH2–O) e1 –CH2– part, where the definition of e1 is the same as that of e; -K 3 is –OC(O)–, –C(O)O–, –C(O)NH–, –NHC(O)–, –OC(O)NH–, –NHC(O)O–, –C(O)(CH2) m C(O)–、–C(O)(CH2) m C(O)NH– or –(4-Ph)CH2NHC(O)(CH2) m C(O)NH–, preferably where K 3 It is –OC(O)NH–, –C(O)(CH2) m C(O)NH– or –C(O)NH–, where m is an integer in the range of 0–10, preferably m = 0, 1, 2, 3, 4, 5 or 6, and most preferably m = 2 or 3; - Preferably, where L 1 Via (K) 3 ) k Connected to Q and via (C(O)) g Preferably connected to L via C(O) 2L 3 Or D, preferably connected to L 2 ; -BM is a branch portion, preferably selected from carbon atom, nitrogen atom, phosphorus atom, (hetero)aromatic ring, (hetero)cyclic or polycyclic portion, more preferably BM is nitrogen atom.

[0142] In the context of this embodiment, the wavy line in structure (23) indicates a group (such as (K)) adjacent to a group (such as (K) 3 ) k 、(K 2 ) e and (C(O)) g The connection of K. Preferably, K 1 It is based on structure (23), where a = 1 and R 13 = H or C1–C 20 Alkyl groups, more preferably R 13 = H or methyl, most preferably R 13 = H.

[0143] Preferred connector L 1 Having structure –(K 3 ) k –(K 1 ) d –(K 2 ) e –(K 1 ) f –(C(O)) g -,in: (a)k = 0; d = 1; g = 1; f = 0; K 2 = –CH2–CH2–O–; e = 1, 2, 3 or 4, preferably e = 2.

[0144] (b) k = 1; K 3 = –C(O)(CH2) m C(O)NH–; m = 2; d = 0; (K 2 ) e = –(CH2–CH2–O) e1 –CH2–CH2–; f = 0; g = 1; e1 = 1, 2, 3 or 4, preferably e = 1.

[0145] (c) k = 1; K 3 = –OC(O)NH–;d = 0;K 2 = –CH2–CH2–O–; g = 1; f = 0; e = 1, 2, 3 or 4, preferably e = 2.

[0146] (d)k = 1;K 3 = –C(O)(CH2) m C(O)NH–; m = 2; d = 0; (K 2 ) e = –(CH2–CH2–O) e1 –CH2–CH2–; f = 0; g = 1; e1 = 1, 2, 3 or 4, preferably e1 = 4.

[0147] (e)k = 1;K 3 = –OC(O)NH–;d = 0;(K 2 ) e = –(CH2–CH2–O) e1 –CH2–CH2–; g = 1; f = 0; e1 = 1, 2, 3 or 4, preferably e1 = 4.

[0148] (f)k = 1;K 3 = –(4-Ph)CH2NHC(O)(CH2) m C(O)NH–, m = 3; d = 0; (K 2 ) e = –(CH2–CH2–O) e1 –CH2–CH2–; g = 1; f = 0; e1 = 1, 2, 3 or 4, preferably e1 = 4.

[0149] (g)k = 0; d = 0; g = 1; f = 0; K 2 = –CH2–CH2–O–; e = 1, 2, 3 or 4, preferably e = 2.

[0150] (h)k = 1;K 3 = –C(O)NH–; d = 0; g = 1; f = 0; K 2 = –CH2–CH2–O–; e = 1, 2, 3 or 4, preferably e = 2.

[0151] In this paper, it is preferred that a = 1 and R = 1 when d and / or f = 1. 13 = H. The optimal choice is that the connector is structure (a).

[0152] In a preferred embodiment, connector L 1 It contains a branched nitrogen atom located at Q. 1 or Z 1 With (L) 2 )o The main chain between them contains another part Z. 1 Or Q 1 As a substituent, this portion is preferably linked to the branched nitrogen atom via a linker. An example of a branched nitrogen atom is the nitrogen atom NR in structure (23). 13 , where R 13 The branched nitrogen atom connects to the second D atom via a spacer portion. Alternatively, the branched nitrogen atom can be located according to the structure –(K 3 ) k –(K 1 ) d –(K 2 ) e –(K 1 ) f –(C(O)) g – of L 1 Inside. In one embodiment, L 1 By [–(K 3 ) k –(K 1 ) d –(K 2 ) e –(K 1 ) f –(C(O)) g –]2BM–(K 1 ) d –(K 2 ) e –(K 1 ) f –(C(O)) g – indicates that K 1 K 2 K 3 , d, e, f, g, and k are as defined above and are selected individually each time they appear, and BM is the branch portion, preferably branching nitrogen atoms, –(K 1 ) d –(K 2 ) e –(K 1 ) f –(C(O)) g – Two instances are connected to this branch. In this paper, two (C(O)) g All of them are related to –(L) 2 ) o –(L 3 ) p –(L 4 ) q –D connection, where L 2 L 3 L 4, o, p, q, and D are as defined above and each is selected individually. In a preferred embodiment, L 2 L 3 L 4 Each of , o, p, q and D for (C(O)) g Both parts of the connection are identical.

[0153] Preferred linker L containing branched atoms 1 Having structure [–(K 3 ) k –(K 1 ) d –(K 2 ) e –(K 1 ) f –(C(O)) g –]2N–(K 1 ) d’ –(K 2 ) e’ –(K 1 ) f’ –(C(O)) g’ -,in: (i)k = d = g = e' = 1; f = d' = g' = 0; K 3 = –C(O)–;K 2 = –CH2–CH2–O–;K 1 It is based on structure (23), where a = 0 and R 13 = H; e = 1, 2, 3 or 4, preferably e = 2.

[0154] (j)k = d = g = e' = g' = 1; f = d' = 0; K 3 = –C(O)–;K 2 = –CH2–CH2–O–;K 1 It is based on structure (23), where a = 0 and R 13 = H; e = 1, 2, 3 or 4, preferably e = 2.

[0155] Connector L 2 Connector L 2 It is a peptide spacer. Linker L 2 It either does not exist (o = 0) or it exists (o = 1). Peptide spacer L 2 and cuttable connector L 3 The combination of these is well known in the art. Connector L2 More preferably, peptides are recognized and cleaved by specific cleavage enzymes, serving as recognition and cleavage sites. This allows cleavage to occur in specific environments (such as specific tumors) where these cleavage enzymes are expressed. Because different peptide sequences are cleaved by different enzymes, L... 2 The group also allows for the customization of conjugates for specific treatments.

[0156] Peptide spacers can also be defined as (NH–CR) 17 –CO) n , where R 17 This refers to an amino acid side chain as known in the art. This definition also covers proline, which has an R atom linked to a nitrogen atom to form a cyclic moiety. 17 In this document, amino acids can be natural or synthetic. Preferably, one or more amino acids are in their L-configuration. n is an integer in the range of 1–5, preferably in the range of 2–4. Therefore, the peptide spacer contains 1–5 amino acids. Preferably, the peptide is a dipeptide (n = 2), a tripeptide (n = 3), or a tetrapeptide (n = 4), and most preferably, the peptide spacer is a dipeptide. R 17 The term "amino acid side chain" is preferably selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolidone, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine, and citrulline. Preferred amino acid side chains are those of Val, Cit, Ala, Lys, Arg, AcLys, Phe, Leu, Ile, Trp, Glu, Asp, and Asn, more preferably from Val, Cit, Ala, Glu, and Lys. In other words, R 17 Preferably selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp) and CH2(1 H -Indole-3-yl) (Trp). R 17Particularly preferred embodiments are CH3 (Ala), CH2CH2CH2NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), CH2CH2C(O)OH (Glu), and CH(CH3)2 (Val). Most preferably, R 17 It is CH3 (Ala), CH2CH2CH2NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), or CH(CH3)2 (Val).

[0157] Although any peptide spacer can be used, preferably, the peptide spacer is selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Glu-Gly-Cit, Glu-Gly-Val, Asp-Val-Cit, iGlu-Val-Cit, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Asn-Asn, Ala-Ala-Asn, Ala-Asn, Asn-Ala, Phe-Phe, Gly, Gly-Gly, Gly-Gly-Gly, Gly-Gly-Gly (SEQ ID NO: 100000). NO: 19), Leu-Gly, Tyr-Gly, Ala-Gly, Pro-Gly, Phe-Gly, Phe-Gly, Ser-Gly, Gly-Phe-Gly, Gly-Gly-Phe-Gly (SEQ. ID NO: 20), Gly-Phe-Gly-Gly (SEQ. ID NO: 21), Phe-Gly-Gly-Gly (SEQ. ID NO: 22), Gly-Gly-Gly-Phe (SEQ. ID NO: 23), Phe-Phe-Gly-Gly (SEQ. ID NO: 24), Gly-Gly-Phe-Phe (SEQ. ID NO: 25), Gly-Gly-Gly-Phe-Gly (SEQ. ID NO: 25) 26) and Lys, more preferably selected from Val-Cit, Val-Ala, Glu-Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably selected from Glu-Gly-Cit, Val-Cit, Val-Ala, Asn-Asn, Ala-Ala-Asn, Asn-Ala, and most preferably selected from Glu-Gly-Cit, Val-Cit, Val-Ala, or Asn-Ala. In this document, AcLys is eN-acetyllysine and iGlu is isoglutamate. In one embodiment, L 2 = Val-Cit. In another embodiment, L 2 =Val-Ala. In another embodiment, L 2 = Asn-Ala. In another embodiment, L 2= Glu-Gly-Cit.

[0158] R 17 The term "amino acid side chain" is preferably selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolidone, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine, and citrulline. Preferred amino acid side chains are those of Val, Cit, Ala, Lys, Arg, AcLys, Phe, Leu, Ile, Trp, Glu, Asp, and Asn, more preferably from Val, Cit, Ala, Glu, and Lys. In other words, R 17 Preferably selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp) and CH2(1 H -Indole-3-yl) (Trp). R 17 Particularly preferred embodiments are CH3 (Ala), CH2CH2CH2NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), CH2CH2C(O)OH (Glu), and CH(CH3)2 (Val). Most preferably, R 17 It is CH3 (Ala), CH2CH2CH2NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), or CH(CH3)2 (Val).

[0159] In one embodiment, amino acid side chain R 17 The substance is replaced by a polar group, preferably selected from oxo groups, (poly)ethylene glycol diamine, (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, carboxylic acid groups, carbonate groups, carbamate groups, cyclodextrins, crown ethers, sugars (e.g., monosaccharides, oligosaccharides), phosphoric acid or its esters, phosphonic acid or esters, hypophosphonic acid or esters, sulfoxides, sulfones, sulfonic acids or esters, sulfinic acids or hyposulfonic acids.

[0160] In a particularly preferred embodiment, L 2Contains peptide spacers represented by a universal structure (25), preferably L 2 Represented by the general structure (25): (25) In this article, R 17 As defined above, preferably R 17 It is CH3(Ala) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line indicates that it is related to (L) 1 ) n and (L) 3 ) p The connection, preferably, is based on the L of structure (25). 2 Connected to (L) via NH 1 ) n And connected to (L) via C(O) 3 ) p .

[0161] L 2 It may contain certain peptide sequences that can be cleaved by specific enzymes. If those enzymes are expressed or overexpressed only in the tumor microenvironment or endosome / lysosomal compartments and leak into the tumor microenvironment of cancer cells, the probability of targeted release of cytokines in tumors increases, while release in healthy tissues decreases.

[0162] Connector L 3 Connector L 3 It is a self-cutting spacer, also known as a self-destructing spacer. Connector L 3 It either doesn't exist (p = 0) or it exists (p = 1). L 2 The cutting caused the connector L 3 The elimination of 1,6-β in L leads to decarboxylation and the release of payload D. This advantageously allows for the cleavage of L 2 The region of enzyme overexpression increases the probability of payload release. Furthermore, payload release can induce bystander killing, which is advantageous because not all cancer cells in a tumor overexpress the target receptor. Therefore, it is preferable that the linker L... 2 and L 3 Both exist (o = p = 1) or neither exists (o = p = 0).

[0163] Preferably, L 3 It is a p-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (L3a): (L3a) In this article, the wavy line indicates the L 1 or L 2 and with L 4 Or the connection of D. Parentheses indicate an optional carbonyl group. Preferably, a carbonyl group is present. Typically, PABC derivatives are derived via NH and L 1 or L 2 Connection, preferably with L 2 Connect, and via OC(O) to L 4 Or D connection.

[0164] Ring A is a 5- or 6-membered aromatic ring or a heteroaromatic ring, preferably a 6-membered aromatic ring or a heteroaromatic ring. Suitable 5-membered rings are oxazoles, thiazoles, and furans. Suitable 6-membered rings are phenyl and pyridyl groups. Ring A can be selected from halogens, X... 2 R 4 、N(R 4 2. C 1-4 Alkyl groups and NO2 substituents are used for substitution. In this paper, X... 2 and R 4 As defined above, including preferred embodiments thereof. In preferred embodiments, optional substituents are selected from F, Cl, Br, OH, OR. 4 SH, NH2, Et, Me, and NO2. In a particularly preferred embodiment, ring A contains 0–2 substituents, more preferably 0 or 1 substituent, and most preferably ring A is unsubstituted. In a preferred embodiment, ring A is 1,4-phenyl, 1,2-phenyl, 2,5-pyridyl, or 3,6-pyridyl. Most preferably, A is 1,4-phenyl.

[0165] R 21 Selected from H, R 26 C(O)OH and C(O)R 26 , where R 26 It is C1–C 24 (Hetero)alkyl groups, C3–C 10 (Hetero)cycloalkyl groups, C2–C 10 (Hetero)aryl group, C3–C 10 alkyl(hetero)aryl groups and C3–C 10 (Hetero)arylalkyl groups, which are optionally substituted and optionally selected from O, S, and NR. 28 One or more heteroatoms are interrupted, where R 28 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups. Preferably, R 26 It is C3–C 10 (Hetero)cycloalkyl or polyalkylene glycols. The polyalkylene glycols are preferably polyethylene glycol or polypropylene glycol, more preferably –(CH2CH2O).s H or –(CH2CH2CH2O) s H. Polyalkylene glycols are most preferably polyethylene glycol, preferably –(CH2CH2O). s H, where s is an integer in the range of 1–10, preferably 1–5, and most preferably s = 1, 2, 3, or 4. More preferably, R 21 Is it H or C(O)R 26 , where R 26 = 4-methylpiperazine or morpholine. Most preferably, R 21 It's H.

[0166] In an alternative embodiment, connector L 3 There exists (p = 1) and connector L 2 It does not exist (o = 0), and L 3 It is a glucuronide-m-amide-benzyloxycarbonyl derivative, preferably a glucuronide derivative according to structure (L3b): (L3b) In this article, the wavy line indicates the L 1 and with L 4 Or the connection of D. Parentheses indicate an optional carbonyl group. Preferably, a carbonyl group is present. Typically, the glucuronide derivative is linked to L via NH. 1 And connected to L via (O)CO 4 Or D. Rings A and R 21 As defined for PABC derivatives according to structure (L3a). Preferably, ring A is a 6-membered aromatic or heteroaromatic ring, such as oxazole, thiazole, furan, phenyl, and pyridyl. In preferred embodiments, ring A is 1,3,4-phenyl, 2,4,5-pyridyl, or 2,5,6-pyridyl. Most preferably, A is 1,3,4-phenyl. Preferably, R... 21 Is it H or C(O)R 26 , where R 26 = 4-methylpiperazine or morpholine. Most preferably, R 21 It's H.

[0167] According to the connector L of structure (L3b) 3It can be cleaved by β-glucuronidase, similar to the mechanism in PABC, which leads to the self-destruction, decarboxylation, and release of the payload of the p-hydroxybenzyloxy group. ADCs comprising glucuronide derivatives according to structure (L3b) are particularly useful for treating cancers with β-glucuronidase overexpression. It has been reported that many solid tumors (including lung cancer, breast cancer, and gastrointestinal cancer) and the tumor microenvironment exhibit higher concentrations of β-glucuronidase than in normal tissues, and the enzyme is not detected in systemic circulation. Therefore, it is preferred that conjugates according to the invention comprising glucuronide derivatives according to structure (L3b) be used to treat patients with lung cancer, breast cancer, and gastrointestinal cancer.

[0168] Connector L 4 Connector L 4 It either doesn't exist (q = 0) or it exists (q = 1). Connector L 4 Selected from: -According to the structure –NR 22 –(C x -alkylene)–C(O)– aminoalkanoic acid spacers, where x is an integer in the range of 1–20, and R 22 It is an H or C1–C4 alkyl group; -According to the structure –NR 22 –(CH2–CH2–O) e6 –(CH2) e7 –C(O)– ethylene glycol spacer, where e6 is an integer in the range of 1–10, e7 is an integer in the range of 1–3, and R 22 It is H or C1–C4 alkyl; and -Based on structure NR 22 –(SO2–NH–) j (C(O)) h – amine spacer, where R 22 It is H or C1–C4 alkyl, z is an integer in the range of 1–10, j is 0 or 1, and h is 0 or 1.

[0169] Connector L 4 It can be an aminoalkanoic acid spacer, i.e., –NR 22 –(C x -alkylene)–C(O)–, where x is an integer in the range of 1 to 20, preferably 1–10, and most preferably 1–6. In this document, the aminoalkanoic acid spacer is typically located via a nitrogen atom and an L… 3 It is connected to D via a carbonyl moiety. Preferred linker L. 4The aminocaproic acid is selected from 6-aminocaproic acid (Ahx, x = 5), β-alanine (x = 2), and glycine (Gly, x = 1), and even more preferably 6-aminocaproic acid or glycine. In one embodiment, L 4 = 6-Aminohexanoic acid. In one embodiment, L 4 = Glycine. In this article, R 22 It is H or C1–C4 alkyl, preferably R 22 It is H or methyl, most preferably R. 22 It's H.

[0170] Alternatively, connector L 4 It can be based on the structure – NR 22 –(CH2–CH2–O) e6 –(CH2) e7 –(C(O)– ethylene glycol spacer, wherein e6 is an integer in the range of 1–10, preferably in the range of 2–6, and e7 is an integer in the range of 1–3, preferably e7 is 2. In this document, R 22 It is H or C1–C4 alkyl, preferably R 22 It is H or methyl, most preferably R. 22 It's H.

[0171] Alternatively, connector L 4 It can be based on the structure – NR 22 –(C x -alkylene)–NR 22 –(C(O)) h – a diamine spacer, wherein h is 0 or 1, x is an integer in the range of 1–20, preferably an integer in the range of 2–6, even more preferably x = 2 or 5, and most preferably x = 2. R 22 It is H or C1–C4 alkyl. In this article, R 22 It is H or C1–C4 alkyl, preferably R 22 It is H or methyl, most preferably R. 22 It is a methyl group. In this text, h is preferably 1, in which case the linker L... 4 It is particularly suitable for coupling via phenolic hydroxyl groups present on payload D.

[0172] Preferred connector Preferably, the connector L is selected from any of the following structures: In this document, it is preferred that x is in the range of 0-100, more preferably x is in the range of 0-50, more preferably x is in the range of 1-30, and most preferably x is in the range of 1-18. In this document, parentheses “()” indicate the optional presence of oxygen atoms.

[0173] In another preferred embodiment, α is preferably 0 and the connector L is based on one of the following structures: or .

[0174] In this article, L 2 It is a peptide sequence that serves as the recognition site for proteases.

[0175] Method for synthesizing conjugates according to general structures (1) or (2) In another aspect, the present invention relates to a method for preparing a coupling compound according to the invention, the method comprising: -Make AB(F) 1 ) x With Q 1 -LQ 2 or (Q) 1 )2–L–Q 2 reaction; - Make the resulting compound with F 2 -D reaction; Alternatively, the method may include the following steps: -Make DF 2 With Q 1 -LQ 2 or (Q) 1 )2–L–Q 2 reaction, - Make the resulting compound react with AB(F) 1 ) x reaction Where AB(F) 1 ) x It contains x reaction fractions F 1 The functionalized antibody and x is an integer in the range of 1-10, where Q 1 and F 1 They are responsive to each other, and Q 2 and F 2 They are reactive to each other, where Q 1 With F 1 Between and Q 2 With F 2 The reaction between them forms covalent bonds between the reactants.

[0176] In a preferred embodiment, x = 2. The method according to this embodiment can be represented according to schemes 1-4, and preferably the method is according to scheme 2 or 4.

[0177] Option 1 In this paper, immune cell conjugating peptides are represented by D. The reaction portion Q... 1 With F 1 The coupling reaction between them provides the linking group Z. 1 And the reaction part Q 2 With F 2 The coupling reaction between them provides the linking group Z. 2 .

[0178] Alternatively, x = 2 and the antibody-cytokine conjugate is obtained according to scheme 2.

[0179] Option 2 In yet another embodiment, x = 2 and the antibody-cytokine conjugate is obtained according to scheme 3.

[0180] Option 3 Alternatively, x = 2 and the antibody-cytokine conjugate is obtained according to scheme 4.

[0181] Option 4 Reaction part Q Reaction part Q 1 and Q 2 For F respectively 1 and F 2 It is reactive. In the context of this invention, Q refers to Qreactive. 1 and Q 2 In the context of this invention, the term "reactive moiety" can refer to the chemical moiety containing the reactive group or the reactive group itself. For example, a cyclooctyne group is a reactive group containing a reactive group, namely a C-C triple bond. However, a reactive group (e.g., an azide reactive group) may also be referred to herein as a reactive moiety. Q is reactive with and complementary to F. In this document, a reactive group is referred to as being “complementary” to another reactive group when the reactive group selectively reacts with it (optionally in the presence of other functional groups). Complementary reaction moieties are known to those skilled in the art and are described in more detail below. The exact properties of Q and F depend on the type of reaction employed, which is a nucleophilic reaction, such as Michael addition or nucleophilic substitution, or a cycloaddition reaction, such as a click reaction. The click probe is reactive in cycloaddition (click reaction) and is preferably selected from azides, tetrazides, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxothiophene, sylene, alkene moieties, and alkyne moieties. Preferably, the click probe Q comprises either an alkene moiety or an alkyne moiety, more preferably wherein the alkene is a (hetero)cyclic alkene and / or the alkyne is a terminal alkyne or a (hetero)cyclic alkyne.

[0182] In a particularly preferred embodiment, Q comprises a cyclic (heteroyne)alkynyl moiety. The alkynyl group may also be referred to as a (heteroyne)cyclic alkynyl group, i.e., a heterocyclic alkynyl group or a cycloalkynyl group, wherein the (heteroyne)cyclic alkynyl group is optionally substituted. Preferably, the (heteroyne)cyclic alkynyl group is a (heteroyne)cyclic heptyneyl group, a (heteroyne)cyclic octyneyl group, a (heteroyne)cyclic nonyneyl group, or a (heteroyne)cyclic decanyneyl group. In this document, the (heteroyne)cyclic alkyne may optionally be substituted. Preferably, the (heteroyne)cyclic alkynyl group is an optionally substituted (heteroyne)cyclic heptyneyl group or an optionally substituted (heteroyne)cyclic octyneyl group. Most preferably, the (heteroyne)cyclic alkynyl group is a (heteroyne)cyclic octyneyl group, wherein the (heteroyne)cyclic octyneyl group is optionally substituted.

[0183] In a particularly preferred embodiment, Q comprises a (hetero)cyclic alkynyl or (hetero)cyclic alkenyl group and is according to structure (Q1): (Q1) In this article: - Described as The key is a double or triple bond; -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups; -Y 2 It is C(R) 31 )2、O、S、S (+) R 31 S(O)R 31 S(O)=NR 31 or NR 31 S (+) Is with B (-) Mutually balanced cation sulfur atoms, of which B (-) It is an anion, and each R in it is an anion. 31 R alone 15 Or a connection to D (via L); -u is 0, 1, 2, 3, 4 or 5; -u' is 0, 1, 2, 3, 4 or 5, where u + u' = 0, 1, 2, 3, 4, 5, 6, 7 or 8; v = an integer in the range 0–16; Typically, v = (u + u') x 2 (when the connection with L (described by a tilde) is via Y) 2 (when) or [(u + u') x 2] – 1 (when the connection with L (described by a tilde) is via one of the carbon atoms in u and u').

[0184] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cyclic alkynyl group and is according to structure (Q1a): (Q1a) In this article, -R 15 and Y 2 As defined above -u is 0, 1, 2, 3, 4 or 5; -u' is 0, 1, 2, 3, 4 or 5, where u + u' = 4, 5, 6, 7 or 8; v = an integer in the range of 8–16.

[0185] In a preferred embodiment, u + u' = 4, 5, or 6, more preferably u + u' = 5.

[0186] In a preferred embodiment, v = 8, 9 or 10, more preferably v = 9 or 10, and most preferably v = 10.

[0187] In a preferred embodiment, Q is a (hetero)cyclic alkynyl group selected from the group consisting of (Q2) – (Q20c) described below. In this paper, the connection with L (depicted as a tilde) can be a connection with any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14), and (Q15) can have connections with L, or can contain hydrogen atoms or be optionally functionalized. (–) It is an anion, preferably selected from... (–) OTf, Cl (–) ,Br (–) or I (–) The most preferred option is B. (–) yes (–) OTf. B (+) It is a cation, preferably a pharmaceutically acceptable cation. In the coupling reaction, B (–) It does not need to be a pharmaceutically acceptable anion, because B (–) In any case, it will exchange with the anions present in the reaction mixture. If (Q19) is used as Q, then the negatively charged counterion is preferably pharmaceutically acceptable when separating the conjugate according to the invention, so that the conjugate can be readily used as a pharmaceutical agent. R 36 It is a halogen selected from fluorine, chlorine, bromine and iodine, preferably R. 36 It's fluorine. Y 4 It is a heteroatom, preferably Y. 4 It is O or NH. R 35 Choose from the following groups: hydrogen, C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups, these C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups are optionally substituted and optionally selected from O, Si, S and NR.14 One or more heteroatoms are interrupted, where R 14 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups, preferably, R 35 Selected from H, C5H 11 CH3, CH2CH3, CH2OH or CH2OTBS.

[0188] In another preferred embodiment, Q is a (hetero)cyclic alkynyl group selected from the group consisting of (Q21) – (Q38a) described below. In structure (Q38), B (–) It is an anion, preferably selected from... (–) OTf, Cl (–) ,Br (–) or I (–) The most preferred option is B. (–) yes (–) OTf. In structure (Q28), B (+) It is a cation, preferably a pharmaceutically acceptable cation. The R group on (Q38b), (Q38c), and (Q38d) 35 and R 36 Defined elsewhere and equally applicable to this embodiment.

[0189] In a preferred embodiment, Q comprises a (hetero)cyclic octynylene moiety or a (hetero)cyclic heptaynylene moiety, preferably according to structures (Q8), (Q26), (Q27), (Q28), (Q37), or (Q38a), which may optionally be substituted. Each of these preferred options for Q is further defined below.

[0190] Therefore, in a preferred embodiment, Q comprises the heterocyclic heptyne moiety according to structure (Q37), also known as TMTHSI, which is optionally substituted. Preferably, the heterocyclic heptyne moiety according to structure (Q37) is not substituted.

[0191] In an alternative preferred embodiment, Q comprises a cyclooctynyl moiety according to structure (Q8), more preferably according to (Q29), also referred to as a bicyclic [6.1.0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctynyl moiety according to structure (Q8) or (Q29) is not substituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctynyl moiety according to structure (Q39) as shown below, wherein V is (CH2). lAnd l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, even more preferably l is 0, 1 or 2, and most preferably l is 0 or 1. In the context of group (Q39), l is most preferably 1. Most preferably, Q is further defined below according to structure (Q42).

[0192] In alternative preferred embodiments, Q comprises a (hetero)cyclic octylene moiety according to structure (Q26), (Q27), or (Q28), also known as a DIBO, DIBAC, DBCO, or ADIBO group, which may optionally be substituted. In the context of this embodiment, Q is preferably a (hetero)cyclic octylene moiety according to structure (Q40) or (Q41), as shown below, wherein Y 1 Is it O or NR? 11 , where R 11 Independently selected from the following groups: hydrogen, straight-chain or branched C1–C 12 alkyl groups or C4–C 12 (Hetero)aryl groups. The aromatic ring in (Q40) is optionally O-sulfonated at one or more positions, while the ring in (Q41) may be halogenated at one or more positions. Preferably, the (hetero)cyclic octylene moiety according to structure (Q40) or (Q41) is not further substituted. Most preferably, Q is according to structure (Q43), as further defined below.

[0193] In an alternative preferred embodiment, Q comprises a heterocyclic heptynyl group and is according to structure (Q37). In a particularly preferred embodiment, Q comprises a cyclooctyne group and is according to structure (Q42): (Q42) In this article: -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups; -R 18 Independently select from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups; -R 19 Choose from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups, which are optionally interrupted by one or more heteroatoms selected from the group consisting of: O, N, and S, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups, and (hetero)arylalkyl groups are independently and optionally substituted, or R 19 It is the second occurrence of Q or D connected via a gap; and -l is an integer in the range of 0 to 10.

[0194] In a preferred embodiment of the reactive group according to structure (Q42), R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 C1–C6 alkyl groups, C5–C6 (hetero)aryl groups, wherein R 16 It is hydrogen or C1–C6 alkyl, more preferably R 15 Independently selected from the group consisting of hydrogen and C1–C6 alkyl groups, most preferably all R 15 All are H. In a preferred embodiment of the reactive group according to structure (Q42), R 18 Independently selected from the group consisting of hydrogen and C1–C6 alkyl groups, most preferably two R groups. 18 All are H. In a preferred embodiment of the reactive group according to structure (Q42), R 19 It is H. In a preferred embodiment of the reactive group according to structure (Q42), l is 0 or 1, more preferably l is 1.

[0195] In a particularly preferred embodiment, Q comprises a (hetero)cyclic octyryl group and is according to structure (Q43): (Q43) In this article: -R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1–C 24 Alkyl groups, C5–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups, wherein these alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)aromatic substituents, and wherein R 16 Independently select from the following groups: hydrogen, halogens, C1–C 24 alkyl groups, C6–C 24 (Hetero)aryl group, C7–C 24 alkyl(hetero)aryl groups and C7–C 24 (Hetero)arylalkyl groups; -Y is N or CR 15 ; - The carbon atom in the fused aromatic ring can be replaced by a nitrogen atom as shown in (Q6a) – (Q6d), preferably where Y is a CR 15 .

[0196] In a preferred embodiment of the reactive group according to structure (Q43), R 15 Independently select from the following groups: hydrogen, halogen, -OR 16 -S(O)3 (-) C1–C6 alkyl groups, C5–C6 (hetero)aryl groups, wherein R 16 It is hydrogen or C1–C6 alkyl, more preferably R 15 Independently select from the following groups: hydrogen and -S(O)3 (-) In a preferred embodiment of the reactive group according to structure (Q43), Y is N or CH, more preferably Y = N.

[0197] In alternative preferred embodiments, Q comprises a cyclic olefin moiety. The alkenyl group Q may also be referred to as a (hetero)cyclic alkenyl group, i.e., a heterocyclic alkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, wherein the (hetero)cyclic alkenyl group is optionally substituted. Preferably, the (hetero)cyclic alkenyl group is a (hetero)cyclic propenyl group, a (hetero)cyclic butenyl group, a norbornenyl group, a norbornadienyl group, a trans-(hetero)cyclic heptenyl group, a trans-(hetero)cyclic octenyl group, a trans-(hetero)cyclic nonenyl group, or a trans-(hetero)cyclic decenyl group, all of which may be optionally substituted. Particularly preferred are (hetero)cyclic propenyl groups, trans-(hetero)cyclic heptenyl groups, trans-(hetero)cyclic octenyl groups, trans-(hetero)cyclic nonenyl groups, or trans-(hetero)cyclic decenyl groups, all of which may be optionally substituted. - (Hetero)cycloheptenyl group or trans - (Hetero)cyclooctenyl group, wherein the (hetero)cyclopropenyl group, the trans-[…] - (hetero)cycloheptenyl group or the trans-[…] - The heterocyclic octenyl group is optionally substituted. Preferably, Q comprises a cyclopropenyl moiety according to structure (Q44), a heterocyclic butene moiety according to structure (Q45), a norbornene or norbornediene group according to structure (Q46), and a trans-[…] group according to structure (Q47). - (Hetero)cycloheptenyl moiety or, according to structure (Q48), trans - (Hetero)cyclooctenyl moiety. In this paper, Y 3 Selected from C(R) 23 2. NR 23 Or O, where each R 23 It is hydrogen alone, C1–C6 alkyl, or linked to L (optionally via a spacer), and is labeled with – – – The bond is a single or double bond. In another preferred embodiment, the cyclopropenyl group is according to structure (Q49). In yet another preferred embodiment, the trans-propenyl group... - The (hetero)cycloheptenyl group is based on structure (Q50) or (Q51). In another preferred embodiment, the trans... - The (hetero)cyclic octene group is based on the structure (Q52), (Q53), (Q54), (Q55), or (Q56). In this paper, the R group on Si in (Q50) and (Q51) is typically alkyl or aryl, preferably C1-C6 alkyl.

[0198] In an alternative preferred embodiment, Q is a thiol reaction probe. In this embodiment, Q is a reactive group compatible with cysteine ​​coupling. Such probes are known in the art and can be selected from the group consisting of: maleimide moiety, haloacetamide moiety, acrylamide moiety, phosphoramidide moiety, cyanoacetylene moiety, vinyl sulfone moiety, vinylpyridine moiety, or methanesulfonylphenyloxadiazole moiety. Most preferably, Q comprises a maleimide moiety. The reagent can be of the monoalkylation type or can be a cross-linker that reacts with two cysteine ​​side chains.

[0199] In a preferred embodiment, the click probe QZ includes a portion selected from (Q1) – (Q56), more preferably a portion selected from (Q1) – (Q56).

[0200] Reaction part F Reactive part F 1 and F 2 For Q respectively 1 and Q 2 It is reactive. In the context of this invention, F refers to Freactive. 1 and F 2 F is reactive to and complementary to Q. In this document, a reactive group is referred to as "complementary" to another reactive group when the reactive group selectively reacts with it (optionally in the presence of other functional groups). Complementary reactive click probes are known to those skilled in the art and are described in more detail below. The exact properties of Q and F depend on the type of click reaction employed. Click probes are reactive in cycloaddition (click reactions) and are preferably selected from azides, tetrazides, triazides, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxothiophenes, sine ketones, olefinic moieties, and alkyne moieties. Preferably, click probe F comprises either an azide moiety or a tetrazide moiety.

[0201] F is reactive to Q in the coupling reaction defined below, preferably wherein the coupling reaction is a cycloaddition reaction or a nucleophilic reaction. Those skilled in the art will understand that the options for F are the same as those for Q, provided that F and Q are reactive to each other. Therefore, F preferably comprises a click probe, a thiol, a thiol reaction moiety, an amine, or an amine reaction moiety; more preferably, F is a click probe, a thiol, or an amine; and most preferably, F is a click probe. The click probe is reactive in the cycloaddition (click reaction) and is preferably selected from azides, tetrazides, triazides, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxothiophenes, syringones, olefinic moieties, and alkyne moieties. Preferably, the click probe comprises or is composed of azides, tetrazides, triazides, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxothiophenes, or syringones, most preferably azides. Typical thiol reaction moieties are selected from maleimide moieties, haloacetamide moieties, acrylamide moieties, phosphoramidide moieties, cyanoacetylene moieties, o-quinone moieties, vinyl sulfones, vinylpyridine moieties, or methanesulfonylphenyloxadiazole moieties. Most preferably, the thiol reaction moieties comprise or contain maleimide moieties. Typical amine reaction moieties are selected from N-hydroxysuccinimide esters, p-nitrophenyl carbonates, pentafluorophenyl carbonates, isocyanates, isothiocyanates, and benzoyl halides. In preferred embodiments, F is a click probe or a thiol, more preferably F is an azide or a thiol, and most preferably F is an azide.

[0202] The reactive group F on an antibody is typically introduced via specific techniques (e.g., (bio)chemical or genetic techniques). The reactive group incorporated into the antibody is prepared by chemical synthesis, such as an azide or terminal alkyne. Methods for preparing modified antibodies are known in the art, for example from WO 2014 / 065661, WO 2016 / 170186, and WO 2016 / 053107, which are incorporated herein by reference. From the same literature, the coupling reaction between the modified antibody and the linker-toxin-construct is known to those skilled in the art.

[0203] Preferably, F is a click probe reactive to (hetero)cyclic alkenes and / or (hetero)cyclic alkynes, and is typically selected from the group consisting of: azides, tetrazides, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxothiophenes, and syl ketones. The preferred structure of the reactive group is the structure (F1) – (F10) described below. In this paper, a tilde represents a connection to either AB or D. For (F3), (F4), (F8), and (F9), the payload can be attached to any one of the tildes. Other tildes can then be attached to R groups selected from: hydrogen, C1–C… 24 alkyl groups, C2–C24 Acyl group, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups, C3–C 24 (Hetero)arylalkyl groups and C1–C 24 The sulfonyl groups, each of which (except for hydrogen) may optionally be substituted and optionally selected from O, S, and NR. 32 One or more heteroatoms are interrupted, where R 32 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups. Those skilled in the art will understand which R groups can be applied to each of group F. For example, the R group attached to the nitrogen atom in (F3) can be selected from alkyl and aryl groups, while the R group attached to the carbon atom in (F3) can be selected from hydrogen, alkyl, aryl, acyl, and sulfonyl groups. Preferably, the reactive moiety F is selected from azides, nitroketones, or tetraazines.

[0204] In a particularly preferred embodiment, F is a tetrazine according to structure (F8a): (F8a) In this article, R 29 Selected from hydrogen, C 1-6 Alkyl, aryl, C(O)-C 1-6 Alkyl, C(O)-aryl, C(O)-OC 1-6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1-6 Alkyl and C(O)-NR 33 -Aryl, where R 33 Is it H or C? 1-4 Alkyl group. Preferably, R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl, and pyrimidinyl. It has been found that when R... 29 When the hydrogen is present, the reactivity in the cycloaddition reaction is optimal. Therefore, in a preferred embodiment, the cycloaddition ring, particularly F, produces the best results. 2 , is (F8a), where R 29 Selected from hydrogen, methyl, phenyl, pyridyl, pyridyl and pyrimidinyl, more preferably R 29 It is hydrogen.

[0205] In another particularly preferred embodiment, F is a nitroketone according to structure (F3a). (F3a) R 1 It is L 10 XR4 In this paper, X is a heteroatom with a lone pair of electrons, capable of capturing the imine intermediate (d) by reacting with the imine carbon atom (see Scheme 3). This capture of the imine intermediate (d) is effective and will halt the rearrangement reaction of Scheme 2 if the reaction forms a 5-membered or 6-membered ring. Therefore, L 10 It should be a linker with two or three carbon atoms.

[0206] More specifically, L 10 It has a structure (C(R) 3 )2) z The connector, where z is 2 or 3. Each R 3 Selected individually from H and C 1-4 Alkyl group. Alternatively, R appearing twice. 3 They can be linked together to form oxo groups or C 3-6 (Hetero)cycloalkyl groups. Preferably, L 10 It has the structure CH2–C(R) 3 )2 or CH2–CH2–C(R 3 The connector of )2, more preferably L 10 = CH2–C(R 3 )2. In the two occurrences of R 3 When the rings are connected together to form a screw connection, it is preferable that such rings are C3–C6 rings, preferably C4 or C5 rings.

[0207] L 10 The preferred embodiment is (L) 10 A) – (L 10 P): In this paper, tilde bonds marked with * are connected to the nitrogen atom of the nitroketone group, and tilde bonds marked with * are connected to XR 4 Connection. The ring (L) screw is connected to L. 10 The main chain atoms. The ring (L) is preferably a cyclobutyl ring or a cyclopentyl ring, most preferably a cyclobutyl ring. Particularly preferred is (L) 10 A), (L 10 B), (L) 10 C) and (L) 10 G), where ring L is a cyclobutyl ring.

[0208] X is S, O, or NH. Most preferably, X is O. 4 Selected from H and C 1-4 Alkyl group. Typically, when X is O or NH, R 4 It is H, and when X is S, R 4 Is it H or C?1-4 Alkyl group. Therefore, XR 4 Typically selected from OH, NH2, SH and S–C 1-4 Alkyl group. In a preferred embodiment, XR 4 The preferred compounds are SH, OH, and NH2, with XR being the most suitable. 4 It is OH.

[0209] R 2a and R 2b The properties of are not critical to this invention, and any suitable substituents of the nitrone compound can be used. 2a and R 2b They can be the same or different; typically they are different. In R... 2a and R 2b Under different conditions, the configuration of the double bond between the nitrogen and carbon atoms in the nitroketone group can be as follows: E - configuration or Z - Configuration. The exact configuration has no effect on the operation of the invention. In this document, the connection with AB or D is typically via R. 2a or R 2b .

[0210] Typically, R 2a Selected from H and C1–C6 (cyclo)alkyl groups. In a preferred embodiment, R 2a Selected from H and C1–C5 (cyclo)alkyl, more preferably, R 2a It is H, Me, or Et, with the most preferred option being R. 2a It's H.

[0211] Typically, R 2b Choose from the following groups: C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups, these C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups may be optionally substituted and may be optionally selected from O, S, and NR. 14 One or more heteroatoms are interrupted, where R 14 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups. Alternatively, R 2a and R 2bConnect to form a (hybrid) ring-shaped portion. In an alternative embodiment, R 2b It is L(D) r , where r is an integer in the range of 1–10, and L is a linker that covalently links D to the nitroketone group. In another alternative embodiment, R 2b It is L 6 AB, where L 6 It is a linker that covalently links AB to a nitroketone group. Antibody AB, payload D, linkers L and L 6 Preferred embodiments of the integer r are defined elsewhere.

[0212] In a preferred embodiment, R 2b It is hydrogen, C1–C 20 Alkyl groups (preferably C1–C) 16 Alkyl groups, more preferably C1–C 10 Alkyl groups), L(D) r or L 6 AB. In this document, alkyl groups are optionally substituted and optionally selected from O, S, and NR. 14 Preferably, one or more heteroatoms of O are interrupted, wherein R 14 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups. In another preferred embodiment, R 2b Choose from the group consisting of: hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, L(D). r or L 6 AB, more preferably selected from the group consisting of: hydrogen, methyl, ethyl, n-propyl, isopropyl, L(D). r or L 6 AB, and even more preferably selected from the group consisting of: hydrogen, methyl, ethyl, L(D). r or L 6 AB.

[0213] Particularly preferred is the use of the nitrone compound according to the invention for preparing bioconjugates, wherein antibody AB is covalently linked to payload D. Therefore, R is particularly preferred. 2b It is L(D) r or L 6 AB. In one embodiment, R 2b It is L(D) r Furthermore, the nitrone compound will be coupled to a (hetero)cyclic alkyne compound containing antibody AB. In one embodiment, R 2b It is L 6 B, and the nitrone compound will be coupled to a (hetero)cyclic alkyne compound containing the payload D. Particularly preferred is that the nitrone is coupled to the payload, and therefore R2b It is L(D) r .

[0214] In a preferred embodiment, click probe F 1 and F 2 The following groups are selected: azides, tetrazides, triazines, nitrones, nitriles, nitriles, nitrile oxides, nitrile imines, diazo compounds, dioxothiophene, syringones, iminosyringones, and catechols. It should be noted that catechols are oxidized in situ to an ortho-quinone group, which is reactive as a click probe. Similarly, the term "tetrazine" also covers "hydrotetrazine," a known precursor that forms a tetrazine upon in situ oxidation. This invention also covers such precursors for click probes that form reactive groups in situ. Figure 12 shows some known examples of click probes formed in situ through oxidation. In a preferred embodiment, F... 1 It is an azide, and F 2 It is an iminosydinoketone, catechol (forming an ortho-quinone group in situ), or tetraazine. More preferably, F 2 It is an iminosydinoketone, a catechol (forming structure (F10) in situ), or a tetrazine according to structure (F8). Most preferably, F... 1 It is an azide based on the structure (F1), and F 2 It is a tetrazine based on the structure (F8a).

[0215] In this invention, it is preferred that Q 1 and Q 2 The exact structure is different, making Q 1 For F 2 It is not reactive, while Q 2 For F 2 It is reactive.

[0216] In a preferred embodiment, F 1 It is an azide and Q 1 It is a benzo-cyclic or tetramethylated (hetero)cyclic alkyne, while F 2 It is tetrazine, and Q 2 It is a bicyclic nonyne. In this paper, Q... 1 Preferably, it is based on structures (Q5), (Q6), (Q7), (Q11), (Q17), (Q18), (Q19), or (Q19a); more preferably, it is based on structures (Q26), (Q27), (Q28), (Q32), (Q37), (Q38), or (Q38a); and most preferably, it is based on structures (Q40), (Q41), or (Q43). In this document, Q... 2 Preferably according to structure (Q8), more preferably according to structure (Q29), and most preferably according to structure (Q42).

[0217] In another preferred embodiment, F 1 It is an azide and Q 1 It is a cyclic alkyne, and Q 2 It is trans-cyclooctene and F 2 It is either nitroketone or tetrazine.

[0218] In a preferred embodiment, DF 2 The ligase is obtained through a ligase-mediated step. Preferably, the ligase is selected from microtubule tyrosine ligase, transglutaminase, lipoic acid ligase, farnesyltransferase, glycosyltransferase, formyl-glycine synthase (FGE), trypsiligase / subtiligase, and more preferably, the ligase is a sorting enzyme. Preferably, the sorting enzyme-mediated step is as follows: ,or In this article, D* represents the remainder of D, L represents leucine, P represents proline, T represents threonine, and G represents glycine. X 1 It is any amino acid, X 2 It is any hydrophobic amino acid, and n is an integer in the range of 1-20, preferably n is 3–15 (SEQ.ID NO: 27–30). F 2 It may contain PEG chains. More preferably, LPX 1 TGX 2 n It is LPETGGH 10 (SEQ. ID NO: 31) and n' is 3. In a particularly preferred embodiment, F 2 It is an azide, and the coupling occurs via the SPAAC reaction, or F... 2 It is a tetrazine, and the coupling occurs via the reverse demand Diels-Alder reaction.

[0219] In another preferred embodiment, F is nitroketone, and DF 2 It is obtained by converting the terminal serine or threonine to a nitrone. This conversion can be achieved by oxidizing the serine or threonine side chain and then converting it to a nitrone, as shown below: Such a conversion of serine or threonine to nitrone can be carried out by conventional chemical transformation. In this paper, [ox] is an oxidizing agent, such as NaIO4. R corresponds to R as defined above for (F3a). 1 Its exact nature is irrelevant in the context of this invention, although it is preferably defined as R above. 1The conjugates thus obtained have improved stability because they can rearrange into stable conjugates, as explained above.

[0220] Particularly preferred is that the oxidation of the terminal amino acid, its conversion to a nitroketone, and its coupling with the linker are carried out in a single-pot synthesis. Preferably, Q 2 It is trans-cyclooctene, or Q 2 It is a cyclic alkyne, and the coupling reaction is a strain-promoted alkyne-nitroketone cycloaddition (SPANC).

[0221] application The conjugates of the present invention are suitable for medical treatment, particularly for the treatment of cancer or autoimmune diseases. Alternatively or additionally, the conjugates of the present invention are used to treat or reduce blood disorders such as neutropenia, anemia, and thrombocytopenia. Such blood disorders are common side effects of treatment with immune cell conjugates (such as IL-15). The conjugates according to the present invention avoid the serious side effects typically associated with treatment with immune cell conjugates. Common side effects include nausea, vomiting, abdominal pain, fatigue, leukopenia, neutropenia, and thrombocytopenia, which are avoided or reduced when the immune cell conjugate is shielded by the spatial volume of the antibody and activated only upon cleavage at the target site.

[0222] Therefore, in a preferred embodiment, the invention further relates to a method for treating cancer, the method comprising administering a conjugate according to the invention to a subject in need. The subject in need is typically a cancer patient. The use of conjugates (such as antibody-drug conjugates) is well known in the field of cancer treatment, and the conjugate according to the invention is particularly suitable for this purpose. The described method is typically suitable for treating cancer. In the method according to this aspect, the antibody-drug conjugate is typically administered at a therapeutically effective dose. The preferred dose for administering the conjugate according to the invention is in the range of 3–20 mg / kg body weight every three weeks, or every two weeks, or weekly, preferably every three weeks. This aspect of the invention can also be expressed as the use of the conjugate according to the invention in the treatment of cancer. In other words, this aspect relates to the use of the conjugate according to the invention in the preparation of a pharmaceutical agent or pharmaceutical composition for use in the treatment of cancer. In this context, cancer treatment is considered to encompass treating tumors, imaging tumors, diagnosing tumors, preventing tumor proliferation, preventing tumor containing tumor, and reducing tumor size.

[0223] This aspect of the invention can also be described as a method for targeting tumor cells expressing a specific extracellular receptor, the method comprising contacting a conjugate according to the invention with cells that may express the extracellular receptor, wherein an antibody specifically targets the extracellular receptor. These tumor cells expressing the extracellular receptor may be present in a subject, in which case the method comprises administering the conjugate according to the invention to the subject in need. Alternatively, the method may occur in vitro or ex vivo. In a preferred embodiment, the cells that may express the extracellular receptor are cells expressing the extracellular receptor. Targeting tumor cells preferably includes treating tumor cells, preventing tumor cell proliferation, preventing tumor cell spread, and reducing one or more of the following in tumor cells:

[0224] In the context of diagnosis, it is typically unknown whether the cells being contacted actually express the specific extracellular receptor being studied. For example, in the diagnosis of HER2-positive breast cancer, a conjugate containing an antibody targeting HER2 (such as trastuzumab) can be brought into contact with the cells. If the tumor cells do express HER2, the conjugate will target those cells; if the tumor cells do not express HER2, the conjugate will not. Similarly, in treating cancer cells that specifically express an extracellular receptor, the technician will understand that a cell-binding agent (such as an antibody) targeting that specific extracellular receptor should be used.

[0225] In the method of the present invention, preferably, the extracellular receptor is selected from the group consisting of: 5T4 (TPBG), ADAM9, ALPP, ALPPL2, AMHRII, ASCT2 (SLC1A5), ASLG659, ASPHD1, av-integrin, avb3-integrin / ITGAV / CD51, Axl, B7-H3 (CD276), B7-H4 (VTCN1), BAFF-R, BCMA (CD269), BMPR1B, short proteoglycan, c-KIT (CD117), c-Met, C4.4a (LYPD3), CA-IX (CA9) / MN, cadherin-6, CanAg, CCR7, CD117 (c-KIT), CD123 (IL-3Rα), CD13, CD133, CD 138 / Glucan-1, CD166 (ALCAM), CD19, CD20, CD203C, CD205, Ly75, CD21, CD22, CD228 (P79, SEMF), CD25 (IL-2R-α), CD30 (TNFRSF8), CD324 (CDH1 / E-cadherin), CD33, CD352 (SLAMF6, NTB-A), CD37, CD38, CD44v6, CD45, CD46, CD47, CD48a (SLAMF2), CD56 (NCAM), CD70, CD71 (TF-R), CD72, CD74, CD79a, CD79b, CDH6, cadherin-6, CEACAM5 (CD66e), sealing protein, CLDN18.2. CLDN6 (Skullin), CLEC12A, CLL-1 / CLEC12A, Cripto, CS1 (SLAMF7, CD319), CXCR5, DKL-1, DLL3 (δ-like 3), DPEP3, E16, EGFR, EGFRvIII, ENPP3, CD203c (AGS-16), EpCAM, EphA2, EphB2R, liver glycoside A4 (EFNA4), ETBR, FAP, FGFR2, FGFR3, fibronectin EDB, FLT3, FOLR1 (FR-a), Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, GPC3 (phosphatidylinositol proteoglycan-3), gpNMB, GPR172A, GPR19, GPR54, GRP20, guanylate cyclase C (GCC), HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Ra, integrin avb6, KAAG-1, LAMP-1 (CD107a), Lewis Y (CD174), LGR5, LIV-1 (SLC39A6, ZIP6), LRRC15, LY64, Ly6E (lymphocyte antigen 6), Ly6G6D, LY6K, mesothelin ( MSLN), MFI2 (TAA), MICA / B, MOSPD2, MPF, MUC1 (CA6), MUC16 / CA-125, MUC1c, NaPi2b (SLC34A2), NCA, conjugin-4 (PVRL4), Notch3, P-cadherin (pCAD, CDH3), P2X5, PD-L1 (CD274, B7-H1), PMEL17, PRLR (prolactin), PSCA, PSMA, PTK7 (CCK4), RET, RNF43, RON, ROR1, ROR2, Serna 5b, SEZ6, SLITRK6 (SLC44A4), STEAP1, STEAP2, STn, TAG72, TENB2, TF (CD142, prothrombin kinase), TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, TNF-α, TROP-2 (TACSTD2), TWEAKR, receptor tyrosine kinase (RTK), tendinin, or antibodies specific to extracellular proteins and / or tumor-associated carbohydrate antigens (TACA) induced by viral infection. Similarly, it is preferred that tumor cells express extracellular receptors selected from the same group. Those skilled in the art can match the desired extracellular receptor with a suitable cell binder capable of targeting that extracellular receptor. In preferred embodiments, the extracellular receptor is selected from PD-L1, HER2, and conjugation-4, more preferably from PD-L1 and HER2.

[0226] The conjugates of the present invention are also particularly suitable as antibiotics, antivirals, anti-inflammatory agents, and anti-autoimmune agents. Therefore, in alternative embodiments, the present invention relates to a method for treating infection, inflammation of autoimmune disorders, comprising administering the conjugate according to the invention to a subject in need. In the method according to this aspect, the antibody-conjugate is typically administered at a therapeutically effective dose. This aspect of the invention can also be expressed as the use of the conjugate according to the invention in treating infection, inflammation of autoimmune disorders. In other words, this aspect relates to the use of the conjugate according to the invention in the preparation of pharmaceutical agents or pharmaceutical compositions for use in treating infection, inflammation of autoimmune disorders. In this context, infection can be bacterial or viral.

[0227] In this embodiment, the antibody is preferably specific to extracellular proteins and / or tumor-associated carbohydrate antigens caused by viral infection. In this document, extracellular proteins caused by viral infection may be human poliovirus (HPV), human cytomegalovirus (HCMV), or human papillomavirus (HPV). Tumor-associated carbohydrate antigens (TACA) may be selected from the group consisting of: Tn, STn, T antigen, LDN, Lewis antigen, etc. c (Le) c ), sialic acid-Lewis c (SLe) c ), 6-sialic acid-Lewis c (6SLe) c ), LN, α-Gal, 3SLN, 6SLN, H antigen, A antigen, B antigen, Lewis antigen a (Le) a ), sialic acid-Lewis a (SLe) a ), 6-sialic acid-Lewis a (6SLe) a Louis b (Le) b ), sialic acid-Lewis b (SLe) b ), 6-sialic acid-Lewis b (6SLe) b Louis x (Le) x ), sialic acid-Lewis x (SLe) x ), 6-sialic acid-Lewis x (6SLe) x Louis y (Le) y ), sialic acid-Lewis y (SLe)y ), 6-sialic acid-Lewis y (6SLe) y Antibodies can be specific to both extracellular proteins and TACA.

[0228] In a preferred embodiment, the present invention relates to a conjugate for use in the treatment of autoimmune disorders. Those skilled in the art understand which combination of target and payload is best suited for a particular autoimmune disease. Cytokines may have inflammatory activity, such as IL-17 and interferon-γ, or have a regulatory role in inflammation, such as IL-10 and TGF-β. In the treatment of autoimmune diseases, it is preferred that the conjugate of the present invention comprises a regulatory cytokine, more preferably selected from IL-4, IL-6, IL-9, IL-10, IL-11, and IL-13, IFN-α, and TGF-β. Alternatively, the conjugate comprises an antibody that specifically targets abnormal cells causing autoimmune diseases; preferably herein, the cytokine promotes inflammation, and the payload is preferably selected from IL-1, IL-2, IL-6, IL-12, IL-15, IL-17, IL-18, IFN-γ, TNF-α, and GM-CFS. In view of this, the present invention also relates to a pharmaceutical composition comprising a conjugate according to the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition typically contains a pharmaceutically effective dose of the conjugate according to the invention. Attached Figure Description

[0229] Figure 1 illustrates a representative (but not exhaustive) set of functional groups (F) naturally present or engineered in biomolecules, which, upon reaction with reactive groups, yield linker groups (Z). Functional group F can be artificially introduced (engineered) into biomolecules at any chosen location. The pyridazine linker group (bottom line) is a product of the rearrangement of the tetraazabicyclo[2.2.2]octane linker group, formed by the reaction of a tetraazine with an alkyne and the loss of N2. Linker group Z having the structure (10a)–(10j) is the preferred linker group used in this invention.

[0230] Figure 2 illustrates preferred embodiments of cyclooctyne and reaction motif Q suitable for metal-free click chemistry. This list is not exhaustive; for example, alkynes can be further activated by fluorination, substitution of aromatic rings, or introduction of heteroatoms into aromatic rings.

[0231] Figure 3 illustrates several structures of UDP sugar derivatives of galactosamine, which can be modified at the 2-position with, for example, a 3-mercaptopropionyl group (11a), an azide acetyl group (11b), or an azide difluoroacetyl group (11c), or at the 6-position of N-acetylgalactosamine with an azide group (11d) or with a thiol group (11e). The monosaccharide (i.e., the UDP-removed portion) is the preferred Su for use in this invention.

[0232] Figure 4 illustrates a general method for non-genetically converting monoclonal antibodies into antibodies containing probes (F) for click conjugation. The click probes can be located at different positions on the antibody, depending on the technique used. For example, the antibody can be converted into an antibody containing two click probes (left structure), four click probes (bottom structure), or eight probes (right structure) for click conjugation.

[0233] Figure 5 illustrates how an IgG antibody modified with two click probes (F) can react with a polypeptide modified with a complementary click probe (Q) to form a stable bond (Q) after the reaction, wherein the polypeptide is selected from any polypeptide capable of binding to immune cells to form a bispecific antibody. Modification of the polypeptide with a single click probe Q can be achieved by any selective genetic or non-genetic method. The probes used for click coupling can be selected from any suitable combination depicted in Figure 1. The stoichiometry of the resulting bispecific antibody depends on the number of click probes F attached in the first modification of the antibody. Symmetrical bivalent IgG (CDR1 = CDR2) can be used, resulting in a bispecific antibody with a 2:2 molecular form (2x attachment of the polypeptide). Asymmetric antibodies (CDR1 ≠ CDR2) can also be used, resulting in a trispecific antibody with a 1:1:2 molecular form. If more than two click probes F are attached, the molecular form can be further varied, resulting in, for example, a 2:4 molecular form (4x F attached to the symmetric antibody) or a 1:1:8 molecular form (8x F attached to the asymmetric antibody).

[0234] Figure 6 illustrates three alternative methods for attaching a single immune cell-conjugating peptide to a full-length antibody (2:1 molecular form). The full-length antibody is first modified with two click probes F. In one method (downward arrow), IgG (F2) is subjected to a peptide modified with two complementary click probes Q linked via suitable spacers, both of which will react with the previously present F on the antibody. In the second method (right arrow), IgG (F2) is subjected to a trivalent construct containing three complementary probes Q, where two complementary probes will react with IgG (F2), leaving one unit of Q free for subsequent reaction with the F-modified peptide. In the third method (diagonal arrow), IgG (F2) is subjected to a trivalent construct containing two complementary probes Q and one non-reactive click probe F2 (which is also different from F). The two click probes Q will react with IgG (F2), leaving F2 for subsequent reaction with the Q2-modified peptide.

[0235] Figure 7 illustrates a specific example of the formation of a 2:2 bispecific antibody based on the glycan remodeling and azide-cyclooctyne click chemistry of full-length IgG. First, IgG is enzymatically remodeled by glycosylase-mediated pruning of all different glycoforms, followed by glycosyltransferase-mediated transfer of azide sugars to the core GlcNAc released by the glycosylase. In the next step, the azide-remodeled IgG is subjected to an immune cell-conjugating peptide, which has been modified with a single cyclooctyne for metal-free click chemistry (SPAAC), resulting in a 2:2 bispecific antibody. The figure also depicts that the cyclooctyne-peptide construct will have a specific spacer between the cyclooctyne and the peptide, allowing for adjustment of the IgG-peptide distance or conferring other properties to the resulting bispecific antibody.

[0236] Figure 8 illustrates how an azide-glycomatured antibody can be converted into a bispecific antibody with a 2:1 molecular form by first undergoing a trivalent cyclooctyne construct suitable for splicing onto a bis-azide antibody, leaving a free cyclooctyne for subsequent SPAAC with an azide-modified peptide, effectively attaching only one peptide to IgG. The latter peptide can also be modified with other complement click probes to react with the cyclooctyne, such as the tetrazine moiety for anti-electron-demanding Diels-Alder cycloaddition. Any combination of F and Q can be envisioned here (Figure 1).

[0237] Figure 9 illustrates various options for trivalent constructs used in the reaction with diazidose-modified mAbs. The trivalent constructs can be isotrivalent or isotrivalent (2+1 form). The isotrivalent construct (X = Y) can consist of 3x cyclooctyne, 3x acetylene, 3x maleimide, or 3x other thiol reactive groups. The isotrivalent construct (X ≠ Y) can, for example, consist of two cyclooctyne groups and one maleimide group, or two maleimide groups and one trans-cyclooctene group. The isotrivalent construct can exist as any combination of X and Y unless X and Y are reactive with each other (e.g., maleimide + thiol).

[0238] Figure 10 shows a series of divalent BCN reagents (105, 107, 118, 125, 129, 134), trivalent BCN reagents (143, 145, 150), and monovalent BCN reagents (154, 157, 161, 163, 168) for sorting and labeling.

[0239] Figure 11 shows a series of divalent or trivalent crosslinking agents (XL01–XL13).

[0240] Figure 12 shows a series of antibody variants that served as starting materials for subsequent conversion into antibody-drug conjugates. Figure 13 shows the structures of various IL-15 mutants (PF18) or IL-15R-IL-15 fusion proteins (207, 208, PF26, and PF57–PF59, IL-15R = Sushi domain of the IL-15 receptor), corresponding to (SEQ. ID NO: 36)–IL15Rα-IL-15 (207), GGG–IL15Rα-IL-15 (208), (SEQ. ID NO: 37)–IL15Rα-IL-15 (PF26), SEQ. ID NO: 13 (PF57), SEQ. ID NO: 14 (PF58), and SEQ. ID NO: 15 (PF59). Also shown are those equipped with suitable click probes (BCN, tetrazine, or azide) or maleimide derivatives, each modified at its N-terminus for site-specific modification. The peptide sequences correspond to SEQ. ID NO: 38 (for structures (PF10), (209), (PF11), (PF12) and (PF13)) and SEQ. ID NO: 39 (for structures (PF14), (PF15), (PF16) and (PF17)).

[0241] Figure 14 shows the structures of IL-15 variants with uncut spacers. The peptide sequences correspond to SEQ. ID NO:5 (structure (PF18)), SEQ. ID NO:10 (structure (PF54)), and SEQ. ID NO:11 (structure (PF55)). The peptide sequence YR(G4S)3 (in structure (PF19)) corresponds to SEQ. ID NO:39, and the peptide sequence R(G4S)3 (in structure (PF21)) corresponds to SEQ. ID NO:41.

[0242] Figure 15 shows the structure of hydroxylamine HO1–HO4.

[0243] Figure 16 shows the structures of IL-15 variants (PF50–52) and IL-15 mutants (PF53 and PF56) with different N-terminal sequence tags. The peptide sequences correspond to SEQ. ID NO: 6 (structure (PF50)), SEQ. ID NO: 7 (structure (PF51)), SEQ. ID NO: 8 (structure (PF52)), SEQ. ID NO: 9 (structure (PF53)), and SEQ. ID NO: 12 (structure (PF56)).

[0244] Figure 17 shows the structures of various nitrone variants of the IL-15R-IL-15 fusion protein (PF57) obtained by incorporating hydroxylamine HO1–HO4 (Figure 17A) and various nitrone variants of various IL-15 mutants (PF18, PF50, PF51, PF53, and PF56) obtained by incorporating hydroxylamine HO1 and HO2 (Figure 17B). The peptide sequence YR(G4S)8 corresponds to SEQ. ID NO: 40, and the peptide sequence YR(G4S)3 corresponds to SEQ. ID NO: 39.

[0245] Figure 18 shows the SDS-page analysis of various antibody-cytokine conjugates based on mPD-L1 and IL-15 variants PF51–PF54 and FPF56 under reducing conditions.

[0246] Figure 19 shows the RP-UPLC analysis under non-reducing conditions, demonstrating the formation of trastuzumab and IL-15 variants with different N-terminal sequences in a 2:1 ratio.

[0247] Figure 20 shows the RP-UPLC analysis under non-reducing conditions, demonstrating the formation of trastuzumab and the IL-15 variant modified with hydroxylamine HO2–HO4 in a 2:1 ratio.

[0248] Figure 21 shows the aggregation of antibody-cytokine conjugates after 0–5 freeze-thaw cycles, as measured by SE-HPLC.

[0249] Figure 22 shows the cleavage of mPD-L1-v1a-218-NHO2-PF52 (2:2) by matrix protease.

[0250] Figure 23 illustrates the cleavage of various antibody-cytokine conjugates by matrix proteases.

[0251] Figure 24 shows the in vitro IL-15 activity assays of various intact antibody-cytokine conjugates. mPD-L1-v1a-218-NHO2-PF52 (2:2) treated with matrix protease is included as a reference.

[0252] Figure 25 shows the in vitro IL-15 activity assay of various matrix protease-treated antibody-cytokine conjugates.

[0253] Figure 26 shows the in vitro IL-15 activities of mPD-L1-v1a-218-NHO2-PF52 (2:2) and mPD-L1-v1a-218-NHO2-PF56 (2:2) with and without pretreatment with matrix protease MT-SP1. Free PF52 is included as a reference.

[0254] Figure 27 shows the in vitro IL-15 activity of tras-v1a-145-NHO1-PF50 (2:1) with and without urokinase pretreatment. Free PF50 and trastuzumab are included as references.

[0255] Figure 28 shows the in vivo tolerability of mPD-L1-v1a-218-NHO1-PF51, mPD-L1-v1a-218-NHO2-PF52, mPD-L1-v1a-218-NHO2-PF54, and IC1 in female BALB / c mice. Single doses of 6 mg / kg (top), 12 mg / kg (middle), and 18 mg / kg (bottom) were administered subcutaneously to mice.

[0256] Figure 29 illustrates the in vivo tolerability of the antibody-cytokine conjugate in female BALB / c mice. The conjugate consisted of different IL-15 mutants and different molecular forms (2:2 and 2:1). A single subcutaneous dose was administered to mice at the specified dose level.

[0257] Figure 30 illustrates the in vivo efficacy in a mouse CT26 syngeneic colon cancer model. Antibody-cytokine conjugates mPD-L1-v1a-218-NHO1-PF51, mPD-L1-v1a-218-NHO2-PF52, mPD-L1-v1a-218-NHO2-PF54, and IC1 were administered at 6 mg / kg on days 0 and 7, and tumor volume in mice was monitored. Data are plotted as the standard error of the mean + / - the mean (top panel) and the median tumor volume (bottom panel).

[0258] Figure 31 illustrates the in vivo efficacy in a mouse CT26 syngeneic colon cancer model, comparing antibody-cytokine conjugates based on different IL-15 mutants and different molecular forms (2:2 and 2:1). In all cases, immunocytokines were administered at specified dose levels of 3 or 6 mg / kg on days 0 and 7. Tumor volume in mice was monitored. Data are plotted as mean + / - standard error of mean (top) and median tumor volume (bottom).

[0259] Figure 32 illustrates the in vivo efficacy in a mouse MC38 syngeneic colorectal cancer model, comparing mice treated with the vector with those treated with 3 and 6 mg / kg mPD-L1-v1a-218-NHO2-PF52. In all cases, mice were administered the vector at specified dose levels of 3 or 6 mg / kg on days 0, 7, and 14. Tumor volume was monitored in the mice. Data are plotted as mean + / - standard error of the mean.

[0260] Example The present invention is illustrated by the following examples.

[0261] Synthesis of hydroxylamine compounds Example 1. Synthesis of compound HO2 2-O-acetic acid hydrate (1.24 g, 1 Eq, 13.5 mmol) and hydroxylamine hydrochloride (936 mg, 1 Eq, 13.5 mmol) were stirred in water (60 mL) at room temperature. 1 M NaOH was added to raise the pH to 5, followed by the addition of sodium cyanoborohydride (2.12 g, 2.5 Eq, 33.7 mmol). The reaction was stirred for 3 h. The mixture was concentrated under vacuum, and the resulting crude product was purified by crystallization from a small amount of water to give HO₂ (754 mg, 8.28 mmol, 62%) as a white solid. ¹H NMR (400 MHz, D₂O) δ 3.77 (s, 1H). ¹³C NMR (101 MHz, DMSO) δ 172.40, 55.10. Example 2. Synthesis of compound HO3 Add 2-nitroethanol (500 mg, 394 μL, 1 Eq, 5.49 mmol) and methanol (12 mL) to a solution of ammonium chloride (793 mg, 2.7 Eq, 14.8 mmol) in water (3 mL). Stir the resulting solution and add zinc (729 mg, 2.03 Eq, 11.1 mmol) in small portions, keeping the temperature below 65°C. Stir the mixture at RT for 30 min and filter. Wash the precipitate with methanol and H2O. Add hydrogen chloride (400 mg, 2.75 mL, 4 mol, 2 Eq, 11.0 mmol) in 1,4-dioxane to the filtrate and concentrate the solution under vacuum. Grind the resulting crude product with IPA to remove insoluble NH4Cl crystals. Concentrate the filtrate under vacuum and place the resulting crude product in H2O. The aqueous phase was extracted with Et₂O and EtOAc to remove residual 2-nitroethane-1-ol. The aqueous phase was concentrated under vacuum to give HO₃ (444 mg, 3.91 mmol, 71%) as a yellow oil. ¹H NMR (400 MHz, D₂O) δ 3.82 – 3.78 (m, 2H), 3.32 – 3.28 (m, 2H). ¹³C NMR (101 MHz, D₂O) δ 54.26, 52.38.

[0262] Example 3. Synthesis of compound HO4 Add 1-(nitromethyl)cyclobutanol (500 mg, 1 Eq, 3.81 mmol) and methanol (12 mL) to a solution of ammonium chloride (551 mg, 2.7 Eq, 10.3 mmol) in water (3 mL). Stir the resulting solution and add zinc (506 mg, 2.03 Eq, 7.74 mmol) in small portions, keeping the temperature below 65°C. Stir the mixture at RT for 15 min and filter. Wash the precipitate with methanol and H2O. Add hydrogen chloride in dioxane (278 mg, 1.91 mL, 4 mol, 2 Eq, 7.63 mmol) to the filtrate and concentrate the solution under vacuum. Grind the resulting crude product with IPA to remove insoluble NH4Cl crystals. Concentrate the filtrate under vacuum and place the resulting crude product in H2O. The aqueous phase was extracted with Et₂O and EtOAc to remove residual 1-(nitromethyl)cyclobut-1-ol. The aqueous phase was concentrated under vacuum to give HO₄ (495 mg, 3.22 mmol, 85%) as a yellow oil. ¹H NMR (400 MHz, D₂O) δ 3.49 (s, 2H), 2.24–2.09 (m, 4H), 1.89–1.50 (m, 2H). ¹³C NMR (101 MHz, D₂O) δ 70.80, 57.41, 33.44, 11.41.

[0263] Synthesis of BCN-compounds Example 4. Synthesis of compound 218 BCN-OPNP (339.2 mg, 1.07 mmol, 2.2 eq) and triethylamine (100 µL, 0.71 mmol, 1.5 eq) were added to a solution of 2-[2-(2-aminoethoxy)ethoxy]ethyl-1-amine (71 mg, 0.48 mmol, 1 eq) in anhydrous DCM (1 mL). After stirring at room temperature for 3 h, the reaction mixture was purified by silica gel rapid column chromatography (0% → 30% EtOAc, in DCM (to remove p-nitrophenol), followed by 0% → 20% MeOH, in DCM) to give 218 (191.5 mg, 0.36 mmol, 74%) as a colorless oil. For C48H41N2O6S+ (M+H+), the calculated value of LCMS (ESI+) is 501.63, and the measured value is 501.56. ¹H NMR (400 MHz, CDCl3) δ (ppm) 4.16 (d, J = 8.0 Hz, 4H), 3.62 (s, 4H), 3.57 (dd, J = 5.6, 4.7 Hz, 4H), 3.40 (q, J = 5.4 Hz, 4H), 2.36 – 2.17 (m, 12H), 1.61 (m, 4H), 1.37 (p, J = 8.6 Hz, 2H), 1.01 – 0.90 (m, 4H).

[0264] Example 5. Synthesis of compounds 127 and 128 Add 4-nitrophenol chloroformate (1.4 g, 7.07 mmol) and Et3N (3.3 mL, 2.4 g, 23.6 mmol) to a solution of diethylene glycol 126 (446 µL, 0.50 g, 4.71 mmol) in DCM (20 mL). Stir the mixture, filter, and concentrate under vacuum (at 55°C). Purify the residue by silica gel chromatography (15% → 75% EtOAc, in heptane) and separate the two products. Product 127 (511 mg, 1.17 mmol, 25%) is given as a white solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.31–8.23 (m, 4H), 7.43–7.34 (m, 4H), 4.54–4.44 (m, 4H), 3.91–3.83 (m, 4H). Product 128 (321 mg, 1.18 mmol, 25%) was given as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.32–8.24 (m, 2H), 7.43–7.36 (m, 2H), 4.50–4.44 (m, 2H), 3.86–3.80 (m, 2H), 3.81–3.74 (m, 2H), 3.69–3.64 (m, 2H).

[0265] Example 6. Synthesis of Compound 145 Triethylamine (41.6 μL, 0.30 mmol) and tris(2-aminoethyl)amine 144 (14.9 μL, 0.10 mmol) were added to a solution of 128 (200 mg, 0.45 mmol) in DCM (1 mL). The mixture was stirred for 150 min and then concentrated under vacuum. The residue was purified by silica gel column chromatography (25% → 100% EtOAc, in DCM, then 0% → 10% MeOH, in DCM) to give 145 (45.4 mg, 42.5 μmol) in a 43% yield as a yellow oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 5.68–5.18 (m, 6H), 4.32–4.18 (m, 6H), 4.18–4.11 (d,J = 7.9 Hz, 6H), 3.74–3.61 (m, 6H), 3.61–3.51 (m, 6H), 3.43–3.29 (m, 6H), 3.29–3.15 (m, 6H), 2.65–2.47 (m, 6H), 2.37–2.16 (m, 18H), 1.69–1.49 (m, 6H), 1.35 (quintet, J = 8.9 Hz, 3H), 1.03–0.87 (m, 6H).

[0266] Example 7-10: Synthesis of compound XL14 Example 7. Synthesis of Compound 219 Di-tert-butyl dicarbonate (775 mg, 1.2 Eq, 3.55 mmol) was added to a solution of sulfonyl-D-alanine hydrate (554 mg, 1 Eq, 2.96 mmol) and triethylamine (599 mg, 825 μL, 2 Eq, 5.92 mmol) in DMF (15 mL). The reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated under vacuum. The residue was redissolved in CH2Cl2 (10 mL) and treated with Et2O (30 mL) under vigorous stirring. The organic solvent was poured off and discarded. The oily residue was washed with Et2O (20 mL) and dried under vacuum to give 219 (1134 mg, 2.9 mmol, 100%, 70% purity) as a colorless oil. Calculated LCMS (ESI–) for C8H14NO7S– (M–H–): 268.1, Found: 268.3.

[0267] Example 8. Synthesis of Compound 220 To a solution of compound 219 (100 mg, 1 Eq, 371 μmol) in DMF (1 mL), 2,2'-(ethane-1,2-diylbis(oxy))bis(ethyl-1-amine) (550 mg, 542 μL, 10 Eq, 3.71 mmol), HATU (155 mg, 1.1 Eq, 409 μmol), and DIPEA (144 mg, 194 μL, 3 Eq, 1.11 mmol) were added. The reaction mixture was stirred for 18 hours and subsequently purified by reversed-phase chromatography (10% --> 100% MeCN, in H2O + 1% AcOH) to give compound 220 (42 mg, 0.11 mmol, 28%) as a colorless oil. For C14H30N3O8S+ (M+H+), the calculated value of LCMS (ESI+) is 400.2, and the measured value is 400.5.

[0268] Example 9. Synthesis of Compound 221 A solution of compound 220 (50 mg, 1 Eq, 0.13 mmol) in DMF (1000 μL) was added to TMTHSI-OSu (64 mg, 1.5 Eq, 0.19 mmol) and triethylamine (51 mg, 70 μL, 4 Eq, 0.5 mmol), and the mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by reversed-phase chromatography (10% --> 100% MeCN, in H2O + 1% AcOH) to give a colorless oily Boc intermediate (41 mg, 66 μmol, 52%). Calculated LCMS (ESI+) for C25H45N4O10S2+(M+H+) was 625.3, and the measured value was 625.5.

[0269] TFA (150 mg, 101 μL, 20 Eq, 1.31 mmol) was added to a solution of the Boc intermediate (41.0 mg, 1 Eq, 65.6 μmol) in DCM (1000 μL), and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum to give compound 221 (41.9 mg, 65.6 μmol, 100%) as a colorless oil. Calculated LCMS (ESI+) for C20H37N4O8S2+ (M+H+) was 525.2, and the measured value was 525.5.

[0270] Example 10. Synthesis of compound XL14 A solution of compound 221 (41.9 mg, 1 Eq, 65.6 μmol) in DMF (1000 μL) was supplemented with (2,5-dioxopyrrolidone-1-yl)carbonate ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)methyl ester (28.7 mg, 1.5 Eq, 98.4 μmol) and triethylamine (26.6 mg, 36.6 μL, 4 Eq, 262 μmol), and the mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by silica gel rapid column chromatography (0%–30% MeOH, in DCM) to give XL14 (11.9 mg, 17.0 μmol, 25.9%) as a white solid. For C31H49N4O10S2+ (M+H+), the calculated value of LCMS (ESI+) is 701.3, and the measured value is 701.5.

[0271] Cytokine expression and isolation A general procedure for mass spectrometry analysis of cytokines, antibodies, and antibody-cytokine fusions. Prior to mass spectrometry analysis, IgG was treated with IdeS for Fc / 2 fragment analysis. For Fc / 2 fragment analysis, 20 µg of (modified) IgG solution was incubated with IdeS / Fabricator™ (1.25 U / µL) in 10 µL of PBS (pH 7.4) at 37°C for 1 hour. The sample was then diluted to 80 µL with PBS (pH 7.4). For cytokine analysis, 4 µg of (modified) cytokine solution was diluted to 80 µL with PBS (pH 7.4). MS analysis was performed on a JEOL AccuTOF LC-plus JMS-T100LP system (ESI-TOF) along with an HPLC system (Agilent 1100 series, Hewlett-Packard). The HPLC system was equipped with a MassPREP™ online desalting column (Waters P / N186002785). For cytokine analysis, a solution of 4 µg (modified) cytokines was diluted to 80 µL with PBS, and the electrospray ionization time of flight (ESI-TOF) was analyzed on a JEOL AccuTOF instrument. Deconvolution spectra were obtained using Magtran software.

[0272] A general procedure for cytokine expression and inclusion body isolation. Cytokine expression was initiated by transforming the corresponding cytokines into BL21(DE3) cells (Novagen). Transformed cells were plated on LB agar containing ampicillin and incubated overnight at 37°C. Single colonies were picked and used to inoculate small cultures in TB medium + ampicillin, followed by overnight incubation at 37°C. Next, larger volumes were inoculated using the overnight cultures by a 20-fold dilution in TB medium + ampicillin. Cultures were incubated at 37°C at 160 RPM and induced with 1 mM IPTG (with 1 mL of 1 M stock solution added) when OD600 reached 2.5–4.0. After induction at 37°C at 160 RPM for >16 h, the cultures were pelleted by centrifugation (5000 xg, 5 min). The cell pellet was lysed in BugBuster™ containing Benzonase and incubated in a drum magazine at room temperature for 30 min. Following lysis, the insoluble fraction was separated from the soluble fraction by centrifugation (15 min, 15000 x g). Half of the insoluble fraction was dissolved in BugBuster™ containing lysozyme (final concentration 200 pg / mL) and incubated on a drum magazine for 10 min. Next, the solution was diluted with 10 volumes of miliQ and centrifuged at 15000 x g for 15 min. The precipitate was washed with a 1:10 dilution of BugBuster™ solution and centrifuged at 12000 x g for 10 min. This washing cycle was performed 1–3 times until a very white inclusion body precipitate was obtained.

[0273] A universal program for cytokine refolding The purified inclusion bodies were dissolved in 5 M guanidine containing 40 mM cysteamine and 20 mM Tris (pH 8.0) and denatured to a final concentration of 1 mg / mL. The solution was then incubated on a roller drum at RT for 2 hours. Subsequently, the 1 mg / mL solution was added dropwise to 10 volumes of refolding buffer (50 mM Tris, 10.53 mM NaCl, 0.44 mM KCl, 2.2 mM MgCl2, 2.2 mM CaCl2, 0.055% PEG-4000, 0.55 M L-arginine, 4 mM cysteamine, 4 mM cystamine, pH 8.0) at 4°C with stirring. The solution was then allowed to stand at 4°C for at least 24 hours. The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 8.0) using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing (12–14 kDa MWCO), 1x overnight and 2x 4 h. The refolded product was loaded onto an equilibrated Q-trap anion exchange column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). The column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 8.0). The retained protein was eluted with buffer B (20 mM Tris buffer, 1 M NaCl, pH 8.0) in a 60 mL gradient from buffer A to buffer B. Mass spectrometry was performed to analyze the product.

[0274] Example 11. Design of IL-15 variants Cleavable IL-15 variants were designed consisting of chemically modifiable N-terminal sequences of (M)SYR-, (M)ST-, or (M)S-, fused with a protease-cleavable linker (amino acid sequences identified by SEQ. ID NO:1 and SEQ. ID NO:2) and wild-type or mutant human IL-15 (amino acid sequence of wild-type IL-15 identified by SEQ. ID NO:3). The N-terminal methionine residue was cleaved after expression, leaving the N-terminal serine residue required for chemical modification. Both cleavable linkers were designed to be recognized by several tumor-specific proteases, including matrix protease (MT-SP1), podase, and urokinase (UpA). Uncleavable variants have been designed by replacing the cleavable linkers with –(G4S)8- (SEQ. ID NO:33), –(G4S)3- (SEQ. ID NO:34), or –(G4S)3GGS- (SEQ. ID NO:35) linkers. In some cases, the sushi domain of IL-15Rα is fused to IL-15 via a flexible linker (the amino acid sequence of IL-15Rα-linker-IL-15 is identified by SEQ. ID NO: 4). Mutated IL-15 includes the N72D mutation with enhanced IL-15Rβ binding (amino acid sequence identified by SEQ. ID NO: 8) and the E46G,V49R mutation lacking IL-15Rα binding (amino acid sequence identified by SEQ. ID NO: 11).

[0275] Example 12. Gene synthesis and cloning into expression vectors Codon-optimized DNA sequences encoding PF18 (amino acid sequence identified by SEQ. ID NO: 5), PF50 (amino acid sequence identified by SEQ. ID NO: 6), PF51 (amino acid sequence identified by SEQ. ID NO: 7), PF52 (amino acid sequence identified by SEQ. ID NO: 8), PF53 (amino acid sequence identified by SEQ. ID NO: 9), PF54 (amino acid sequence identified by SEQ. ID NO: 10), PF55 (amino acid sequence identified by SEQ. ID NO: 11), PF56 (amino acid sequence identified by SEQ. ID NO: 12), PF57 (amino acid sequence identified by SEQ. ID NO: 13), PF58 (amino acid sequence identified by SEQ. ID NO: 14), and PF59 (amino acid sequence identified by SEQ. ID NO: 15) were obtained from Genscript, Piscataway, USA. All codon-optimized DNA sequences were cloned into the pET15b vector for tag-free protein expression in *E. coli*.

[0276] Example 13. Expression and refolding of cytokines PF18 and PF50-PF59 according to A general procedure for cytokine expression and inclusion body isolation. Expression and inclusion body isolation of each cytokine mentioned in Table 1 were performed. A general program for cytokine refolding (With minor modifications) Cytokine refolding was performed. For cytokines PF51, PF52, PF53, PF54, and PF56, normal refolding was performed. For cytokines PF18, PF50, and PF55, the cytokines were dissolved in 5 M guanidine containing 40 mM cysteine ​​and 20 mM Tris (pH 8.0) and denatured to a final concentration of 2 mg / mL instead of 1 mg / mL. For cytokines PF57 and PF59, the cytokines were dissolved in 5 M guanidine containing 40 mM cysteine ​​and 20 mM Tris (pH 8.0) and denatured to a final concentration of 3 mg / mL instead of 1 mg / mL. For cytokine PF58, the cytokine was dissolved in 5 M guanidine containing 40 mM cysteine ​​and 20 mM Tris (pH 8.0) and denatured to an unknown concentration instead of 1 mg / mL.

[0277] Table 1. Expression and refolding conditions of purified cytokines. Preparation of modified cytokines Example 14. N-terminal nitroketone functionalization of PF18 to obtain NHO1-PF18 Add 2 eq NaIO4 (16.4 µL of 50 mM stock solution in PBS) to IL15 PF18 (8070 µL, 50 µM). Incubate the reaction at 4°C for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 14109 Da). Purify the reaction mixture using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and elute with PBS. Add 160 eq NHO1 (558 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (558 µL of 100 mM stock solution in PBS) to the eluent (11500 µL, 32 µM, PBS). Incubate the reaction mixture at 25°C for 3 h. Mass spectrometry analysis revealed a single peak (observed mass 14119 Da), corresponding to NHO1-PF18. The reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and eluted with PBS.

[0278] Example 15. N-terminal nitroketone functionalization of PF50 to obtain NHO1-PF50 Add 4 eq NaIO4 (7.8 µL of 100 mM stock solution in PBS) to PF50 (2500 µL, 80 µM). Incubate the reaction mixture at 4°C for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 15294 Da). Purify the reaction mixture using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and elute with PBS. Add 160 eq NHO1 (308 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (308 µL of 100 mM stock solution in PBS) to the eluent (3500 µL, 50 µM). Incubate the reaction mixture at 25°C overnight. Mass spectrometry analysis showed a single peak (observed mass 15306 Da) corresponding to NHO1-PF50. The reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and eluted with PBS.

[0279] Example 16. N-terminal nitroketone functionalization of PF51 to obtain NHO1-PF51 Add 2 eq NaIO4 (68.5 µL of 100 mM stock solution in PBS) to PF51 (56100 µL, 61 µM). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 14594 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (2260 µL, 10 mM, in DMF). Add 160 eq NHO1 (5500 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (5500 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 14604 Da) corresponding to NHO1-PF51. Dialyze the reaction mixture into PBS using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing (12-14 kDa MWCO), 1x overnight and 2x 3h.

[0280] Example 17. N-terminal nitroketone functionalization of PF55 to obtain NHO1-PF55 Add 2 eq NaIO4 (16.5 µL of 100 mM stock solution in PBS) to PF55 (12807 µL, 61 µM). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 15683 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (546 µL, 10 mM, in DMF). Add 160 eq NHO1 (1366 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (1366 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 15694 Da) corresponding to NHO1-PF55. On the AKTAexplorer (GE Healthcare), PBS (pH 7.5) was used as the mobile phase, and the reaction mixture was directly loaded onto the desalting column (Stopvan, HiPrep). TM On the 26 / 10 desalination column.

[0281] Example 18. N-terminal nitroketone functionalization of PF57 to obtain NHO1-PF57 Add 2 eq NaIO4 (11 µL of 100 mM stock solution in PBS) to PF57 (8000 µL, 69 µM). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 25707 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (364 µL, 10 mM, in DMF). Add 160 eq NHO1 (870 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (870 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 25720 Da) corresponding to NHO1-PF57. On the AKTA explorer (GE Healthcare), PBS (pH 7.5) was used as the mobile phase, and the reaction mixture was directly loaded onto a desalting column (Stopvan, HiPrep). TM On the 26 / 10 desalination column.

[0282] Example 19. N-terminal nitroketone functionalization of PF58 to obtain NHO1-PF58 Add 2 eq NaIO4 (18.8 µL of 10 mM stock solution in PBS) to PF58 (1443 µL, 65 µM). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 25388 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (62 µL, 10 mM, in DMF). Add 160 eq NHO1 (150 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (150 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 25401 Da) corresponding to NHO1-PF58. On the AKTA explorer (GE Healthcare), PBS (pH 7.5) was used as the mobile phase, and the reaction mixture was directly loaded onto the Superdex™ 75Increase (Stopfan).

[0283] Example 20. N-terminal nitroketone functionalization of PF59 to obtain NHO1-PF59 Add 2 eq NaIO4 (6 µL of 10 mM stock solution in PBS) to PF59 (1000 µL, 30 µM). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 25494 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (19.8 µL, 10 mM, in DMF). Add 160 eq NHO1 (49 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (49 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 25503 Da) corresponding to NHO1-PF59. The reaction mixture was purified by performing five rounds of buffer exchange into PBS (pH 7.5) using an Amicon Ultra-0.5 centrifugal filter unit (10 kDa MWCO).

[0284] Example 21. N-terminal nitroketone functionalization of PF52 to obtain NHO2-PF52 Add 2 eq NaIO4 (1113 µL of 10 mM stock solution in PBS) to PF52 (88679 µL, 61 µM, in PBS). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 14375 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (3672 µL, 10 mM, in DMF). Add 160 eq NHO2 (8901 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (8901 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 14429 Da) corresponding to NHO2-PF52. The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 7.5) using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing with a 12–14 kDa MWCO solution, 2 x 1 h. For purification, the dialysis product was directly loaded onto AEX columns (Spectrum™ 2 x 5 mL, HiTrap) on an AKTA explorer (GE Healthcare). TMThe product was loaded onto a Q HP column. After loading, the column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 7.5). The retained protein was then eluted with a linear gradient of buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). On an AKTA explorer (GE Healthcare) column, PBS (pH 7.5) was used as the mobile phase, and the eluent was loaded directly onto a desalting column (HiPrep, Sitopan). TM On the 26 / 10 desalination column.

[0285] Example 22. N-terminal nitroketone functionalization of PF53 to obtain NHO2-PF53 Add 2 eq NaIO4 (278 µL of 10 mM stock solution in PBS) to PF53 (22170 µL, 61 µM, in PBS). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 14376 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (918 µL, 10 mM, in DMF). Add 160 eq NHO2 (2225 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (2225 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 14430 Da) corresponding to NHO2-PF53. The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 7.5) using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing with a 12–14 kDa MWCO solution, 2 x 1 h. For purification, the dialysis product was directly loaded onto AEX columns (Spectrum™ 2 x 5 mL, HiTrap) on an AKTA explorer (GE Healthcare). TM The product was loaded onto a Q HP column. After loading, the column was first washed with buffer A (20 mM Tris buffer, 10 mM NaCl, pH 7.5). The retained protein was then eluted with a linear gradient of buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). On an AKTA explorer (GE Healthcare) column, PBS (pH 7.5) was used as the mobile phase, and the eluent was loaded directly onto a desalting column (HiPrep, Sitopan). TM On the 26 / 10 desalination column.

[0286] Example 23. N-terminal nitroketone functionalization of PF54 to obtain NHO2-PF54 Add 2 eq NaIO4 (85 µL of 100 mM stock solution in PBS) to PF54 (69660 µL, 61 µM, in PBS). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 14090 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (2807 µL, 10 mM, in DMF). Add 160 eq NHO2 (6800 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (6800 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 14146 Da) corresponding to NHO2-PF54. The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 7.5) using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing with a 12–14 kDa MWCO solution, 2 x 1 h. For purification, the dialysis product was directly loaded onto AEX columns (Spectrum™ 2 x 5 mL, HiTrap) on an AKTA explorer (GE Healthcare). TM The product was loaded onto a Q HP column. After loading, the column was first washed with buffer A (20 mM Tris buffer, 10 mM NaCl, pH 7.5). The retained protein was then eluted with a linear gradient of buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). On an AKTA explorer (GE Healthcare) column, PBS (pH 7.5) was used as the mobile phase, and the eluent was loaded directly onto a desalting column (HiPrep, Sitopan). TM On the 26 / 10 desalination column.

[0287] Example 24. N-terminal nitroketone functionalization of PF56 to obtain NHO2-PF56 Add 2 eq NaIO4 (55.7 µL of 100 mM stock solution in PBS) to PF56 (45629 µL, 61 µM). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 14359 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (1837 µL, 10 mM in DMF). Add 160 eq NHO2 (4453 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (4453 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 143414 Da) corresponding to NHO2-PF56. The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 7.5) using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing with a 12–14 kDa MWCO solution, 2 x 1 h. For purification, the dialysis product was directly loaded onto AEX columns (Spectrum™ 2 x 5 mL, HiTrap) on an AKTA explorer (GE Healthcare). TM The product was loaded onto a Q HP column. After loading, the column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 7.5). The retained protein was then eluted with a linear gradient of buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). On an AKTA explorer (GE Healthcare) column, PBS (pH 7.5) was used as the mobile phase, and the eluent was loaded directly onto a desalting column (HiPrep, Sitopan). TM On the 26 / 10 desalination column.

[0288] Example 25. N-terminal nitroketone functionalization of PF57 to obtain NHO2-PF57 Add 2 eq NaIO4 (16.3 µL of 10 mM stock solution in PBS) to PF57 (1350 µL, 61 µM). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 25708 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (54 µL, 10 mM, in DMF). Add 160 eq NHO2 (133 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (133 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 25763 Da) corresponding to NHO2-PF57. The reaction mixture was purified by performing five rounds of buffer exchange into PBS (pH 7.5) using an Amicon Ultra-0.5 centrifugal filter unit (10 kDa MWCO).

[0289] Example 26. N-terminal nitroketone functionalization of PF57 to obtain NHO3-PF57 Add 2 eq NaIO4 (16.3 µL of 10 mM stock solution in PBS) to PF57 (1350 µL, 61 µM). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 25708 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (54 µL, 10 mM, in DMF). Add 160 eq NHO3 (133 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (133 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 25749 Da) corresponding to NHO3-PF57. The reaction mixture was purified by performing five rounds of buffer exchange into PBS (pH 7.5) using an Amicon Ultra-0.5 centrifugal filter unit (10 kDa MWCO).

[0290] Example 27. N-terminal nitroketone functionalization of PF57 to obtain NHO4-PF57 Add 2 eq NaIO4 (16.3 µL of 10 mM stock solution in PBS) to PF57 (1350 µL, 61 µM). Incubate the reaction at RT for 5 min. Mass spectrometry analysis showed that serine was oxidized to the corresponding hydrate (observed mass 25708 Da). To quench the remaining NaIO4 in the reaction, add 6.6 eq p-methoxybenzylthiol (54 µL, 10 mM, in DMF). Add 160 eq NHO4 (133 µL of 100 mM stock solution in PBS) and 160 eq p-anisidine (133 µL of 100 mM stock solution in PBS) to the quenched reaction. Incubate the reaction mixture at RT overnight. Mass spectrometry analysis showed a single peak (observed mass 25789 Da) corresponding to NHO4-PF57. The reaction mixture was purified by performing five rounds of buffer exchange into PBS (pH 7.5) using an Amicon Ultra-0.5 centrifugal filter unit (10 kDa MWCO).

[0291] Example 28. N-terminal BCN functionalization of NHO1-PF51 by SPANC to obtain 218-NHO1-PF51. Add 10 eq BCN-PEG2-BCN 218 (8562 µL, 4 mM, in DMF) to NHO1-PF51 (77055 µL, 44 µM, in PBS). Incubate the reaction overnight at room temperature. Dialyze the solution to 10 mM NaCl and 20 mM Tris (pH 8.0) using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing (12–14 kDa MWCO) for 2 x 1 h. For purification, the dialysis product was directly loaded onto AEX columns (2 x 5 mL, HiTrap, GE Healthcare) on an AKTA explorer. TM The column was loaded with Q HP. After loading the product, the column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 8.0). The retained protein was eluted with a linear gradient of buffer B (20 mM Tris buffer, 1 M NaCl, pH 8.0). The eluent was dialyzed into PBS using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing (12–14 kDa MWCO) for 1x overnight and 2x 3 h. Mass spectrometry showed the correct product (observed mass 15105 Da), corresponding to 218-NHO1-PF51. Example 29. N-terminal BCN functionalization of NHO2-PF52 by SPANC to obtain 218-NHO2-PF52. Add 10 eq BCN-PEG2-BCN 218 (17335 µL, 2 mM, in DMF) to NHO2-PF52 (173349 µL, 22 µM, in PBS). Incubate the reaction overnight at room temperature with stirring. Dialyze the solution to 100 mM NaCl and 20 mM Tris (pH 7.5) using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing (12–14 kDa MWCO) for 2 x 1 h. For purification, the dialysis product was directly loaded onto AEX columns (2 x 5 mL, HiTrap, GE Healthcare) on an AKTA explorer. TM The product was loaded onto a Q HP column. After loading, the column was first washed with buffer A (20 mM Tris, 100 mM NaCl, pH 7.5). The retained protein was then eluted with a linear gradient of buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). On an AKTA explorer (GE Healthcare) column, PBS (pH 7.5) was used as the mobile phase, and the eluent was loaded directly onto a desalting column (HiPrep, Sitopan). TM On a 26 / 10 desalting column. Mass spectrometry showed the correct product (observed mass 14929 Da), corresponding to 218-NHO2-PF52.

[0292] Example 30. N-terminal BCN functionalization of NHO2-PF53 by SPANC to obtain 218-NHO2-PF53. Add 10 eq of bis-BCN-PEG2 218 (6934 µL, 2 mM, in DMF) to NHO2-PF3 (62408 µL, 22 µM, in PBS). Incubate the reaction overnight at room temperature with stirring. Dialyze the solution to 100 mM NaCl and 20 mM Tris (pH 7.5) using Spectrum™ Spectra / Por™ 2 RC dialysis membrane tubing (12–14 kDa MWCO) for 2 x 1 h. For purification, the dialysis product was directly loaded onto AEX columns (Spirovan 2 x 5 mL, HiTrap) on an AKTA explorer (GE Healthcare). TM The product was loaded onto a Q HP column. After loading, the column was first washed with buffer A (20 mM Tris, 100 mM NaCl, pH 7.5). The retained protein was then eluted with a linear gradient of buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). On an AKTA explorer (GE Healthcare) column, PBS (pH 7.5) was used as the mobile phase, and the eluent was loaded directly onto a desalting column (HiPrep, Sitopan).TM On a 26 / 10 desalting column. Mass spectrometry showed the correct product (observed mass 14930 Da), corresponding to 218-NHO2-PF53.

[0293] Example 31. N-terminal BCN functionalization of NHO2-PF54 by SPANC to obtain 218-NHO2-PF54. Add 10 eq of bis-BCN-PEG2 218 (17857 µL, 2 mM, in DMF) to NHO2-PF54 (160712 µL, 22 µM, in PBS). Incubate the reaction overnight at room temperature with stirring. Dialyze the solution to 100 mM NaCl and 20 mM Tris (pH 7.5) using Spectrum™ Spectra / Por™2 RC dialysis membrane tubing (12–14 kDa MWCO) for 2 x 1 h. For purification, the dialysis product was directly loaded onto AEX columns (Spirovan 2 x 5 mL, HiTrap) on an AKTA explorer (GE Healthcare). TM The product was loaded onto a Q HP column. After loading, the column was first washed with buffer A (20 mM Tris, 100 mM NaCl, pH 7.5). The retained protein was then eluted with a linear gradient of buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). On an AKTA explorer (GE Healthcare) column, PBS (pH 7.5) was used as the mobile phase, and the eluent was loaded directly onto a desalting column (HiPrep, Sitopan). TM On a 26 / 10 desalting column. Mass spectrometry showed the correct product (observed mass 14646 Da), corresponding to 218-NHO2-PF54.

[0294] Example 32. N-terminal BCN functionalization of NHO1-PF55 by SPANC to obtain 218-NHO1-PF55 Add 10 eq of bis-BCN-PEG2 218 (2000 µL, 5 mM, in DMF) to NHO1-PF55 (22000 µL, 45 µM, in PBS). Incubate the reaction overnight at room temperature. On an AKTA explorer (GE Healthcare), using PBS (pH 7.5) as the mobile phase, directly load the reaction mixture onto a desalting column (Stopvan, HiPrep). TM On a 26 / 10 desalting column. Mass spectrometry showed the correct product (observed mass 16196 Da), corresponding to 218-NHO1-PF55.

[0295] Example 33. N-terminal BCN functionalization of NHO2-PF56 by SPANC to obtain 218-NHO2-PF56. Add 10 eq of bis-BCN-PEG2 218 (10345 µL, 2 mM, in DMF) to NHO2-PF56 (93103 µL, 22 µM, in PBS). Incubate the reaction overnight at room temperature with stirring. Dialyze the solution to 100 mM NaCl and 20 mM Tris (pH 7.5) using Spectrum™ Spectra / Por™2 RC dialysis membrane tubing (12–14 kDa MWCO) for 2 x 1 h. For purification, the dialysis product was directly loaded onto AEX columns (Spirovan 2 x 5 mL, HiTrap) on an AKTA explorer (GE Healthcare). TM The product was loaded onto a Q HP column. After loading, the column was first washed with buffer A (20 mM Tris, 100 mM NaCl, pH 7.5). The retained protein was then eluted with a linear gradient of buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). On an AKTA explorer (GE Healthcare) column, PBS (pH 7.5) was used as the mobile phase, and the eluent was loaded directly onto a desalting column (HiPrep, Sitopan). TM On a 26 / 10 desalting column. Mass spectrometry showed the correct product (observed mass 14914 Da), corresponding to 218-NHO2-PF56.

[0296] Preparation of modified antibodies A universal procedure for analytical RP-UPLC of cytokines, monoclonal antibodies, and antibody-cytokine conjugates. Prior to RP-UPLC analysis, protein samples were diluted to approximately 0.1 mg / mL using PBS (pH 7.4). RP-UPLC analysis was performed on a Waters Acquity UPLC-SQD column. Samples (5 µL) were injected at 0.4 mL / min onto a Bioresolve RP mAb 2.1 × 150 mm 2.7 μm (Waters) column at 70°C. A linear gradient of 0.1% TFA and acetonitrile in water was applied from 30% to 54% over 9 minutes.

[0297] A universal SE-HPLC program for the analysis of cytokines, monoclonal antibodies, and antibody-cytokine conjugates. HPLC-SEC analysis was performed on an Agilent 1100 series (HP) column using an Xbridge BEH200A (3.5 µM, 7.8 x 300 mm, PN 186007640, Waters Corporation) column. Samples were diluted to 1 mg / mL in PBS and measured at 0.86 mL / min isocratic rate (0.1 M sodium phosphate buffer (pH 6.9) (NaHPO4 / Na2PO4) containing 10% isopropanol) for 16 minutes.

[0298] Example 34A. Transient expression of mPD-L1 in CHO mPD-L1 is an antibody targeting mouse PD-L1 as described in US 2016 / 0340429 A1, consisting of the LC sequence identified by SEQ. ID NO: 16 and the HC sequence identified by SEQ. ID NO: 17. mPD-L1 was transiently expressed in CHO Kl cells at a scale of 500 mL by Evitria (Zurich, Switzerland). The antibody was purified using two tandemly linked HiTrapMabSelect Sure 5 mL columns. After loading the supernatant, the columns were washed with TBS (pH 7.5) at 10 CV. IgG was eluted with 0.1 M sodium acetate (pH 3.0) and neutralized with 2.5 M Tris-HCl (pH 7.2). After three dialysis cycles to 20 mM histidine-HCl and 150 mM NaCl (pH 7.5), IgG was concentrated to 19.3 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) and obtained in a final yield of 87 mg.

[0299] Example 34B. Transient expression of enrofloxacin in CHO Enfortumab, an antibody targeting conjugation-4, was transiently expressed in CHO Kl cells at a scale of 3 L by Evitria (Zurich, Switzerland). The antibody was purified using a 20 mL Protein A column (Captiva PriMAb). After loading the supernatant, the column was washed with TBS (pH 7.5) at 10 CV. IgG was eluted with 0.1 M sodium acetate (pH 3.0) and neutralized with 2.5 M Tris-HCl (pH 7.2). After three dialyses to 20 mM histidine-HCl and 150 mM NaCl (pH 7.5), the IgG was concentrated to 19.96 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) and obtained in a final yield of 99 mg.

[0300] Example 34C. Enroximate remodeling of enroximate to enroximate (6-N)3 -GalNAc) 2 (enfo-v1a) Enroximate (3509 µL, 80 mg, 17.7 mg / mL, in 20 mM histidine-HCl, 150 mM NaCl, pH 7.5) was incubated with EndoSH (1% w / w) (as described in PCT / EP2017 / 052792), His-TnGalNAcT (3% w / w) (as described in PCT / EP2016 / 059194), alkaline phosphatase (commercially available from Roche, 0.01% w / w), and UDP-6-N3-GalNAc (25 eq compared to IgG) (prepared according to PCT / EP2016 / 059194) at 30°C in 20 mM histidine-HCl (pH 7.5), 150 mM NaCl (pH 7.5), and 6 mM MnCl2 for 16 h. Next, the functionalized IgG was purified using two tandemly connected HiTrap MabSelect Sure 5 mL columns. After loading the reaction mixture, the columns were washed with TBS + 0.2% Triton at 10 CV, followed by washing with TBS (pH 7.5) at 10 CV. The IgG was eluted with 0.1 M sodium acetate (pH 3.0) and neutralized with 2.5 M Tris-HCl (pH 7.2). After three dialysiss to TBS (pH 7.5), the IgG was concentrated to approximately 25 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). Mass spectrometry analysis of the sample after IdeS treatment revealed a major Fc / 2 product (observed mass 24365 Da, approximately 90% of the total Fc / 2), corresponding to a 6-N3-GalNAc-GlcNAc(Fuc)-substituted Fc / 2 fragment.

[0301] Example 35. Trastuzumab enzymatic remodeling into tras(6-N) 3 -GalNAc) 2 (tras-v1a) Trastuzumab (Herceptin) obtained from the pharmacy was reconstituted and exchanged with 20 mM histidine-HCl and 150 mM NaCl (pH 7.5) using a HiPrep™ 26 / 10 desalting column (Sentofan) on an AKTA Purifier-10 (Sentofan). Trastuzumab (31.3 mL, 1128 mg, 36.0 mg / mL, in 20 mM histidine-HCl, 150 mM NaCl, pH 7.5) was incubated with EndoSH (1% w / w) (as described in PCT / EP2017 / 052792), His-TnGalNAcT (as described in PCT / EP2016 / 059194) (3% w / w), alkaline phosphatase (commercially available from Roche, 0.01% w / w), and UDP-6-N3-GalNAc (10 eq compared to IgG) (prepared according to PCT / EP2016 / 059194) at 30°C in 20 mM histidine-HCl (pH 7.5), 150 mM NaCl (pH 7.5), and 6 mM MnCl2 for 16 h. Next, the functionalized IgG was purified using a Protein A column (Captiva PriMab, CV = 50 mL). After loading the reaction mixture, the column was washed with TBS + 0.2% Triton at 10 CV, followed by washing with TBS (pH 7.5) at 10 CV. The IgG was eluted with 0.1 M sodium acetate (pH 3.0) and neutralized with 2.5 M Tris-HCl (pH 7.2). After three dialyses to TBS (pH 7.5), the IgG was concentrated to approximately 25 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). Mass spectrometry analysis of the sample after IdeS treatment revealed a major Fc / 2 product (observed mass 24366 Da, approximately 90% of the total Fc / 2) (corresponding to the 6-N3-GalNAc-GlcNAc(Fuc) substituted Fc / 2 fragment) and a minor Fc / 2 product (observed mass 24220 Da, approximately 10% of the total Fc / 2) (corresponding to the 6-N3-GalNAc-GlcNAc substituted Fc / 2 fragment).

[0302] Example 36. Intramolecular crosslinking of tras-v1a with trivalent linker 145 to give tras-v1a-145 Towards tras-v1aThe solution (1698 µL, 35 mg, 20.61 mg / mL, in TBS (pH 7.5)) was supplemented with TBS (pH 7.5) (1802 µL), propylene glycol (3427 µL), and trivalent linker 145 (23.3 µL, 40 mM in DMF, 4 equivalents compared to IgG). The reaction was incubated overnight at RT, followed by purification on a Superdex 200 Increase 16 / 600 column (Stenofan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stenofan). Mass spectrometry analysis of the IdeS digested sample revealed a major product (calculated mass 49799 Da, observed mass 49799 Da) corresponding to an intramolecularly cross-linked trastuzumab derivative. tras-v1a-145 .

[0303] Example 37. Enzymatic remodeling of mPD-L1 to mPD-L1(6-N) 3 -GalNAc) 2 (mPD-L1-v1a) mPD-L1 (4528 µL, 87 mg, 19.3 mg / mL, in 20 mM histidine-HCl, 150 mM NaCl, pH 7.5) was incubated with EndoSH (1% w / w) (as described in PCT / EP2017 / 052792), His-TnGalNAcT (as described in PCT / EP2016 / 059194) (4% w / w), alkaline phosphatase (commercially available from Roche, 0.01% w / w), and UDP-6-N3-GalNAc (25 eq compared to IgG) (prepared according to PCT / EP2016 / 059194) at 30°C in 20 mM histidine-HCl (pH 7.5), 150 mM NaCl (pH 7.5), and 6 mM MnCl2 for 16 h. Next, the functionalized IgG was purified using two tandemly connected HiTrap MabSelect Sure 5 mL columns. After loading the reaction mixture, the columns were washed with TBS + 0.2% Triton at 10 CV, followed by washing with TBS (pH 7.5) at 10 CV. The IgG was eluted with 0.1 M sodium acetate (pH 3.0) and neutralized with 2.5 M Tris-HCl (pH 7.2). After three dialyzes to PBS (pH 7.4), the IgG was concentrated to approximately 25 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). Mass spectrometry analysis of the sample after IdeS treatment revealed a major Fc / 2 product (observed mass 24368 Da, approximately 90% of the total Fc / 2), corresponding to a 6-N3-GalNAc-GlcNAc(Fuc)-substituted Fc / 2 fragment.

[0304] Example 38. Intramolecular crosslinking of mPD-L1-v1a with trivalent linker 145 to give mPD-L1-v1a-145 Towards mPD-L1-v1a A solution (1569 µL, 50 mg, 31.86 mg / mL, in PBS (pH 7.4)) was supplemented with TBS (pH 7.5) (3431 µL), propylene glycol (4967 µL), and trivalent linker 145 (33.3 µL, 40 mM solution in DMF, 4 equivalents compared to IgG). The reaction was incubated overnight at RT, followed by purification on a Superdex 200 Increase 16 / 600 column (Stopvan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stopvan). Mass spectrometry analysis of the IdeS digested sample revealed a major product (calculated mass 49799 Da, observed mass 49799 Da) corresponding to an intramolecularly crosslinked mPD-L1 derivative. mPD-L1-v1a-145 .

[0305] Example 39. mPD-L1-v1a coupled with the bivalent linker XL14 to give mPD-L1-v1a-XL14 Towards mPD-L1-v1a A solution (1883 µL, 60 mg, 31.86 mg / mL, in PBS (pH 7.4)) was prepared by adding TBS (pH 7.5) (1617 µL) and the divalent linker XL14 (500 µL, 20 mM in DMF, 25 equivalents to IgG). The reaction was incubated overnight at RT, followed by purification on a Superdex 200 Increase 16 / 600 column (Stenofan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stenofan). Mass spectrometry analysis of the IdeS digested sample revealed a major product (observed mass 25063 Da, approximately 90% of the total Fc / 2), corresponding to the desired product where XL14 was coupled only to a single Fc / 2 fragment, leaving a second reactive stem for further coupling. A minor product was observed (observed mass 49427 Da, approximately 10% of total Fc / 2), corresponding to an undesirable intramolecular crosslinked derivative.

[0306] Preparation of antibody-cytokine conjugates General procedures for purifying antibody-cytokine conjugates Unless otherwise specified, purify the antibody-cytokine conjugate according to the following procedure. Dilute the reaction with 2 volumes of 20 mM Tris-HCl (pH 8.0) to reduce the salt concentration to 50 mM NaCl. Next, load the sample onto a 5 mL HiTrap Q HP column (Stenofan) using 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl as the mobile phase. After loading, wash the column with ≥ 20 column volumes (CV) of 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 0.2% Triton-X100, followed by ≥ 20 CV of 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. Next, elute the product using a linear gradient from 20 CV to 20 mM Tris-HCl (pH 8.0) and 500 mM NaCl. Fractions containing the desired product were collected, combined, and concentrated to approximately 5 mL using a Vivaspin Turbo 15 ultrafiltration system (Sartorius). The sample was then purified on a Superdex 200 Increase 16 / 600 column (Stenofan) using PBS (pH 7.4) as the mobile phase. The final antibody-cytokine conjugate was flash-frozen and stored at -80°C until further use.

[0307] Example 40. Transient expression of gene fusion IC1 in CHO The gene fusion IC1 consisted of the same mPD-L1 antibody targeting mouse PD-L1, fused at the C-terminus to the same cleavable linker and IL-15 sequence. The LC sequence was identified by SEQ. ID NO: 16 and the HC sequence by SEQ. ID NO: 18. IC1 was transiently expressed in CHO Kl cells at a scale of 1100 mL by Evitria (Zurich, Switzerland). The antibody was purified using two tandemly linked HiTrap MabSelect Sure 5 mL columns. After loading the supernatant, the column was washed with TBS (pH 7.5) at 10 CV. The gene fusion protein was eluted with 0.1 M sodium acetate (pH 3.0) and neutralized with 2.5 M Tris-HCl (pH 7.2) to produce 22 mg IC1, with a monomer level of 77% according to SE-HPLC analysis. The sample was concentrated to 5 mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) and then purified on a Superdex 200 Increase 16 / 600 column (Sartorius) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stopvan). IC1 was obtained with a final yield of 12 mg and a monomer level of 95% according to SE-HPLC analysis. The separation of IC1 was confirmed by reducing SDS-PAGE analysis (Figure 18). The broadband observed for the heavy chain (HC) fused with IL-15 indicates heterogeneity, which is likely due to glycosylation on the IL-15 moiety.

[0308] Example 41. Coupling tras-v1a-145 with NHO1-PF50 to obtain the conjugate tras-v1a-145-NHO1-PF50 （2:1） Add NHO1-PF50 (150 µL, 3.7 mg / mL, in PBS (pH 7.4), 2.6 equivalents to IgG) to a solution of tras-v1a-145 (247 µL, 2 mg, 8.1 mg / mL, in PBS (pH 7.4)). Incubate the reaction overnight at room temperature. Dilute the reaction 20-fold with 20 mM Tris-HCl (pH 8.0) and load the mobile phase of 20 mM Tris-HCl (pH 8.0) and 10 mM NaCl onto a 1 mL HiTrap Q HP column (Stenofan). Elute the product using a linear gradient of 50 CV to 20 mM Tris-HCl (pH 8.0) and 1 M NaCl. Collect the fraction containing the desired 2:1 fraction and concentrate it to 1 mL using a centrifuge filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Next, the sample was purified on a Superdex 200 Increase 10 / 300 GL column (Stenofan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stenofan). Mass spectrometry analysis of the intact sample revealed a major product (observed mass 162697 Da) corresponding to the conjugate tras-v1a-145-NHO1-PF50 (2:1).

[0309] Example 42. mPD-L1-v1a is coupled with 218-NHO1-PF51 to obtain the conjugate mPD-L1-v1a-218-NHO1- PF51 (2:2) 218-NHO1-PF51 (1352 µL, 7.8 mg / mL, in PBS (pH 7.4), 5 equivalents to IgG) was added to a solution of mPD-L1-v1a (659 µL, 21 mg, 31.9 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at 37°C, followed by purification according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO1-PF51 (2:2) (Figure 18).

[0310] Example 43. mPD-L1-v1a is coupled with 218-NHO2-PF52 to obtain the conjugate mPD-L1-v1a-218-NHO2- PF52 (2:2) 218-NHO2-PF52 (2077 µL, 6.0 mg / mL, in PBS (pH 7.4), 5 equivalents to IgG) was added to a solution of mPD-L1-v1a (785 µL, 25 mg, 31.9 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at 37°C, followed by purification according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO2-PF52 (2:2) (Figure 18). Additionally, mass spectrometry analysis of the IdeS-digested sample revealed a major product (observed mass 39295 Da) corresponding to the conjugated Fc / 2 fragment.

[0311] Example 44. mPD-L1-v1a is coupled with 218-NHO2-PF53 to obtain the conjugate mPD-L1-v1a-218-NHO2- PF53 (2:2) 218-NHO2-PF53 (2639 µL, 3.64 mg / mL, in PBS (pH 7.4), 5 equivalents to IgG) was added to a solution of mPD-L1-v1a (606 µL, 19.3 mg, 31.9 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at 37°C, followed by purification according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO2-PF53 (2:2) (Figure 18). Additionally, mass spectrometry analysis of the IdeS-digested sample revealed a major product (observed mass 39296 Da) corresponding to the conjugated Fc / 2 fragment.

[0312] Example 45. mPD-L1-v1a is coupled with 218-NHO2-PF54 to obtain the conjugate mPD-L1-v1a-218-NHO2- PF54 (2:2) 218-NHO2-PF54 (3261 µL, 5.39 mg / mL, in PBS (pH 7.4), 6 equivalents to IgG) was added to a solution of mPD-L1-v1a (942 µL, 30 mg, 31.9 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at 37°C, followed by purification according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO2-PF54 (2:2) (Figure 18). Additionally, mass spectrometry analysis of the IdeS-digested sample revealed a major product (observed mass 39012 Da) corresponding to the conjugated Fc / 2 fragment.

[0313] Example 46. mPD-L1-v1a is coupled with 218-NHO2-PF56 to obtain the conjugate mPD-L1-v1a-218-NHO2- PF56 (2:2) 218-NHO2-PF56 (1667 µL, 6.56 mg / mL, in PBS (pH 7.4), 5 equivalents to IgG) was added to a solution of mPD-L1-v1a (691 µL, 22 mg, 31.9 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at 37°C, followed by purification according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO2-PF56 (2:2) (Figure 18). Additionally, mass spectrometry analysis of the IdeS-digested sample revealed a major product (observed mass 39280 Da) corresponding to the conjugated Fc / 2 fragment.

[0314] Example 47. mPD-L1-v1a-145 is coupled with NHO2-PF52 to obtain the conjugate mPD-L1-v1a-145-NHO2- PF52 (2:1) NHO2-PF52 (910 µL, 7.47 mg / mL, in PBS (pH 7.4), 3 equivalents to IgG) was added to a solution of mPD-L1-v1a-145 (869 µL, 23.6 mg, 27.1 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at room temperature, followed by purification according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-145-NHO2-PF52 (2:1) (Figure 18). Additionally, mass spectrometry analysis of the IdeS-digested sample revealed a major product (observed mass 64226 Da) corresponding to a cross-linked and coupled Fc / 2 fragment (Fc / 2-IL-15-Fc / 2).

[0315] Example 48. mPD-L1-v1a-XL14 was coupled with NHO2-PF52 to obtain the conjugate mPD-L1-v1a-XL14- NHO2-PF52 (2:2) NHO2-PF52 (1739 µL, 7.47 mg / mL, in PBS (pH 7.4), 3 equivalents to IgG) was added to a solution of mPD-L1-v1a-XL14 (1541 µL, 45 mg, 29.21 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at 37°C, followed by purification according to standard procedures. Mass spectrometry analysis of the IdeS-digested sample revealed a major product (observed mass 39495 Da) corresponding to the conjugated Fc / 2 fragment.

[0316] Example 49. Coupling tras-v1a-145 with NHO1-PF57 to obtain the conjugate tras-v1a-145-NHO1-PF57 （2:1） Add NHO1-PF57 (727 µL, 6.20 mg / mL, in PBS (pH 7.4), 1.6 equivalents to IgG) to a solution of tras-v1a-145 (1600 µL, 16 mg, 10 mg / mL, in PBS (pH 7.4)). Incubate the reaction overnight at room temperature. Dilute the reaction 20-fold with 20 mM Tris-HCl (pH 8.0) and load it onto a 1 mL HiTrap Q HP column (Stenofan) using 20 mM Tris-HCl (pH 8.0) and 10 mM NaCl as the mobile phase. Elute the product using a linear gradient of 50 CV to 20 mM Tris-HCl (pH 8.0) and 1 M NaCl. Collect the fraction containing the desired 2:1 form and concentrate it to 1 mL using a centrifuge filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Next, the sample was purified on a Superdex 200 Increase 10 / 300 GL column (Stenofan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stenofan). Mass spectrometry analysis of the intact sample revealed a major product (observed mass 173099 Da) corresponding to the conjugate tras-v1a-145-NHO1-PF57 (2:1).

[0317] Example 50. Coupling tras-v1a-145 with NHO1-PF58 to obtain the conjugate tras-v1a-145-NHO1-PF58 （2:1） NHO1-PF58 (42 µL, 7.94 mg / mL, in PBS (pH 7.4), 3 equivalents to IgG) was added to a solution of tras-v1a-145 (86 µL, 0.7 mg, 8.1 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at room temperature. Next, the sample was purified on a Superdex 200 Increase 10 / 300 GL column (Stenofan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stenofan). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO1-PF58 (2:1) (Figure 19).

[0318] Example 51. Coupling tras-v1a-145 with NHO1-PF59 to obtain the conjugate tras-v1a-145-NHO1-PF59 （2:1） NHO1-PF59 (140 µL, 2.16 mg / mL, in PBS (pH 7.4), 2 equivalents to IgG) was added to a solution of tras-v1a-145 (85 µL, 0.9 mg, 10 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at room temperature. Next, the sample was purified on a Superdex 200 Increase 10 / 300 GL column (Stenofan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stenofan). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO1-PF59 (2:1) (Figure 19).

[0319] Example 52. Coupling tras-v1a-145 with NHO2-PF57 to obtain the conjugate tras-v1a-145-NHO2-PF57 （2:1） NHO2-PF57 (69 µL, 7.49 mg / mL, in PBS (pH 7.4), 3 equivalents to IgG) was added to a solution of tras-v1a-145 (95 µL, 1.0 mg, 10.6 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at room temperature. Next, the sample was purified on a Superdex 200 Increase 10 / 300 GL column (Stenofan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stenofan). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO2-PF57 (2:1) (Figure 20).

[0320] Example 53. Coupling tras-v1a-145 with NHO3-PF57 to obtain the conjugate tras-v1a-145-NHO3-PF57 （2:1） NHO3-PF57 (86 µL, 5.96 mg / mL, in PBS (pH 7.4), 3 equivalents to IgG) was added to a solution of tras-v1a-145 (95 µL, 1.0 mg, 10.6 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at room temperature. Next, the sample was purified on a Superdex 200 Increase 10 / 300 GL column (Stenofan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stenofan). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO3-PF57 (2:1) (Figure 20).

[0321] Example 54A. tras-v1a-145 is coupled with NHO4-PF57 to obtain the conjugate tras-v1a-145-NHO4- PF57 (2:1) NHO4-PF57 (73 µL, 7.06 mg / mL, in PBS (pH 7.4), 3 equivalents to IgG) was added to a solution of tras-v1a-145 (95 µL, 1.0 mg, 10.6 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at room temperature. Next, the sample was purified on a Superdex 200 Increase 10 / 300 GL column (Stenofan) using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Stenofan). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO4-PF57 (2:1) (Figure 20).

[0322] Example 54B. tras-v1a is coupled with 218-NHO2-PF52 to obtain the conjugate tras-v1a-218-NHO2- PF52 (2:2) 218-NHO2-PF52 (1285 µL, 5.57 mg / mL, in PBS (pH 7.4), 5 equivalents to IgG) was added to a solution of tras-v1a (607 µL, 14.4 mg, 23.72 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at 37°C, followed by purification according to standard procedures. Mass spectrometry analysis of the IdeS digested sample revealed a major product (observed mass 39294 Da) corresponding to the conjugated Fc / 2 fragment of tras-v1a-218-NHO2-PF52 (2:2).

[0323] Example 54C. Enfo-v1a is coupled with 218-NHO2-PF52 to obtain the conjugate enfo-v1a-218-NHO2- PF52 (2:2) 218-NHO2-PF52 (964 µL, 5.57 mg / mL, in PBS (pH 7.4), 5 equivalents to IgG) was added to a solution of enfo-v1a (470 µL, 10.8 mg, 22.97 mg / mL, in PBS (pH 7.4)). The reaction was incubated overnight at 37°C, followed by purification according to standard procedures. Mass spectrometry analysis of the IdeS digested sample revealed a major product (observed mass 39294 Da) corresponding to the conjugated Fc / 2 fragment of enfo-v1a-218-NHO2-PF52 (2:2).

[0324] Evaluation of antibody-cytokine conjugates Example 55: Freeze-thaw stability study The antibody-cytokine conjugates mPD-L1-v1a-218-NHO1-PF51 (2:2), mPD-L1-v1a-218-NHO2-PF52 (2:2), mPD-L1-v1a-145-NHO2-PF52 (2:1), and IC1 were diluted to 1 mg / mL in PBS (pH 7.4). The samples were then rapidly frozen in liquid nitrogen and thawed again. This step was performed five times. Aggregate levels were determined by SE-HPLC after 0, 1, 3, and 5 cycles (Figure 21). Compared to the gene fusion IC1, the conjugates mPD-L1-v1a-218-NHO1-PF51 (2:2), mPD-L1-v1a-218-NHO2-PF52 (2:2), and mPD-L1-v1a-145-NHO2-PF52 (2:1) were less prone to aggregation.

[0325] Example 56: Antibodies prepared using IL-15 variants with variable N-terminal IL-15 sequences and different hydroxylamines. Stability study of cytokine conjugates The antibody-cytokine conjugates were diluted to 1 mg / mL in PBS (pH 7.4) and incubated at 37°C. Stability was determined by RP-UPLC analysis at specified time points (Table 2). Variants prepared with hydroxylamine HO2, HO3, and HO4 showed improved stability compared to the variant prepared using HO1.

[0326] Table 2. Stability of antibody-cytokine conjugates as determined by RP-UPLC. Stability is plotted as a percentage of the cytokine to antibody ratio compared to T0. Example 57: Cleavage of antibody-cytokine conjugates via matrix protease (MT-SP1) mPD-L1-v1a-218-NHO2-PF52 (2:2) was incubated at a final concentration of 1 mg / mL with 0.5, 1.0, 2.0, and 4.0 (w / w)% recombinant human matrix protease (commercially available from Bio-techne) at 37°C for 2 hours, followed by RP-UPLC analysis. Complete cleavage was observed using 1.0% matrix protease (Figure 22).

[0327] Example 58. Treatment of antibody-cytokine conjugates with MT-SP1 prior to in vitro evaluation A set of antibody-cytokine conjugates (35 µg each), as shown in Figure 23, were incubated at 37°C for 2 h with 2 (w / w)% recombinant human matrix protease (commercially available from Andy Biotech) at a final concentration of 1 mg / mL, followed by RP-UPLC analysis. Complete cleavage was observed in all cases (Figure 23). IL-15 activity was evaluated in an in vitro IL-15 bioassay using the cleaved constructs.

[0328] Example 59: Cleavage of antibody-cytokine conjugates via urokinase (uPA) Tras-v1a-145-NHO1-PF50 (2:1) was incubated at a final concentration of 1 mg / mL with 0.1%, 1.0%, and 10.0% (w / w) of recombinant human urokinase (uPA, commercially available from Andy Biotech) at room temperature for 3 hours. Whole samples were analyzed by MS. For the 10% (w / w) uPA condition, mass spectrometry analysis of whole samples showed a major product (observed mass 149118 Da) (corresponding to an IL-15-free cleavage antibody) and a minor product (observed mass 13598 Da) (corresponding to free IL-15). For the 0.1% and 1.0% (w / w) uPA conditions, mass spectrometry analysis of whole samples showed uncleaved tras-v1a-145-NHO1-PF50 (2:1) as the major product (observed mass 162708 Da). IL-15 activity was evaluated in an in vitro IL-15 bioassay using samples treated with 10% (w / w) uPA.

[0329] In vitro evaluation General Procedure for In Vitro IL-15 Bioassay IL-15 activity was monitored using the IL-15 Bioassay (Promega, catalog number JA2015). This is a bioluminescent cell-based assay designed to measure IL-15 stimulation or inhibition using a STAT-5 response element as a readout. When IL-15 binds to its receptor, receptor-mediated pathway signaling induces luminescence, which can be detected and quantified with a photometer after substrate addition. The assay was performed according to the manufacturer's instructions. In brief, unless otherwise specified, the cleaved or intact antibody-cytokine conjugate or control was added in duplicate to IL-15 cells at a 10-fold dilution serially, square root, to obtain a final concentration ranging from 0.1 to 1000 pM (in the case of cleaved constructs, the concentration refers to the initial antibody-cytokine conjugate). IL-15 cells were incubated for 6 hours at 37°C and a humidified atmosphere of 5% CO2. The assay reagent (Bio-Glo™ luciferase assay reagent) was then added, and the chemiluminescence intensity was recorded using a microplate reader (Envision, PerkinElmer). Data analysis was performed using Graphpad Prism software. EC was calculated using nonlinear regression. 50 The maximum induction fold was calculated by dividing the relative luminescent units (RLU) of the treated cells by the RLU of the untreated cells (both minus the background).

[0330] Example 60. In vitro IL-15 activity assay of intact and cleaved antibody-cytokine conjugates The activity of five intact antibody-cytokine conjugates, including mPD-L1-v1a-218-NHO2-PF52 (2:2), was measured via an in vitro IL-15 bioassay for comparison, with and without MT-SP1 pretreatment. MT-SP1 treatment was performed according to Example 58. All intact antibody-cytokine conjugates showed only minimal activation in the range of 1.0 to 2.5 fold induction, while the cleaved variant of mPD-L1-v1a-218-NHO2-PF52 (2:2) showed an induction fold of 7.2 (Figure 24 and Table 3), demonstrating that IL-15 is inactivated or attenuated upon linker conjugation with the antibody but is activated upon cleavage of the linker by MT-SP1.

[0331] Table 3. EC50 of intact and cleaved antibody-cytokine conjugates 50 Values ​​and maximum induction folds, as determined by IL-15 bioassay. Example 61. In vitro IL-15 activity assay of cleaved antibody-cytokine conjugates According to Example 58, antibody-cytokine conjugates were pretreated with MT-SP1 to obtain free cytokines. The activities of five cleaved antibody-cytokine conjugates were measured via an in vitro IL-15 bioassay according to a standard procedure. Both mPD-L1-v1a-218-NHO1-PF51 (2:2) and mPD-L1-v1a-218-NHO2-PF52 (2:2) showed significant activity in ECG compared to the gene fusion IC1, which contained the same cleavable linker, IL-15 variant, and stoichiometry. 50 It showed higher potency in both value and maximum induction fold (Figure 25 and Table 4). Compared with the 2:2 form, the corresponding 2:1 form mPD-L1-v1a-145-NHO2-PF52 (2:1) showed approximately 2 times higher EC50. 50 The values ​​are as expected based on the amount of IL-15 that can be released. The variant mPD-L1-v1a-218-NHO2-PF53 (2:2), which has enhanced IL-15Rβ binding, showed comparable in vitro potency to mPD-L1-v1a-218-NHO2-PF52 (2:2), which can be explained by the fact that the assay was performed using a mouse cell line and that this mutation binds to human IL-15Rβ with a higher affinity than to mouse IL-15Rβ.

[0332] Table 4. EC5 values ​​of antibody-cytokine conjugates pretreated with MT-SP1 50 Value and maximum induction fold were determined by IL-15 bioassay. Example 62. In vitro IL-15 activity assay of intact and cleaved antibody-cytokine conjugates Following Example 58, mPD-L1-v1a-218-NHO2-PF52 (2:2) and mPD-L1-v1a-218-NHO2-PF56 (2:2) were pretreated with MT-SP1 to obtain free cytokines. The activities of both compounds with and without MT-SP1 pretreatment were measured using an in vitro IL-15 bioassay. Unmodified cytokine PF52 was included as a reference. Cleavage and intact mPD-L1-v1a-218-NHO2-PF56 (2:2) were added at higher final concentrations in the range of 32 pM to 316 nM (square root of the 10-fold dilution series). Both antibody-cytokine conjugates showed activation upon pretreatment with MT-SP1, as demonstrated by the fold increase in induction (Figure 26 and Table 5). mPD-L1-v1a-218-NHO2-PF56 (2:2) showed a high >1000-fold EC50. 50 The value indicates that the high-affinity binding with IL-15Rα has disappeared, while the binding with IL-15Rβ / γ remains.

[0333] Table 5. EC5 of intact and cleaved antibody-cytokine conjugates 50 Values ​​and maximum induction folds, as determined by IL-15 bioassay. Unmodified cytokine p52 was included as a reference. Example 63. In vitro IL-15 activity assay using alternative antibodies and cleavable linkers Following Example 59, Tras-v1a-145-NHO1-PF50 (2:1) was pretreated with urokinase (UpA) to obtain free cytokines. The activity of tras-v1a-145-NHO1-PF50 (2:1) with and without UpA pretreatment was measured using an in vitro IL-15 bioassay. Unmodified cytokine PF50 and unmodified antibody trastuzumab were included as controls. Intact tras-v1a-145-NHO1-PF50 (2:1) showed only minimal activation (Table 6 and Figure 27), while cleaved tras-v1a-145-NHO1-PF50 (2:1) showed a similar response to modified cytokine PF50.

[0334] Table 6. EC5 of intact and cleaved antibody-cytokine conjugates 50 Values ​​and maximum induction folds, as determined by IL-15 bioassay. Unmodified cytokine p52 was included as a reference. In vivo evaluation Example 64. In vivo tolerance in female BALB / c mice Female BALB / c mice (groups of 3 mice, 6 to 8 weeks old at the start of the study, obtained from Vital River Laboratory Animal Technology Co., Ltd., China) were treated with either the medium (PBS pH 7.4) or the specified antibody-cytokine conjugate (6 mg / kg) and compared with IC1 (6 mg / kg). In all cases, via subcutaneous A single dose was administered by injection. Following administration, the body weight of all animals was measured daily (Figures 28 and 29, top). Based on the readings from this initial study, a second tolerability study was conducted according to the same procedure but using concentrations ranging from 3 to 12 mg / kg (Figures 28 and 29, middle). Finally, a third study was conducted according to the same procedure using concentrations ranging from 4.5 to 18 mg / kg (Figures 28 and 29, bottom). The antibody-cytokine conjugates mPD-L1-v1a-218-NHO1-PF51 (2:2) and mPD-L1-v1a-218-NHO2-PF52 (2:2) with IL-15 cleavage showed no signs of toxicity at 6 and 12 mg / kg, but a modest weight loss (≤ 10% mean weight loss) was observed when administered at 18 mg / kg, indicating an MTD of approximately 18 mg / kg (Figure 28). The variant mPD-L1-v1a-218-NHO2-PF56 (2:2), lacking IL-15Rα binding, showed an MTD of approximately 4.5 mg / kg, and for an initial dose of 6 mg / kg, one in three mice died on day 5. Variants mPD-L1-v1a-145-NHO2-PF52 (2:1) and mPD-L1-v1a-218-NHO2-PF53 (2:2) also showed reduced tolerability compared to mPD-L1-v1a-218-NHO2-PF52 (2:2), with estimated MTDs of 9 and 15 mg / kg, respectively (Figure 29).

[0335] Example 65. In vivo efficacy in a CT26 syngeneic colon cancer model Female BALB / c mice (5 to 8 weeks old at the start of the study, obtained from Viton Lever Laboratory Animal Technology Co., Ltd., China) were subcutaneously inoculated in the right posterior flank region with 5 × 10⁶ PBS in 0.1 ml. 5 CT26 tumor cells are used for tumor development. When the tumor volume is between 70 and 120 mm... 3 Within the range, in each group of eight mice subcutaneousInjection of the mediator (PBS pH 7.4) or the specified antibody-cytokine conjugate (3 or 6 mg / kg as specified). In all cases, the dose was administered on days 0 and 7. Tumor volume and body weight were measured 2–3 times weekly after randomization. Compared with the corresponding gene fusion IC1 and the uncleavable variant mPD-L1-v1a-218-NHO2-PF54 (2:2), mPD-L1-v1a-218-NHO2-PF52 (2:2) showed improved antitumor efficacy (Figure 30). Additionally, variants mPD-L1-v1a-145-NHO2-PF52 (2:1), mPD-L1-v1a-218-NHO2-PF53 (2:2), and mPD-L1-v1a-218-NHO2-PF56 (2:2) showed antitumor efficacy compared with the mediator group (Figure 31).

[0336] Example 66. In vivo efficacy in an MC38 syngeneic colon cancer model Female C57BL / 6 mice (6-7 weeks old at the start of the study, obtained from Viton Lever Laboratory Animal Technology Co., Ltd., China) were subcutaneously inoculated in the right posterior flank region with 1 × 10⁻⁶ PBS in 0.1 ml. 6 MC38 colorectal cancer cells were used to study tumor development. When the average tumor volume reached 94 mm... 3 At that time, the mice were divided into groups of 10 mice each, and subcutaneous Injectable mediator (PBS pH 7.4) or mPD-L1-v1a-218-NHO2-PF52 (2:2) (3 or 6 mg / kg as specified). In all cases, the dose was administered on days 0, 7, and 14. Tumor volume and body weight were measured 2–3 times weekly after randomization. Compared with the mediator group, mPD-L1-v1a-218-NHO2-PF52 (2:2) showed a significant reduction in tumor volume (p < 0.001 for both dose levels, as determined by one-way ANOVA on day 16, followed by Tukey HSD test).

[0337] sequence The following list provides a selection of peptide sequences relevant to this invention. A complete list is disclosed in a separate sequence listing file.

[0338] Protease can cleave linker 1 Sequence identifier (SEQ. ID NO: 1): GSSGGSGGSGGSGLSGRSDNHGSSGS Protease can cleave linker 2 Sequence identifier (SEQ. ID NO: 2): GGGGSGGGGSRASRANGS Sequence identifier of human IL-15 (SEQ. ID NO: 3): NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS of IL-15Rα-IL-15 fusion protein Sequence identifier (SEQ. ID NO: 4): ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS of SYR-(G4S)3-IL15 (PF18) Sequence identifier (SEQ. ID NO: 5): SYRGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS of SYR cleavable linker 1-IL15 (PF50) Sequence identifier (SEQ. ID NO: 6): MSYRGSSGGSGGSGGSGLSGRSDNHGSSGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS of SYR cleavable linker 2-IL15 (PF51) Sequence identifier (SEQ. ID NO: 7): MSYRGGGGSGGGGSRASRANGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ST Cleavable Linker 2-IL15 (PF52) Sequence identifier (SEQ. ID NO: 8): STGGGGSGGGGSRASRANGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ST Cleavable Linker 2-IL15 (N72D) (PF53) Sequence identifier (SEQ. ID NO: 9): STGGGGSGGGGSRASRANGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ST-(G4S)3GGS-IL15 (PF54) Sequence identifier (SEQ. ID NO: 10): STGGGGSGGGGSGGGGSGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SYR-(G4S)8-IL15 (PF55) Sequence identifier (SEQ. ID NO: 11): SYRGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ST Cleavable Linker 2-IL15 (E46G, V49R) (PF56) Sequence identifier (SEQ. ID NO: 12): MSTGGGGSGGGGSRASRANGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLGLQRISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS of SYR-(G4S)8-IL15Rα-IL-15 (PF57) Sequence identifier (SEQ. ID NO: 13): (M)SYRGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS of S-(G4S)8-IL15Rα-IL-15 (PF58) Sequence identifier (SEQ. ID NO: 14): SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS of ST-(G4S)8-IL15Rα-IL-15 (PF59) Sequence identifier (SEQ. ID NO: 15): STGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS of the mPD-L1 light chain Sequence identifier (SEQ. ID NO: 16): QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC of the mPD-L1 heavy chain Sequence identifier (SEQ. ID NO: 17): EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQAPGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain of the gene fusion (IC1) Sequence identifier (SEQ. ID NO: 18): EVQLLESSGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQAPGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECV LNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFHVHIVQMFINTS

Claims

1. An antibody-cytokine conjugate having structure (1) or structure (2). in: - AB is an antibody; - L is the connector that connects AB to D; - x is an integer in the range of 1–10; - Z 1 and Z 2 It is a linking group, wherein at least Z 1 Products that include cycloaddition or nucleophilic reactions; - b is 0 or 1; - L 6 Yes – (H) v –S–(L 7 ) w’ -,in: OH is a monosaccharide. ov is an integer in the range of 1–10. o S is a sugar or a sugar derivative. o w' is 0 or 1, and o L 7 It is –N(H)C(O)–(CH2–O) z –(CH2–CH2–O) γ –CH2– or CH2–(O–CH2–CH2) γ – where γ is an integer in the range of 0–100 and z is 0 or 1; - D is based on –(D) 1 ) α –D 2 The polypeptide, wherein: o D 1 It is a cleavable peptide linker; o D 2 It is an immune cell binder polypeptide that is specific to immune cell receptors; o α is 0 or 1; - Where if α = 0, then L is a cuttable connector.

2. The antibody-cytokine conjugate according to claim 1, wherein b = 1, preferably D 2 The atomic chain between AB and AB consists of 10–100 atoms, preferably selected from B, C, O, N, S, and P.

3. The antibody-cytokine conjugate according to any one of the preceding claims, wherein the antibody is specific for extracellular receptors on tumor cells, preferably wherein the extracellular receptors on the tumor cells are selected from the group consisting of: 5T4 (TPBG), ADAM9, ALPP, ALPPL2, AMHRII, ASCT2 (SLC1A5), ASLG659, ASPHD1, av-integrin, avb3-integrin / ITGAV / CD51, Axl, B7-H3 (CD276), B7-H4 (VTCN1), BAFF-R, BCMA (CD269), BMPR1B, short proteoglycan, c-KIT (CD117), c-Met, C4.4a (LYPD3), CA-IX (CA9) / MN, cadherin-6, CanAg, CCR7, CD117 (c-KIT), CD123 (IL-3Rα), CD13, CD133, CD138 / cohesin Glycan-1, CD166 (ALCAM), CD19, CD20, CD203C, CD205, Ly75, CD21, CD22, CD228 (P79, SEMF), CD25 (IL-2R-α), CD30 (TNFRSF8), CD324 (CDH1 / E-cadherin), CD33, CD352 (SLAMF6, NTB-A), CD37, CD38, CD44v6, CD45, CD46, CD47, CD48a (SLAMF2), CD56 (NCAM), CD70, CD71 (TF-R), CD72, CD74, CD79a, CD79b, CDH6, cadherin-6, CEACAM5 (CD66e), sealing protein, CLDN18.2, CLDN6 (sealin-6, Skullin), CLEC12A, CLL-1 / CLEC12A, Cripto, CS1 (SLAMF7,CD319), CXCR5, DKL-1, DLL3 (δ-like 3), DPEP3, E16, EGFR, EGFRvIII, ENPP3, CD203c (AGS-16), EpCAM, EphA2, EphB2R, liver glycoside-A4 (EFNA4), ETBR, FAP, FGFR2, FGFR3, fibronectin EDB, FLT3, FOLR1 (FR-a), Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, GPC3 (phosphatidylinositol proteoglycan-3), gpNMB, GPR172A, GPR19, GPR54, GRP20, guanylate cyclase C (GCC), HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Ra, integrin avb6, KAAG-1, LAMP-1 (CD107a), Lewis Y (CD174), LGR5, LIV-1 (SLC39A6, ZIP6), LRRC15, LY64, Ly6E (lymphocyte antigen 6), Ly6G6D, LY6K, mesothelin ( MSLN), MFI2 (TAA), MICA / B, MOSPD2, MPF, MUC1 (CA6), MUC16 / CA-125, MUC1c, NaPi2b (SLC34A2), NCA, conjugin-4 (PVRL4), Notch3, p-cadherin (pCAD, CDH3), P2X5, PD-L1 (CD274, B7-H1), PMEL17, PRLR (prolactin), PSCA, PSMA, PTK7 (CCK4), RET, RNF43, RON, ROR1, ROR2, Serna 5b, SEZ6, SLITRK6 (SLC44A4), STEAP1, STEAP2, STn, TAG72, TENB2, TF (CD142, prothrombin kinase), TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, TNF-α, TROP-2 (TACSTD2), TWEAKR, receptor tyrosine kinase (RTK), tendinin, or the antibody may be specific for extracellular proteins and / or tumor-associated carbohydrate antigens (TACA) induced by viral infection.and / or wherein the immune cell engaging polypeptide is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15 , IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26, IL-28, IL-29, IL-33, IL-36, IL37, IL-38, IF N-α (including IFN-α1 / 13, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α14, IFN-α16, IFN-α17, and IFN-α21), IFN-β, IFN-γ, IFN-A, TFN-α, TNF-β, TGF-β1, M-CSF, G-CSF, GM-CSF, and CXL10; more preferably, the immune cell conjugating polypeptide is IL-2 or IL-15, more preferably IL-15.

4. The antibody-cytokine conjugate according to any one of the preceding claims, wherein each Z 1 Selected separately from structure (Z2) – (Z20c): The connection with L is represented by a tilde, B (-) It is an anion, B (+) It is a cation, and the nitrogen atoms of (Z10), (Z13), (Z14) and (Z15) can be linked to L, or contain hydrogen atoms or be substituted; R 36 It is a halogen selected from fluorine, chlorine, bromine, and iodine; Y 4 It is a heteroatom; R 35 Choose from the following groups: hydrogen, C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups, these C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups are optionally substituted and optionally selected from O, Si, S and NR. 14 One or more heteroatoms are interrupted, where R 14 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups; Cyclo-Z is selected from triazoles, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazoline, pyrazoline, or piperazine. Preferably, Z 1 Selected from structure (Z21) – (Z38a), more preferably Z 1 Yes (Z29): Even more preferably, Z 1 It can be obtained via a click reaction between cycloalkynes and azides, and Z 2 It can be obtained through a click reaction between cycloalkynes and nitrones.

5. The antibody-cytokine conjugate according to any one of the preceding claims, wherein the linker L is a linker having the following structure: -(L 1 )-(L 2 ) o -(L 3 ) p -(L 4 ) q – in: - L 1 With Z 1 Connect, and if it exists, L 4 With Z 2 connect; - L 1 L 2 L 3 and L 4 Each individually is Z 1 With Z 2 Connectors that are linked together; - o, p, and q are each individually 0 or 1, preferably o = p = q = 0; And preferably, wherein: a) Connector L 1 It is represented by the following: –(K 3 ) k –(K 1 ) d –(K 2 ) e –(K 1 ) f –(K 2 ) e’ –(C(O)) g -,or [–(K 3 ) k –(K 1 ) d –(K 2 ) e –(K 1 ) f –]2BM–(C(O)) g – (K 1 ) d’ –(K 2 ) e’ –(K 1 ) f’ –(C(O)) g’ –, in: - d and d' are either 0 or 1 individually. - e and e' are individually integers in the range of 1–10. - f and f' are either 0 or 1 individually. - g and g' are integers in the range of 0–10, - k = 0 or 1, provided that if k = 1, then d = 0. - K 1 It is based on the sulfonamide group in structure (23) Where a = 0 or 1, and R 13 Choose from the following groups: hydrogen, C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups, these C1–C 24 Alkyl groups, C3–C 24 Cycloalkyl groups, C2–C 24 (Hetero)aryl group, C3–C 24 alkyl(hetero)aryl groups and C3–C 24 (Hetero)arylalkyl groups are optionally substituted and optionally selected from O, S and NR. 14 One or more heteroatoms are interrupted, where R 14 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups; - K 3 is –OC(O)–, –C(O)O–, –C(O)NH–, –NHC(O)–, –OC(O)NH–, –NHC(O)O–, –(O)(CH2) m C(O)–、–C(O)(CH2) m C(O)NH– or –(4-Ph)CH2NHC(O)(CH2) m C(O)NH–, where m is an integer in the range of 0–10; - K 2 It is the –CH2–CH2–O– or –O–CH2–CH2– part, or (K 2 ) e It is –(CH2–CH2–O) e1 –CH2–CH2– or –(CH2–CH2–O) e1 –CH2– part, where the definition of e1 is the same as that of e; - BM is a branch portion, preferably selected from carbon atom, nitrogen atom, phosphorus atom, (hetero)aromatic ring, (hetero)cyclic or polycyclic portion, more preferably BM is nitrogen atom; and / or b) Connector L 2 It is a peptide spacer, preferably wherein L 2 It has a protease recognition sequence, preferably wherein L 2 It contains 1–5 amino acids, including optionally non-natural amino acids, and more preferably dipeptides, tripeptides or tetrapeptides as spacers. and / or c) Connector L 3 It is a spacer associated with the cutting mechanism, preferably L 3 Selected from p-aminobenzyloxycarbonyl derivatives based on structure (L3a) or glucuronic acid-functionalized p-hydroxybenzyloxycarbonyl (L3b): The parentheses indicate an optional carbonyl group, R 21 It is H, R 26 or C(O)R 26 , where R 26 It is C1–C 24 (Hetero)alkyl groups, C3–C 10 (Hetero)cycloalkyl groups, C2–C 10 (Hetero)aryl group, C3–C 10 alkyl(hetero)aryl groups and C3–C 10 (Hetero)arylalkyl groups, which are optionally substituted and optionally selected from O, S, and NR. 28 One or more heteroatoms are interrupted, where R 28 Independently selected from the group consisting of hydrogen and C1–C4 alkyl groups, preferably wherein R 21 Is it H or C(O)R 26 , where R 26 = 4-N-methylpiperazine or morpholine, most preferably wherein R 21 It is H; and / or d) Connector L 4 It is a connecting spacer, preferably L 4 It is based on –NR 22 –(C z -alkylene)–NR 22 –(SO2–NH–) j (C(O)) h – a diamine, where R 22 It is H or C1–C4 alkyl, z is an integer in the range of 1–10, j is 0 or 1, and h is 0 or 1; or spacer L 4 It is based on the structure – NR 22 –(CH2–CH2–O) e6 –(CH2) e7 –C(O)– ethylene glycol spacer, where e6 is an integer in the range of 1–10, e7 is an integer in the range of 1–3, and R 22 It is H or C1–C4 alkyl; or According to structure –NR 22 –(C z -alkylene)–C(O)– aminoalkanoic acid spacers, where x is an integer in the range of 1–20, and R 22 It is H or C1–C4 alkyl; or...

6. The antibody-cytokine conjugate according to any one of the preceding claims, wherein α = 1, and D is transmitted via D 1 N-terminus and Z 2 connect.

7. The antibody-cytokine conjugate according to any one of the preceding claims, wherein D 1 Contains a cleavable site, which is cleaved by a protease overexpressed in the tumor microenvironment, preferably one of the following: serine protease, cysteine ​​protease, aspartic protease, threonine protease, glutamate protease, metalloproteinase, gelatinase and asparagine peptide lyase, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, kallikrein, hK1, hK1O, hKT5, plasmin, collagenase, type IV collagenase, matrix lysozyme, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidin, bromelain, calpain, caspase, caspase-3, miR1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymotrypsin. Renin, pepsin, matrix proteases, membrane serine protease 1 (MT-SP1), podase, plasmepsin, pitcherine, extrametalopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM12, urokinase plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1b convertase, thrombin, FAP (FAP-α), transmembrane serine protease type II (TTSP), neutrophil elastase, protease 3, neutrophil serine protease 4, mast cell chymotrypsin, mast cell trypsin, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

8. The antibody-cytokine conjugate according to any one of the preceding claims, wherein L is based on any one of the following structures: Where x is in the range of 0–100, preferably in the range of 0–50, more preferably in the range of 1–30, and most preferably in the range of 1–18.

9. The antibody-cytokine conjugate according to any one of claims 1–11, wherein LZ 2 as follows: or ， in o and L 1 The tilde key connecting to Z represents the key. 1 The connection is such that ring Z is as defined in claim 4, preferably wherein α is 0 in the definition of D; and o L 1 and L 2 It is as defined in claim 5.

10. The antibody-cytokine conjugate according to any one of the preceding claims, for use in medical treatment.

11. The antibody-cytokine conjugate according to any one of claims 1–9, for use in the treatment of cancer or autoimmune diseases.

12. A pharmaceutical composition comprising a conjugate according to any one of claims 1–9 and a pharmaceutically acceptable carrier.

13. A method for preparing an antibody-cytokine conjugate according to any one of claims 1–9, wherein the method comprises the following steps: - Make AB(F) 1 ) x With Q 1 -LQ 2 or (Q) 1 )2–L–Q 2 reaction; - and make the resulting compound with F 2 -D reaction; Alternatively, the method may include the following steps: - Make DF 2 With Q 1 -LQ 2 or (Q) 1 )2–L–Q 2 reaction, - and make the resulting compound react with AB(F) 1 ) x reaction Where AB(F) 1 ) x It contains x reaction fractions F 1 The functionalized antibody and x is an integer in the range of 1–10, where Q 1 and F 1 They are responsive to each other, and Q 2 and F 2 They are reactive to each other, where Q 1 With F 1 Between and Q 2 With F 2 The reaction between them forms covalent bonds between the reactants.

14. The method of claim 13, wherein F 1 It is an azide, Q 1 Includes cyclic alkynes, Q 2 Contains trans-cyclooctene and F 2 It contains nitroketone or tetrazine.

15. The method according to claim 13 or 14, wherein the method is preceded by: - Convert the terminal serine or threonine of D to nitrone to obtain F. 2 -D steps, where F 2 It is nitroketone, or - Convert D to F 2 -D ligase-mediated ligation step, preferably wherein the ligase is selected from tubulin tyrosine ligase, transglutaminase, lipoate ligase, farnesyltransferase, glycosyltransferase, formyl-glycine synthase (FGE), trypsiligase / subtiligase, more preferably, the ligase is a sorting enzyme. Preferably, D is produced through microbial recombinant expression and then converted into DF. 2 More preferably, the microorganism is a prokaryote, and most preferably Escherichia coli.

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