Severable immune cell engager

Abstract

The present invention relates to an antibody-cytokine conjugate in which the cytokine is conjugated to an antibody through a cleavable linker. The conjugate according to the present invention is a functional antibody Ab(F) containing x reactive moieties F. x An antibody-cytokine conjugate (where x is an integer in the range of 1 to 10) can be prepared by conjugating it with an immune cell-engaging polypeptide containing one or two reactive moieties Q, wherein the antibody is specific to tumor cells and the immune cell-engaging polypeptide is specific to immune cells, and the reaction forms a covalent bond between the functional antibody and the immune cell-engaging polypeptide by the reaction of Q and F. The present invention further relates to antibody-cytokine conjugates that can be obtained by the process according to the present invention and their medical applications.

Description

Detailed description of the invention

[0001] [Field of Invention]

[0001] The present invention relates to immune cell engagers generated from antibodies and other polypeptides. More specifically, the present invention relates to conjugates, compositions and methods suitable for conjugating a target immune cell-binding polypeptide to an antibody via a cleavable linker without requiring genetic manipulation of the antibody before conjugation. The antibody-immune cell engager conjugates and methods obtained as compounds and compositions may be useful, for example, in immunotherapy for cancer patients.

[0002] [Background of the Invention]

[0002] Antibody-drug conjugates (ADCs), considered ideal drugs in therapy, consist of an antibody to which a drug is conjugated. Antibodies (also known as ligands) can be in small protein formats (scFv, Fab fragments, DARPin, aphibodies, etc.), but are generally monoclonal antibodies (mAbs) selected based on their high selectivity and affinity for a given antigen, their long circulating half-life, and their near- or near-absent immunogenicity. Thus, mAbs as protein ligands for carefully selected biological receptors provide an ideal targeting platform for the selective delivery of drugs. For example, monoclonal antibodies known to selectively bind to specific cancer-associated antigens can be used to deliver chemically conjugated cytotoxic drugs to tumors via binding, internalization, intracellular processing, and ultimately the release of active catabolites. Cytotoxic drugs can be in other formats such as small molecule toxins, protein toxins, or oligonucleotides. As a result, tumor cells can be selectively eradicated while avoiding normal cells that are not targeted by the antibody. Similarly, while the chemical conjugation of antimicrobial agents (antibiotics) to antibodies can be applied to the treatment of bacterial infections, the conjugation of anti-inflammatory drugs is under investigation for the treatment of autoimmune diseases. For example, the binding of oligonucleotides to antibodies is a potentially promising technique for the treatment of neuromuscular diseases. Therefore, the concept of targeted delivery of active pharmaceuticals to the best specific cellular locations is a powerful technique for the treatment of a wide range of diseases, having many beneficial aspects compared to systemic delivery of the same drug.

[0003]

[0003] An alternative strategy using monoclonal antibodies for targeted delivery of specific protein drugs involves fusing the latter protein to one or more ends of an antibody, which may be the N-terminus or C-terminus of the light chain or heavy chain (or both). In this case, the biologically active protein of interest, such as Pseudomonas exotoxin A (PE38) or anti-CD3 single-strand variable fragment (scFv), is genetically encoded as a fusion to the antibody, although it may not necessarily require a peptide spacer, and the antibody is expressed as a fusion protein. The peptide spacer may or may not contain a protease-sensitive cleavage site.

[0004]

[0004] Monoclonal antibodies can be genetically modified at the protein sequence level to alter their structure, thereby introducing (or removing) specific properties. For example, mutations may be introduced in the antibody Fc fragment to eliminate binding to the Fc gamma receptor, or binding to the FcRn receptor or a specific cancer target may be regulated, or the antibody may be manipulated to reduce pI and control the clearance rate from circulation.

[0005]

[0005] Novel strategies in therapeutic treatment involve the use of antibodies capable of simultaneously binding to multiple antigens or epitopes, i.e., bispecific antibodies (directed simultaneously to two different antigens or epitopes) or triplicate antibodies (directed to antigens of three different epitopes), as outlined by reference in Kontermann and Brinkmann, Drug Discov. Today 2015, 20, 838-847. Bispecific antibodies with "dual-target" function may interfere with multiple surface receptors or ligands associated with cancer, proliferation, or inflammatory processes, for example. Bispecific antibodies may also position targets in very close proximity to either support protein complex formation on cells or induce intercellular contact. Examples of "forced binding" function are bispecific antibodies that support protein complex formation in coagulation cascades or tumor-targeted immune cell recruiters and / or activators. Depending on the method of production and structure, bispecific antibodies differ in the number of antigen-binding sites, geometric shape, serum half-life, and effector function.

[0006]

[0006] A wide range of different formats for bispecific antibodies have been developed over the years and can be broadly classified into IgG-like (with Fc fragments) formats and non-IgG-like (without Fc fragments) formats, as summarized by Kontermann and Brinkmann, Drug Discov. Today 2015, 20, 838-847 and Yu and Wang, J. Cancer Res. Clin. Oncol. 2019, 145, 941-956, which are incorporated by reference. Most bispecific antibodies are produced by one of three methods: somatic cell fusion (quadroma) of two hybridoma strains, gene (protein / cell) manipulation, or chemical conjugation with a crosslinker, resulting in a total of more than 60 different technical platforms.

[0007]

[0007] IgG-like formats based on the complete IgG molecular structure include, but are not limited to, the dual variable domain (DVD-Ig), Duobody technology, Knob-in-Hole (KIH) technology, general light chain technology, and cross mAb technology. Cleavage IgG versions include, but are not limited to, 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 produced by fusing different antigen-binding sites (e.g., scFv or Fab) to other protein domains, thereby allowing for the inclusion of further functionality. For example, as demonstrated by Mueller et al., J. Biol. Chem. 2007, 282, 12650-12660, incorporated by reference, two scFv fragments are fused to albumin, giving the antibody fragment a long circulation time of serum albumin. Another example is the "dock-and-lock" technique based on the heterodimerization of cAMP-dependent protein kinase A and protein A kinase-immobilized proteins, as reported by Rossi et al., Proc.Nat.Acad.Sci.2006,103,6841-6846, incorporated by reference. As shown by Rossi et al., Bioconj.Chem.2012,23,309-323, these domains can bind to Fab fragments and the overall antibody to form a multivalent bispecific antibody. The dock-and-lock strategy requires the generation of a fusion protein between the targeted antibody and the peptide fragment for docking to the protein A kinase-immobilized protein. The therapeutic Ab fragment (scFv, diabody) can be fused with albumin or an albumin-binding protein, increasing the half-life of the drug in the blood by up to 5-6 times. The construction of such molecules leads to unpredictable results, thereby limiting the application of bispecific antibodies, which are produced as a result of fusions of different Ab fragments or the binding of Ab to other proteins, to the study and development of new therapeutic molecules.

[0008]

[0008] Chemical conjugations for generating non-IgG bispecific antibodies were first used by Brennan et al., Science 1985, 229, 81-83, incorporated by reference, by reducing and then oxidizing two Fab2 fragments obtained by pepsinolysis of rabbit IgG to produce bispecific Fab2. Similarly, homobifunctional reagents that interact with cysteine ​​residues have been reported by Glennie et al. 1987, 139, 2367-2375, incorporated by reference. Chemical conjugations of Ab to CD3 and CD20 (rituximab) have been used to obtain T cells having a bispecific antibody-coated surface, as shown by Gall et al., Exp. Hematol. 2005, 33, 452-459, incorporated by reference. The generation of bispecific CD20×CD3 is ensured by treating OKT3 (anti-CD3) with Traut reagent and subsequently mixing it with maleimide-functionalized rituximab (obtained by pretreatment of rituximab with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC)). Due to random chemical conjugation of both antibodies, followed by random heterodimerization, the bispecific antibody is inevitably obtained as a highly heterogeneous mixture (including polymers). The only chemical method reported to date that is also site-specific is the CovX-Body technique, as reported by Doppalapudi et al., Bioorg. Med. Chem. Lett. 2007, 17, 501-506, incorporated by reference, which is based on placing an aldolase-catalyzed antibody site in a targeted antibody and subsequently treating it with a peptide fragment chemically modified with an azetidinone motif, thereby inducing spontaneous ligation. Bispecific antibodies were produced by adding two short peptides inhibiting VEGF or angiopoietin 2 using a branched linker, as reported by Doppalapudi et al., Proc. Nat. Acad. Sci. 2010, 107, 22611-22616, which is incorporated by reference.

[0009]

[0009] Current formats for bispecific antibody production based on chemical Ab or Ab fragment conjugation have not been adopted due to low yields of (low-purity) products and high commercial costs. Furthermore, advances in recombinant DNA technology have enabled the efficient production of fusion proteins, which have yielded positive clinical results. In any case, non-genetic chemical modification methods can significantly accelerate time to clinical trials if appropriate control of stoichiometric site specificity can be ensured.

[0010]

[0010] Examples of bispecific antibodies currently in or in clinical development include catumakisomab (EpCAM×CD3), blinatumomab (CD19×CD3), GBR1302 (Her2×CD3), MEDI-565 (CEA×CD3), BAY2010112 (PSMA×CD3), RG7221 (angiopoietin×VEGF), RG6013 (FIX×FX), RG7597 (Her1×Her3), MC These are LA128 (Her2 × Her3), MM111 (Her2 × Her3), MM141 (IGF1R × Her3), ABT122 (TNFalpha × IL17), ABT981 (IL1a × IL1b), ALX0761 (IL17A × IL17F), SAR156597 (IL4 × IL13), AFM13 (CD30 × CD16), and LY3164530 (Her1 × cMET).

[0011]

[0011] A common strategy in the field of cancer treatment is to utilize bispecific antibodies that bind to upregulated tumor-associated antigens (TAAs or simply targeted) and receptors present on cancer-destroying immune cells such as T cells or NK cells. Such bispecific antibodies are known as T cell or NK cell redirecting antibodies, respectively. Although immune cell redirection techniques have been around for over 30 years, new technologies are overcoming the limitations of first-generation immune cell redirecting antibodies, particularly extending half-life to allow intermittent administration, reducing immunogenicity, and improving the safety profile. Currently, there is one approved drug (blinatumomab or Blincyto®) and more than 30 other bispecific formats at various stages of clinical development. The basis for blinatumomab approval (2014) was derived from a single-arm trial with a 32% complete remission rate and a minimal residual disease (MRD) response (31%) in all treated patients. Currently, 51 clinical trials of blinatumomab are underway for ALL (39 trials), NHL (10 trials), multiple myeloma (1 trial), and lymphoid cancer with Richter transformation (1 trial). However, blinatumomab has a major drawback due to its short serum half-life (2.11 hours, due to its relatively small size and simple structure), requiring patients to undergo continuous intravenous infusion.

[0012]

[0012] As with other methods of treating serious diseases, therapeutic bispecific antibodies can 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 therapeutic bispecific antibodies appears in the blood during treatment. Most adverse events occur at the start of treatment, and in most cases, side effects normalize during continued treatment. Most of the data on therapeutic BsAb adverse effects is available for blinatumomab and catumakisomab, as these drugs have undergone numerous clinical trials. Common side effects of blinatumomab and catumakisomab therapy include a "cytokine storm," i.e., elevated cytokine levels and some neurological events. Cytokine release-related symptoms are common side effects of many therapeutic mAbs and are caused by specific mechanisms of action, such as the use of cytotoxic T cells as effectors. Minimizing cytokine release syndrome is possible by using lower initial doses of medication in combination with subsequent higher doses and corticosteroids (dexamethasone) and antihistamine premedication.

[0013]

[0013] One method for mitigating adverse events associated with immune cell engagement therapy, particularly cytokine release syndrome, and avoiding the use of step-up dosing regimens, is reported by Bacac et al., Clin. Cancer Res. 2018, 24, 4785-4797, incorporated by reference. Significantly higher potency and safer administration can be achieved by generating a CD20×CD3 T cell engager having a 2:1 molecular format, i.e., bivalent binding to CD20 and monovalent binding to CD3, which has been shown to be achieved by insertion of an anti-CD3 fragment in 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, made possible by the highly binding-active bivalent binding to CD20 and the head-to-tail orientation of the B cell and T cell binding domains in a 2:1 molecular format. The heterodimerized human IgG1 Fc region with the "PG LALA" mutation was incorporated to maintain neonatal Fc receptor (FcRn) binding, enabling a long circulating half-life, while eliminating binding to the Fcg receptor and complementing component C1q. The bispecific CD20 T cell engager exhibits significantly higher efficacy than other CD20-TCB antibodies in clinical development and is effective against tumor cells expressing low levels of CD20. CD20-TCB shows potent activity in primary tumor samples with a low effector-to-target ratio.

[0014]

[0014] For the purpose of T cell engagement, the most studied receptor to date is the CD3 receptor on activated T cells. T cell redirecting bispecific antibodies are the most widely used approach in cancer treatment, and the first report of bispecific antibodies specifically engaging CD3 on T cells and, on the other hand, independently of their T cell receptor (TCR) to antigens on cancer cells was published 30 years ago. T cell redirecting antibodies have made considerable progress in the treatment of malignant hematological disorders and solid tumors over the past decade. Catumaxomab was the first bispecific antibody of its kind targeting epithelial cell adhesion molecule (EpCAM) and CD3, and was approved in Europe in 2009 for the treatment of malignant ascites (however, it was withdrawn in 2017 for commercial reasons). Following this discovery, another bispecific targeting of CD19 and CD3 (blinatumomab) was successfully achieved, and in 2014, it received FDA approval for the treatment of relapsed or refractory precursor B-cell acute lymphoblastic leukemia (ALL). While many patients currently benefit from blinatumomab, several T-cell redirecting antibodies exist with different formats and characteristics that have shown potential antitumor efficacy in clinical trials.

[0015]

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

[0016]

[0016] In the field of CD137 targeting, agonist monoclonal antibodies (not bispecific) have shown considerable preclinical promise, but their clinical development has been delayed due to insufficient therapeutic index, particularly hepatotoxicity. CD137 is expressed on T cells that are already primed to recognize tumor antigens by MHC / TCR interaction. It is a member of the TNFRSF (tumor necrosis factor receptor superfamily) that requires clustering to deliver an activation signal to T cells. Monospecific monoclonal antibodies that can stimulate CD137 are clinically available and are known to be potent T cell activators, but they suffer from treatment-limiting hepatotoxicity due to Fc receptor and multivalent format-driven clustering. Bispecific tumor-targeting antibodies that are monovalent for CD137 cannot induce CD137 clustering in normal tissue. Co-engagement CD137 clustering on tumor-associated T cells is induced only upon binding of bispecific antibodies to tumor-associated antigens on tumor cells. This is very potent, but drives tumor-specific T cell activation. Tumor-targeted crosslinking of Cd137 / 4-1BB may provide a safe and effective method for co-stimulating T cells for cancer immunotherapy, and its combination with T cell bispecific antibodies may provide a convenient "off-the-shelf" systemic cancer immunotherapy approach 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 (glypican-3×CD137), PRS-343 (Her2×CD137), and CB307 (PSMA×CD137). The various bispecific antibodies listed above are intentionally selected as monovalent for CD137 and therefore cannot induce CD137 cluster formation in normal tissue. For example, only after the bispecific binding of CB307 to PSMA on tumor cells does this trigger co-engagement of CD137 clustering on tumor-associated T cells, thereby driving very potent but tumor-specific T cell activation.

[0017]

[0017] Antibodies known to bind to T cells are known in the art and are highlighted by reference in Martin et al., Clin.Immunol.2013, 148, 136-147 and Rossi et al., Int.Immunol.2008, 20, 1247-1258, for example, OKT3, UCHT3, BMA031 and their humanized versions. Antibodies known to bind to Vγ9Vδ2 T cells are also known; see, for example, de Bruin et al., J.Immunol.2017, 198, 308-317, for example.

[0018]

[0018] Similar to T cell engagement, NK cell recruitment into the tumor microenvironment has been widely investigated. NK cell engagement is typically based on binding CD16, CD56, NKp46, or other NK cell-specific antibodies, as outlined in Konjevic et al., 2017, http: / / dx.doi.org / 10.5772 / intechopen.69729, incorporated by reference. NK cell engagers can be generated by the fusion or insertion of an NK-binding antibody (fragment) to complete IgG binding to tumor-associated antigens. Alternatively, given that NK cell antitumor activity is regulated by numerous activating and inhibitory NK cell receptors, specific cytokines may also be utilized, and changes in NK cell receptor expression and signaling underlie cytotoxic NK cell deceleration. Based on this and predictive in vitro findings, cytokines including IFNα, IL-2, IL-12, IL-15, and IL-18 are used systemically or ex vivo to activate and expand NK cells, thereby improving NK cell antitumor activity by increasing the expression of NK cell activating receptors and inducing cytotoxic effector molecules. Furthermore, this cytokine-based therapy has been shown to enhance NK cell proliferation and regulatory functions, inducing NK cells exhibiting cytokine-induced memory-like properties that present a newly defined subset of NK cells with improved NK cell activity and lifespan. Several cytokine payloads have been developed and tested in preclinical trials for therapies in cancer treatment and chronic inflammation. Pro-inflammatory cytokines such as IL-2, TNF, and IL-12 have been found to increase and activate local leukocyte infiltration at tumor sites, and are therefore being investigated for tumor treatment. For example, IL-2 monotherapy, as aldethleukin (Proleukin®), is approved and is in Phase III clinical trials in combination with nivolumab (NKTR-214). Similarly, various recombinant versions of IL-15 are currently undergoing clinical evaluation (rhIL-15 or ALT-803).IL-15-specific mutants, such as the IL-15 receptor (IL-15R) and IL-15 complex, as well as the IL-15 and IL-15R fusion construct (Sushi domain), have also been evaluated for their antitumor activity. See, for example, Bessard et al., Mol. Canc. Ther. 2009, 8, 2736-2745. In contrast, immunosuppressive cytokines such as IL-10 may be considered payloads for the treatment of chronic inflammatory conditions or other diseases (e.g., endometriosis).

[0019]

[0019] Systemic administration of pro-inflammatory cytokines can result in severe off-target adverse effects that can limit doses and prevent increases to therapeutically effective regimens. Certain cytokine products (e.g., IL-2, TNF, IL-12) have recommended doses in the single-digit milligram range (per person) or even less. Adverse effects associated with intravenous administration of pro-inflammatory cytokines may include hypotension, fever, nausea, or flu-like symptoms, but sometimes serious hematological, endocrine, autoimmune, or neurological events may also occur. Given these issues, there is a clear biomedical need for the development of “next-generation” cytokine products that are better tolerable, exhibit preferential action at the lesion site, and help preserve normal tissue, as outlined in Murer and Neri, New Biotechnol. 2019, 52, 42-53, incorporated by reference. Therefore, targeted delivery of cytokines to tumors aims to induce a local pro-inflammatory environment in which immune cells can be activated and mobilized. A list of antibody-cytokine fusions described in the literature is reported by Hutmacher and Neri, Adv. Drug Deliv. Rev. 2018, 141, 67-91 (incorporated by reference). A list of clinical cytokine fusions is provided by Murer and Neri, New Biotechnol. 2019, 52, 42-53 (incorporated by reference). Various IL-15 fusion proteins are undergoing preclinical evaluation, such as OXS-3550 (CD33-IL-15-CD16 fusion) prepared by Trike technology, which is close to Phase I evaluation, as outlined in "T-cell & NK-Cell Engaging Bispecific Antibodies 2019: A Business, Stakeholder, Technology and Pipeline Analysis," 2019, published by La Merie publishing (incorporated by reference).

[0020]

[0020] A common strategy in the field of immune cell engagement is to utilize the elimination or removal of the ability of antibodies to bind to Fc gamma receptors, which has multiple pharmaceutically acceptable implications. The first consequence of the removal of binding to Fc gamma receptors is a decrease in Fc gamma receptor-mediated uptake of antibodies by macrophages or megakaryocytes, for example, which can lead to dose-dependent toxicity, as reported for Kadcyla® (trastuzumab-DM1) and LOP628. Selective deglycosylation of antibodies in vivo offers an opportunity to treat patients with antibody-mediated autoimmunity. Removal of high-mannose glycoforms from recombinant therapeutic glycoproteins is beneficial because, as explained, for example, by Gorovits and Krinos-Fiorotti, Cancer Immunol.Immunother.2013,62,217-223 and Goetze et al, Glycobiology 2011,21,949-959 (both incorporated by reference), high-mannose glycoforms are known to undergo nonspecific uptake by endogenous mannose receptors, resulting in rapid clearance and impaired therapeutic efficacy. In addition, Van de Bovenkamp et al, J.Immunol.2016,196,1435-1441 (incorporated by reference) describes the extent to which high-mannose glycans affect immunity. The possibility that improper glycosylation in monoclonal antibodies may contribute to ineffective production from expressed Ig genes has been explained by Reusch and Tejada, Glycobiology 2015, 25, 1325-1334 (incorporated by reference).

[0021]

[0021] In the field of immunotherapy, the binding of glycosylated antibodies to Fc gamma receptors on immune cells can induce systemic activation of the immune system before the binding of antibodies to tumor-associated antigens, potentially leading to a cytokine storm (cytokine release syndrome, CRS). Therefore, to mitigate the risk of CRS, the majority of immune cell engagers in hospitals are based on Fc silencing antibodies and lack the ability to bind to Fc gamma receptors. In addition, various companies in the field of bispecific antibodies are modifying molecular structures with defined ratios with respect to target binding to the immune cell engagement antibody domain. For example, Roche has developed a T cell engager based on an asymmetric monoclonal antibody that retains divalent binding ability to TAA (e.g., CD20 or CEA) by both CDRs, but for the additional anti-CD3 fragment, it is engineered to only one of the two heavy chains (a 2:1 target binding:CD3 binding ratio). Similar strategies can be used for T cell engagement / activation by anti-CD137(4-1BB), anti-OX40, or anti-CD27, or for NK cell engagement / activation by anti-CD16, CD56, NKp46, or other NK cell-specific receptors.

[0022]

[0022] Inhibition of binding to the Fc gamma receptor can be achieved, for example, by specific mutations in the antibody (specifically, Fc fragments) or by naturally occurring glycans (C) present in the Fc fragments. HThis can be achieved in various ways, such as by removing two domains (near N297). Glycan removal can be achieved, for example, by genetic modification in the Fc domain, such as the N297Q mutation or the T299A mutation, or by enzymatic removal of glycans after recombinant antibody expression, for example, using PNGase F or endoglycosidase. For example, endoglycosidase H is known to prepare high-mannose and hybrid glycoforms, while endoglycosidase S can prepare complex glycans and, to some extent, hybrid glycans. Endoglycosidase S2 can prepare complex, hybrid, and high-mannose glycoforms. Endoglycosidase F2 can prepare complex glycans (but not hybrid), while endoglycosidase F3 can prepare only 1,6-fucosylated complex glycans. Another endoglycosidase, endoglycosidase D, can hydrolyze only Man5 (M5) glycans. An overview of the specific activities of various endoglycosidases is disclosed in Freeze et al. in Curr. Prot. Mol. Biol., 2010, 89:17.13A.1-17, which is incorporated herein by reference. Further advantages of protein deglycosylation for therapeutic applications include improved batch-to-batch consistency and significantly improved uniformity.

[0023]

[0023] The idea can be derived from the field of ADC technology for preparing antibody-protein complexes to generate bispecific antibodies or antibody-cytokine fusions.

[0024]

[0024] Incorporated by reference: GTHermanson, "Bioconjugate Techniques", Elsevier, 3 rdAs outlined in Ed.2013, many techniques are known for bioconjugation. Two main techniques can be recognized for ADC preparation by random conjugation based on either acylation of the lysine side chain or alkylation of the cysteine ​​side chain. Acylation of the ε-amino group of the lysine side chain is typically achieved by exposing the protein to a reagent based on an activated ester or activated carbonate derivative, for example, SMCC is used in the production of Kadcyla®. The main chemical reaction for alkylation of the thiol group in the cysteine ​​side chain is based on the use of maleimide reagents, for example, used in the production of Adcetris®. In addition to standard maleimide derivatives, various maleimide variants are also applicable to more stable cysteine ​​conjugations, as demonstrated, for example, by James Christie et al., J.Contr.Rel.2015, 220, 660-670 and Lyon et al., Nat.Biotechnol.2014, 32, 1059-1062, both incorporated by reference. Another important technique for conjugation to cysteine ​​side chains is via disulfide bonds, which are bioactivatable linkages used to reversibly link protein toxins, chemotherapeutic drugs, and probes to carrier molecules (see, for example, Pillow et al., Chem.Sci.2017, 8, 366-370, incorporated by reference).Other methods for cysteine ​​alkylation include, for example, nucleophilic substitution of haloacetamides (typically bromoacetamide or iodoacetamide) (see, e.g., Alley et al., Bioconj. Chem. 2008, 19, 759-765 incorporated by reference) or various methods based on nucleophilic addition at unsaturated bonds, such as reactions with acrylate reagents (see, e.g., Bernardim et al., Nat. Commun. 2016, 7, DOI:10.1038 / ncomms13128 and Ariyasu et al., Bioconj. Chem. 2017, 28, 897-902, both incorporated by reference), and reactions with phosphoamides (see, e.g., Kasper et al. incorporated by reference). This includes reactions with allenamides (see, for example, Abbas et al., Angew. Chem. Int. Ed. 2019, 58, 11625-11630), reactions with cyanoethynyl reagents (see, for example, Kolodych et al., Bioconj. Chem. 2015, 26, 197-200, incorporated by reference), reactions with vinyl sulfones (see, for example, Gil de Montes et al., Chem. Sci. 2019, 10, 4515-4522, incorporated by reference), or reactions with vinylpyridines (see, for example, https: / / iksuda.com / science / permalink / (accessed January 7, 2020)). The reaction with methylsulfonylphenyloxadiazole has also been reported for cysteine ​​conjugation by Toda et al., Angew. Chem. Int. Ed. 2013, 52, 12592-12596, which is incorporated by reference.

[0025]

[0025] Several processes have been developed that enable the generation of antibody-drug conjugates with a defined drug-antibody ratio (DAR) by site-specific conjugation to one (or more) predetermined sites in an antibody. Site-specific conjugation is typically achieved by manipulating specific amino acids (or sequences) in the antibody, which function as anchor points for payload binding. For example, see Aggerwal and Bertozzi, Bioconj.Chem.2014,53,176-192, incorporated by reference, for the most typical manipulation of cysteine. Furthermore, various other site-specific conjugation techniques have been studied over the past decade, most notably the gene encoding of non-natural amino acids such as p-acetophenylalanine, which is suitable for oximligation, or p-azidomethylphenylalanine, which is suitable for click chemistry conjugation. Most methods based on gene reengineering of antibodies result in ADCs with a DAR of approximately 2.Alternative methods for antibody conjugation without antibody reengineering include reduction of interchain disulfide crosslinks followed by bis-sulfone reagents (see, for example, Balan et al., Bioconj.Chem.2007,18,61-76 and Bryant et al., Mol.Pharmaceutics 2015,12,1872-1879, both incorporated by reference), mono- or bis-bromomaleimides (see, for example, Smith et al., J.Am.Chem.Soc.2010,132,1960-1965 and Schumacher et al., both incorporated by reference). See al., Org. Biomol. Chem. 2014, 37, 7261-7269), bis-maleimide reagent (see, for example, International Publication No. 2014114207), bis(phenylthio)maleimide (see, for example, Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269 and Aubrey et al., Bioconj. Chem. 2018, 29, 3516-3521, both incorporated by reference), bis-bromopyridazinedione (see, for example, Robinson et al., RSC Advances 2017, 7, 9073-9077, incorporated by reference), bis(halomethyl)benzene (see, for example, Ramos-Tomillero et al., incorporated by reference) This involves the addition of a payload conjugated to a cysteine ​​crosslinking reagent, such as al., Bioconj. Chem. 2018, 29, 1199-1208, or a bis(halomethyl) aromatic compound (see, for example, International Publication No. 2013173391). Typically, ADCs prepared by cysteine ​​crosslinking have a drug-antibody loading (DAR4) of approximately 4.

[0026]

[0026] Ruddle et al., ChemMedChem 2019, 14, 1185-1195, states that in recent years, C H 1 and C LWe demonstrated that DAR1 conjugates can be prepared from antibody Fab fragments (prepared by papain digestion or recombinant expression of complete antibodies) by selective reduction of interchain disulfide chains followed by recrosslinking of the fragments with a symmetric PDB dimer containing two maleimide units. The resulting DAR1-type Fab fragments were shown to be highly homogeneous, stable in serum, and exhibit excellent cytotoxicity. Subsequent publications, White et al., MAbs2019, 11, 500-515 (incorporated by reference) and further in International Publication No. 2019034764, demonstrate that DAR1 type conjugates can also be prepared from complete IgG antibodies before or after either an antibody having only one intrachain disulfide crosslink in the antibody:hinge region (Flexmab technology, reported by Dimasi et al., J.Mol.Biol.2009, 393, 672-692 (incorporated by reference)) or an antibody having additional free cysteine ​​obtained by mutation or insertion of a natural amino acid (e.g., HC-S239C) (e.g., HC-i239C, reported by Dimasi et al., Mol.Pharmaceut.2017, 14, 1501-1516). All manipulated antibodies were shown to enable the generation of DAR1 ADCs through the reaction of the resulting cysteine-modified ADCs with bismaleimide-derived PBD dimers. Fleximab-derived DAR1 ADCs were shown to be highly resistant to serum payload loss and exhibited potent antitumor activity in a HER2-positive gastric cancer xenograft model. Furthermore, these ADCs were tolerated in rats at twice the dose compared to site-specific DAR2 ADCs prepared using a single maleimide-containing PBD dimer. However, no improvement in the therapeutic range was observed, as the minimum effective dose (MED) of DAR1 ADCs increased by the same coefficient of 2 compared to DAR2 ADCs.

[0027]

[0027] Both are incorporated by reference in International Publication No. 2014065661 and van Geel et al., Bioconj.Chem. 2015, 26, 2233-2242, in which antibodies can be site-specifically conjugated based on enzymatic remodeling of the native antibody glycan at N297 (modification by endoglycosidase and introduction of an azide-modified GalNAc derivative under the action of glycosyltransferase), followed by binding of a cytotoxic payload using click chemistry. Verkade et al., Antibodies 2018, 7, 12 demonstrated that the introduction of acylated sulfamides further improves the glycan remodeling technique with respect to therapeutic index, and that the DAR of the resulting antibody-drug conjugate can be adjusted to DAR2 or DAR4 by the selection of a specific linker. It has also been demonstrated that glycan modification before conjugation results in the elimination of binding of the resulting antibody-drug conjugate (ADC) to the Fc gamma receptor (Fc silencing). ADCs prepared using this technique were found to exhibit a significantly expanded therapeutic index compared to various other conjugation techniques and glycan remodeling conjugation techniques currently clinically applied, such as ADCT-601 (ADC Therapeutics).

[0028]

[0028] A similar enzymatic method for converting antibodies to azide-modified antibodies accompanied by Fc silencing, reported by Lhospice et al., Mol. Pharmaceut. 2015, 12, 1863-1871, incorporated by reference, utilizes the bacterial enzyme transglutaminase (BTG or TGase). Deglycosylation of the natural glycosylation site N297 using PNGase F has been shown to release the adjacent N295, which serves as a substrate for TGase-mediated introduction, and when applied to an azide-supported molecule in the presence of TGase, converts the deglycosylated antibody to a bisazide antibody. Subsequently, the bisazide antibody was reacted with DBCO-modified cytotoxin to produce an ADC with DAR2. A genetic method based on C-terminal TGase-mediated azide introduction followed by conversion in ADCs via metal-free click chemistry has been reported by Cheng et al., Mol. Cancer Therap. 2018, 17, 2665-2675, incorporated by reference.

[0029]

[0029] In addition to the binding of small molecules, it has also been well demonstrated that various click chemistrys are suitable for the generation of protein-protein conjugates. For example, Witte et al., Proc.Nat.Acad.Sci.2012,109,11993-11998, incorporated by reference, shows that non-natural interterminal and interterminal protein dimers can be obtained by combining the introduction of two complementary click probes (azide and DBCO) into two different proteins via saltase, followed by seamless ligation based on metal-free click chemistry (strain-enhanced azide-alkyne cycloaddition or SPAAC). Wagner et al., Proc.Nat.Acad.Sci.2014,111,16820-16825, incorporated by reference, applied this method to prepare bispecific antibodies based on C-terminal sorting with anti-influenza scFv. This was further extended to metal-free click chemistry based on reverse electron-demanded Diels-Alder cycloaddition with tetrazine, as incorporated by reference in Bartels et al., Methods 2019, 154, 93-101. Tetrazine ligation had also been previously applied, for example, by Angew. Chem. Int. Ed. 2009, 48, 7013-7016 and Robillard et al., Angew. Chem. Ed. Engl. 2010, 49, 3375-3378, both incorporated by reference, and these were for chemical modification by the initial (random) chemical placement of trans-cyclooctene (TCO) to the antibody.In contrast, site-specific introduction of TCO (or other click portions of tetrazine or cyclopropene for tetrazine ligation) into antibodies can be achieved by the aforementioned antibodies as well as by numerous methods based on pre-modification of antibodies, such as those reported by Lang et al., J.Am.Chem.Soc.2012, 134, 10317-10320, Seitchik et al., J.Am.Chem.Soc.2012, 134, 2898-2901 and Oller-Salvia, Angew.Chem.Int.Ed.2018, 57, 2831-2834, all of which are incorporated by reference.

[0030]

[0030] Saltase is a suitable enzyme for site-directed modification of proteins after pre-introduction of a saltase recognition sequence, as first reported in Popp et al., Nat. Chem. Biol. 2007, 3, 707-708. Many other enzyme-enzyme recognition sequence combinations are also known for site-directed protein modification, as outlined by Milczek, Chem. Rev. 2018, 118, 119-141 (both incorporated by reference), and are particularly applicable to antibodies, as outlined by Falck and Mueller, Antibodies 2018, 7, 4 (doi: 10.3390 / antib7010004) and van Berkel and van Delft, Drug Discov. Today:Technol. 2018, 30, 3-10 (both incorporated by reference). Furthermore, a wide range of methods are applicable to non-genetic modification of native proteins, as outlined by Koniev and Wagner, Chem. Soc. Rev. 2015, 44, 5495-5551 (both incorporated by reference), and to N-terminal modification by Rosen and Francis, Nat. Chem. Biol. 2017, 13, 697-705 and Chen et al., Chem. Sci. 2017, 8, 27172722 (both incorporated by reference). Any of the above methods can be used to implant appropriate click probes into polypeptides / proteins and applied to immune cell engagers or cytokines, for example, as outlined by Chen et al., Acc. Chem. Res. 2011, 44, 762-773 and Jung and Kwon, Polymer Chem. 2016, 7, 4585-4598 (both incorporated by reference). When complementary click probes are placed on antibodies targeting tumor-associated antigens, immune cell engagers can be easily generated, but the stoichiometry of tumor-binding antibodies to immune cell conjugates can be adjusted by the appropriate selection of technology.

[0031]

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

[0032]

[0032] Chemical conjugation with affinity peptides for site-directed modification (CCAP) was developed by Kishimoto et al., Bioconj. Chem. 2019, using peptides that bind highly to human IgG-Fc, thereby enabling selective modification of a single lysine in the Fc fragment using a biotin moiety or cytotoxic payload. Similarly, Yamada et al., Angew. Chem. Int. Ed. 2019, 58, 5592-5597 and Matsuda et al., ACS Omega 2019, 4, 20564-20570, both incorporated by reference, demonstrate that a similar method (AJICAP® method) can be applied for site-directed introduction of a thiol group in a single lysine in an antibody heavy chain. CCAP or AJICAP® technology can also be used for site-directed introduction of an azide group or other functionalities.

[0033]

[0033] Chemical conjugation of immune cell engagers to antibodies is applied, but typically results in heterogeneous mixtures. The preparation of homogeneous bispecific antibodies or antibody-cytokine fusions that do not require prior reengineering of full-length IgG and / or allow for adjustment of the number of immune cell engaging polypeptides, as well as the spacer length and the structure between IgG and polypeptides, is reported in International Publication No. 2021144315.

[0034]

[0034] Fusion proteins containing cleavable linkers are described, for example, in International Publication No. 2021198490, International Publication No. 2021188835, and International Publication No. 2019 / 173832. The cleavable linker can link a masking agent to the remainder of the fusion protein, as described in International Publication No. 2021188835 or International Publication No. 2019 / 173832. To date, no method has been reported for preparing homogeneous antibody-cytokine conjugates linked via chemical linkers, in which the cytokines are released upon cleavage by proteases.

[0035] [Overview of the prefecture]

[0035] The present invention relates to an antibody-cytokine conjugate in which the cytokine is linked to the antibody via a chemical linker, thereby enabling targeted delivery of the cytokine by the release of the cytokine at the target. The antibody-cytokine conjugate can be produced without requiring genetic modification of IgG. The present invention makes it possible to adjust the molecular format of the antibody-cytokine conjugate in a 2:1 or 2:2 ratio, i.e., the ratio of the complement-dependent region in the complete IgG CDR (2) to the immune cell-engaging polypeptide (1 or 2).

[0036]

[0036] Surprisingly, the inventors have found that the conjugate according to the present invention has higher efficacy and therapeutic area than the gene fusions of antibodies and immune cell engagers.

[0037]

[0037] Although we do not wish to be bound by theory, the inventors believe that the immune cell-engaging polypeptide of the fusion protein is glycosylated, resulting in heterogeneous products with variable efficacy, and that the average efficacy of the heterogeneous immune cell-engaging polypeptide is lower than the average efficacy of the homogeneous non-glycosylated product.

[0038]

[0038] Accordingly, a first aspect of the present invention relates to a cleavable antibody-cytokine conjugate. A second aspect of the present invention relates to a cleavable antibody-cytokine conjugate for medical procedures. A third aspect of the present invention relates to a process for preparing an antibody-cytokine conjugate.

[0039] [Detailed explanation] [Definition]

[0039] When the verb "includes" and its conjugations are used herein and in the claims, they are used in their non-restrictive sense to mean that the items following the word are included, but not excluded from items not specifically mentioned. In addition, reference to an element with the indefinite article "a" or "an" does not exclude the possibility of two or more elements being present unless the context explicitly requires that only one element be present. Thus, the indefinite article "a" or "an" usually means "at least one".

[0040]

[0040] The compounds disclosed herein and in the claims may contain one or more chiral centers, and different diastereomers and / or enantiomers may exist for the compounds. Unless otherwise specified, the description of any compound herein and in the claims is intended to include all diastereomers and mixtures thereof. In addition, unless otherwise specified, the description of any compound herein and in the claims is intended to include both individual enantiomers and any mixtures of enantiomers, racemates, etc. Where the structure of a compound is shown as a particular enantiomer, it should be understood that the invention of the present application is not limited to that particular enantiomer.

[0041]

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

[0042]

[0042] The compounds disclosed herein and in the claims may further exist as exo and endodiastereoisomers. Unless otherwise specified, any description of any compound herein and in the claims is intended to include both the individual exo and individual endodiastereoisomers of the compound, as well as mixtures thereof. Where the structure of a compound is shown as a particular endo or exodyatereomer, it should be understood that the invention of the present application is not limited to that particular endo or exodyatereomer.

[0043]

[0043] Furthermore, the compounds disclosed herein and in the claims may exist as cis and trans isomers. Unless otherwise specified, any description of a compound herein and in the claims is intended to include both the individual cis and individual trans isomers of the compound, as well as mixtures thereof. For example, if the structure of a compound is shown as a cis isomer, it should be understood that the corresponding trans isomer and mixtures of the cis and trans isomers are not excluded from the invention of the present application. If the structure of a compound is shown as a specific cis or trans isomer, it should be understood that the invention of the present application is not limited to that specific cis or trans isomer.

[0044]

[0044] The compounds according to the present invention may exist in salt form, which is also included in the present invention. A salt is typically a pharmaceutically acceptable salt containing a pharmaceutically acceptable anion. The term “its salt” means a compound formed when an acidic proton, typically a proton of an acid, is substituted with a cation such as a metal cation or an organic cation. Where applicable, a salt is a pharmaceutically acceptable salt, which is not required for salts not intended for administration to a patient. For example, in a salt of a compound, the compound may be protonated with an inorganic or organic acid to form a cation together with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0045]

[0045] The term “pharmaceutically acceptable” salt means a salt that is acceptable for administration to a patient such as a mammal (a salt having a counterion that is mammalian safety acceptable for a given administration regimen). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable salt of a compound, which is derived from a variety of organic and inorganic counterions known in the art, and includes, for example, salts of organic or inorganic acids such as sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, where the molecule contains a basic functional group, such as sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, as well as salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, and oxalate.

[0046]

[0046] The term "protein" is used herein in its usual scientific sense. Herein, a polypeptide containing about 10 or more amino acids is considered a protein. Proteins contain natural amino acids, but may also contain non-natural amino acids.

[0047]

[0047] The term "monosaccharide" is used herein in its usual scientific sense and refers to an oxygen-containing heterocycle resulting from intramolecular hemiacetal formation during cyclization of a chain containing 5 to 9 (hydroxylated) carbon atoms, most commonly 5 carbon atoms (pentose), 6 carbon atoms (hexose), or 9 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]

[0048] The term "cytokine" is used herein in its usual scientific sense and refers to small molecule proteins (5-20 kDa) that regulate the activity of immune cells by binding to their homologous receptors and subsequently triggering cellular signaling. Examples of cytokines include chemokines, interferons (IFNs), interleukins, monokines, lymphokines, colony-stimulating factors (CSFs), and tumor necrosis factor (TNFs). Examples of cytokines include IL-1α (IL1a), IL-1β (IL1b), IL-2 (IL2), IL-4 (IL4), IL-5 (IL5), IL-6 (IL6), IL-8 (IL8), IL-10 (IL10), IL-12 (IL12), IL-15 (IL15), IFN-α (IFNA), IFN-γ (IFN-G), and TNF-α (TNFA).

[0049]

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

[0050]

[0050] In this specification, “antibody fragment” is defined as part of an intact antibody including its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies, minibodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, fragments generated by a Fab expression library, or any of the above epitope-binding fragments that bind immunospecifically to a target antigen (e.g., cancer cell antigen, viral antigen, or microbial antigen).

[0051]

[0051] An "antigen" is defined as an entity to which an antibody specifically binds.

[0052]

[0052] The terms “substantial” or “substantially” are defined herein as a majority of a group of mixtures or samples, 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 group.

[0053]

[0053] A linker is defined herein 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 bonded to each other via a linker. A linker may include one or more linkers and spacer portions that connect various parts within the linker.

[0054]

[0054] In this specification, “polar linker” is defined as a linker containing structural elements that have a specific purpose of increasing the polarity of the linker and thereby improving its water solubility. A polar linker may include, for example, one or more units or combinations thereof selected from ethylene glycol, carboxylic acid moieties, sulfonate moieties, sulfone moieties, acylated sulfamide moieties, phosphate moieties, phosphinate moieties, amino groups, or ammonium groups.

[0055]

[0055] A “spacer” or spacer portion is defined herein as a portion that covalently bonds two or more portions of a linker together with a certain distance between them (i.e., a distance between them). The linker may be part of a linker structure, linker conjugate or bioconjugate, for example, as defined below.

[0056]

[0056] The term "click probe" refers to a functional part that can undergo a click reaction. That is, two compatible click probes undergo a click reaction with each other and covalently bond in the product. Click-compatible probes are known in the art and preferably include (cyclic) alkynes and azides. In relation to the present invention, click probe Q in the compound according to the present invention can react with click probe F on a (modified) protein, and when the click reaction occurs, a conjugate is formed in which the protein is conjugated to the compound of the present invention. In this specification, F and Q are compatible click probes.

[0057]

[0057] The term "(hetero)alkyl" refers to alkyl groups and heteroalkyl groups. A heteroalkyl group is one in which one or more carbon units (e.g., CH2, CH, or C) in the alkyl chain are O, S, S(O), S(O)2, or NR 4 These are alkyl groups substituted with heteroatoms such as . In other words, alkyl chains include O, S, S(O), S(O)2 and NR 4 One or more elements selected from are interposed. Unlike substituents, such interpositions are located within the alkyl chain, and a substituent is, for example, a pendant group monovalently bonded to a carbon atom of the alkyl chain. In preferred embodiments, the (hetero)alkyl group is an alkyl group, such as ethyl (Et), isopropyl (a-Pr), n-propyl (n-Pr), tert-butyl (t-Bu), isobutyl (i-Bu), n-butyl (n-Bu), or n-pentyl.

[0058]

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

[0059]

[0059] In this specification, the “acylsulfamide moiety” is defined as a sulfamide moiety (H2NSO2NH2) that is N-acylated or N-carbamolylated at one end of the molecule and N-alkylated (mono or bis) at the other end of the molecule. In connection with the present invention, and particularly in the examples, this group is also referred to as “HS”.

[0060]

[0060] A “domain” is generally defined based on sequence homology and can be any region of a protein that is often associated with a particular structural or functional entity. Members of the CEACAM family are known to consist of Ig-like domains. The term domain is used herein to refer to individual Ig-like domains, e.g., “N-domains” or groups of consecutive domains, e.g., “A3-B3 domains”.

[0061]

[0061] A "coding sequence" or a sequence that "codes" an expression product such as RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, produces that RNA, polypeptide, protein, or enzyme. That is, a nucleotide sequence codes for the amino acid sequence of that polypeptide, protein, or enzyme. A protein coding sequence may include a start codon (usually ATG) and a stop codon.

[0062]

[0062] The term “gene” means a DNA sequence that codes for or corresponds to a specific amino acid sequence containing all or part of one or more proteins or enzymes, and which may or may not contain regulatory DNA sequences, such as promoter sequences that determine the conditions under which a gene is expressed. Some genes that are not structural genes can be transcribed from DNA to RNA but are 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 may refer to a genomic sequence that codes for a protein, i.e., a sequence that includes regulatory, promoter, intron, and exon sequences.

[0063]

[0063] "Biomolecules" are defined herein as any molecules that can be isolated from any molecules consisting of natural molecules or naturally occurring macromolecular structures, particularly smaller macromolecular structures that are components of 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]

[0064] The term "payload" refers to a portion that is covalently bonded to a targeting site, such as an antibody. Therefore, the payload refers to a monovalent site having one open end that is covalently bonded to the targeting site via a linker. The payload may be a small molecule or a biomolecule. In the present invention, the payload is an immune cell engager polymer.

[0065]

[0065] The terms "complement-dependent region" or "CDR" refer to a variable fragment of an antibody that can bind to a specific receptor or antigen.

[0066] [The present invention]

[0066] The present invention provides an antibody-cytokine conjugate that has an extended in vivo half-life and increased in vitro and in vivo activity, which can activate the immune response in the tumor microenvironment and thereby confer protective antitumor immunity. The antibody-cytokine conjugate according to the present invention enables targeted delivery of cytokines by cytokine release at the target site. The conjugate according to the present invention has higher efficacy and therapeutic area than antibody-cytokine conjugates that do not have gene fusions of antibodies and immune cell engagers, or cleavable sites.

[0067]

[0067] Antibody-cytokine conjugates can be produced without requiring genetic modification of IgG. The present invention makes it possible to adjust the molecular format of the antibody-cytokine conjugate by defining a ratio of 2:1 or 2:2, i.e., the ratio of complement-dependent regions in the complete IgG CDR (2) to the immune cell-engaging polypeptide (1 or 2).

[0068]

[0068] Thus, the first aspect of the present invention relates to a cleavable antibody-cytokine conjugate. In a second aspect, the present invention relates to a cleavable antibody-cytokine conjugate for medical treatment. In a third aspect, the present invention relates to a process for preparing an antibody-cytokine conjugate. A pharmaceutical composition containing the conjugate according to the present invention is also included within the present invention.

[0069] [Conjugate of general structure (1) or structure (2)]

[0069] The present invention relates to an antibody-cytokine conjugate of general structure (1) or (2), with structure (1) or structure (2)

Chemical formula

[0070]

[0070] Preferably, linker L 6 There exists such that β=1.

[0071] [Antibody AB]

[0071] AB is an antibody. Antibodies known in the art include IgA, IgD, IgE, IgG, IgM, Fab, VHH, scFv, diabody, minibody, afibody, affin, affimer, atrimer, finomer, Cys-not, DARPin, adnectin / centrin, notchin, anticarin, 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, for example IgG1, IgG2, IgI3, or IgG4. Preferably, AB is a full-length antibody, but AB can also be an Fc fragment.

[0072]

[0072] Antibody Ab is typically specific to extracellular receptors on tumor cells, preferably 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, Brevican, 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 / Sindecane-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), Claudine, CLDN18.2. CLDN6 (Claudin-6, Skullin), CLEC12A, CLL-1 / CLEC12A, Crypt, CS1 (SLAMF7, CD319), CXCR5, DKL-1, DLL3 (Delta-like 3), DPEP3, E16, EGFR, EGFRvIII, ENPP3, CD203c (AGS-16), EpCAM, EphA2, EphB2R, Ephrin-A4 (EFNA4), ETBR, FAP, FGFR2, FGFR3, Fibronectin EDB, FLT3, FOLR1 (FR-a), Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, GloboH, GPC3 (Glypican-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, Nectin-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 The antibody is selected from the group consisting of 5b, SEZ6, SLITRK6 (SLC44A4), STEAP1, STEAP2, STn, TAG72, TENB2, TF (CD142, thromboplastin), TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, TNF-α, TROP-2 (TACSTD2), TWEAKR, receptor tyrosine kinase (RTK), and tenascin, or the antibody is specific to extracellular proteins and / or tumor-associated glycogen antigens (TACAs) resulting from viral infection. In preferred embodiments, the extracellular receptor is selected from PD-L1, HER2, and nectin-4, more preferably from PD-L1 and HER2.

[0073]

[0073] Antibodies may also be specific to extracellular proteins resulting from viral infections, such as human poliovirus (HPV), human cytomegalovirus (HCMV), or human papillomavirus (HPV). Antibodies may include Tn, STn, T-antigen, LDN, Lewis. c (Le c ), Cialil-Lewis c (SLe c ), 6-Sialil-Lewis c (6SLe c ), LN, alpha-Gal, 3SLN, 6SLN, H antigen, A antigen, B antigen, Lewis a (Le a ), Cialil-Lewis a (SLe a ), 6-Sialil-Lewis a (6SLe a ), Lewis b (Le b ), Ciaril-Lewis b (SLe b ), 6-Sialil-Lewis b (6SLe b ), Lewis x (Le x ), Cialil-Lewis x (SLe x ), 6-Sialil-Lewis x (6SLe x ), Lewis y (Le y ), Ciaril-Lewis y (SLe y ), 6-Sialil-Lewis y (6SLe y The antibody may also be specific to tumor-associated glycosylation antigens (TACAs) selected from the group of ) and / or combinations thereof. The antibody may be simultaneously specific to both extracellular proteins and TACAs.

[0074]

[0074] The number of payloads D bound to a single antibody is known in the art as DAR (drug-antibody ratio). In the present invention, the drug is an immune cell engager polypeptide specific to an immune cell receptor. In the present invention, the DAR value is an integer in the range of 1 to 10, preferably 1, 2 or 4, and more preferably 1 or 2.

[0075]

[0075] A portion of the antibody is the reactive part F 1 or linking group Z 1 Linker L connects to the peptide portion of the cell binding agent. 6 This is possible. Preferably, the linking group Z 1 It is linked to the antibody via glycan.

[0076] [Linker L 6 ]

[0076] Linker L 6 It is preferable that a reactive group F is present. 1 The reactive group F can be introduced at a specific location on the antibody. This can be done by artificially introducing the reactive group F, for example, by using transglutaminase, saltase, or by enzymatic glycan modification (e.g., glycosyltransferase or α-1,3-mannosyl-glycoprotein-2-bN-acetylglucosaminyl-transferase). 1 This is the case of conjugation via a modified sugar residue S(F 1 )2 is introduced into the glycan, allowing the glycan to be extended by one monosaccharide residue S, thereby creating two reactive groups F on the antibody glycan. 1 A conjugation is introduced. In the most preferred embodiment, conjugation occurs via the antibody glycan. The site of conjugation is preferably in the heavy chain of the antibody.

[0077]

[0077] All recombinant antibodies produced in mammalian host systems contain an N-glycosylation site conserved at position 297 (Kabat numbering) or a nearby asparagine residue of the heavy chain modified by a complex-type glycan. While this naturally occurring glycosylation site of the antibody is preferably used, other glycosylation sites, including those introduced artificially, are also used in linker L 6 It can be used to connect L 6 The nucleotide is linked to an amino acid of the antibody located in the range of 250-350 on the heavy chain, preferably in the range of 280-310, more preferably in the range of 295-300, and most preferably at position 297 on the heavy chain. When this conserved glycosylation position of the antibody is used, the resulting conjugate is formed as a symmetric dimer, and each half-antibody has one F 1 It contains. Some antibodies may have a second glycosylation site per half-antibody, which is preferably not used as a conjugation site. Those skilled in the art can perform the enzymatic conversion in such a way that only the primary glycosylation site is used for conjugation. Alternatively, those skilled in the art can perform the enzymatic conversion in such a way that the second glycosylation site is also used for conjugation, thereby doubling the DAR of the antibody-drug conjugate.

[0078]

[0078] L 6 AB to F 1 or Z 1 It is a linker that connects to -(H) v -S-(L 7 ) w’ - is represented by where H is a monosaccharide, v is an integer in the range of 0 to 10, S is a sugar or sugar derivative, w' is 0 or 1, L 7 These are -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-. (H) v It can be linear or branched, and S is (H) v It can be linked to any monosaccharide. Typically, L 6 It is formed, at least partially, by antibody glycans.

[0079]

[0079] L 6 no-(H) v -The part is a glycan or a part thereof. Therefore, the -(H) of the glycan v - Typically originates from the original antibody. (H) v Preferably, the structure is selected from (H1), (H2), and (H3). [ka]

[0080]

[0080] Here, w is 0 or 1, j is an integer in the range of 0 to 9, GlcNAc is the N-acetylglucosamine moiety, Fuc is the fucose moiety, Gal is the galactose moiety, a wavy bond to * represents binding to the antibody peptide, and a wavy bond to ** represents binding to S. Fuc is typically bound to GlcNAc via an α-1,6-glycosidic bond. Typically, the antibody may be fucosylated (w=1) or not (w=0). In relation to the present invention, the presence of the fucosyl moiety is irrelevant, and similar effects can be obtained with fucosylated (w=1) and non-fucosylated (w=0) antibody conjugates. The GlcNAc residue is sometimes called the core-GlcNAc residue and is a monosaccharide that is directly bound to the peptide moiety of the antibody.

[0081]

[0081] S is core-GlcNAc(Fuc) w It can be directly bonded to a portion, i.e., j=0, which means that the rest of the glycan is core-GlcNAc(Fuc) before S is bonded. w This means that a portion is removed. Such trimming of glycans is well known in the art and can be achieved by the action of endoglycosidases. (H3) can be obtained from a prepared glycan by introducing galactose using galactosyltransferase and then introducing S-fucose using fucosyltransferase, as described in International Publication No. 2022037665. Alternatively, core-GlcNAc(Fuc)w There is one or more monosaccharide residues between the moiety and S, that is, j is an integer in the range of 1 to 9, preferably j = 1 to 5. In a preferred embodiment, (G) j is an oligosaccharide fraction containing j monosaccharide residues G, and j is an integer in the range of 2 to 5. In another preferred embodiment, (G) j is an oligosaccharide fraction containing j monosaccharide residues G, and j is 1. (G) j is typically linked to the GlcNAc moiety of GlcNAc(Fuc) via an α-1,4-bond w 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.

[0082]

[0082] Any monosaccharide that may be present in the glycan can be used as G, and each G is preferably individually selected from the group consisting of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose and N-acetylneuraminic acid. More preferred options for G are galactose, N-acetylglucosamine, mannose. When j = 1, it is preferred that G = galactose and S = N-acetylneuraminic acid.

[0083]

[0083] When j is 3 to 10, (G) j can be linear or branched. Preferred examples of the branched oligosaccharide (G) j are (a), (b), (c), (d), (e), (f) and (h) as shown below.

Chemical formula

[0084] 0]

[0084] Here, the wavy line represents the bond to the core GlcNac(Fuc) w For (G) where j > 2 jIf present, it is preferable that it terminates with 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 promotes the synthesis of functional antibodies because the monosaccharide derivative S can be readily introduced to the terminal GlcNAc residue by glycosyl transfer. The presence of the Gal moiety promotes the synthesis of functional antibodies because the monosaccharide derivative S sialic acid can be readily introduced to the terminal Gal residue by sialyltransferase. Alternatively, the presence of the Gal moiety allows for the introduction of the monosaccharide derivative S fucose onto the adjacent GlcNAc by fucosyltransferase, as described in International Publication No. 2022037665. (G) having structures (a) to (h) j In the above preferred embodiment, portion S may be linked to any of the terminal GlcNAc residues, i.e., it does not have a wavy linkage linked to a core GlcNAc residue on the antibody.

[0085]

[0085] Antibodies and antibody conjugates with j=0 or 1 do not bind to the Fc-gamma receptor or their binding is significantly reduced, while antibodies and antibody conjugates with j in the range of 4 to 10 bind to the Fc-gamma receptor. Therefore, by selecting a specific value for j, a desired degree of binding to the Fc-gamma receptor can be obtained. Thus, j=0, 1, 4, 5, 6, 7, 8, 9, or 10 is preferred, more preferably j=0, 1, 4, or 5, and most preferably the antibody is trimmed to j=0.

[0086]

[0086] S is a sugar or a sugar derivative. The term "sugar derivative" is used herein to denote 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 (Fuc), amino sugars and sugar acids, such as glucosamine (GlcNH2), galactosamine (GalNH2), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) also referred to 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.

[0087]

[0087] Linking group Z 1 or reactive group F 1 can be directly linked to S, or S and Z 1 or F 1 there can be a linker L 7 between them. Thus, L 7 may or may not be present (w’ = 1 or 2) or may not be present (w’ = 0). Typically, each moiety Z can be linked to S via a linker L 7 and thus, in one embodiment, is x with w’ = 0. Preferably, L 7 is absent and each linking moiety Z is directly linked to S. When present, L 7 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.

[0088] [Cleavable site]

[0088] The conjugate of the present invention contains a cleavable site to enable the release of an immune cell engager polypeptide at the target site. The cleavable site is part of the payload D (i.e., D 1The following conditions are present and α=1) or in the linker L (preferably L 2 and L 3 As such, i.e., o=p=1), it can exist. In a preferred embodiment, α=1 and o=p=0.

[0089] [Immune cell engager polypeptide D]

[0089] The payload of the conjugate of the present invention is an immune cell engager polypeptide D, also known as a cytokine. Payload D is linked to an antibody via a cleavable linker. The cleavable units may be present in linker L (i.e., in one embodiment, L is cleavable) or in the peptide chain of D (i.e., α=1 and cleavable peptide linker D). 1 (There exists). Typically, when α=1, L is not cleavable.

[0090]

[0090] Polypeptide D or a precursor of polypeptide D is preferably produced by microbial recombinant expression. Recombinant expression is particularly preferably carried out in prokaryotes, in a cell-free protein expression system, by a synthetic method, or in combination with a synthetic method and a protein ligation method. Proteins obtained by microbial expression in prokaryotes, or by a cell-free method, or by a synthetic method are not glycosylated, and therefore when D or a precursor of D is obtained by microbial expression in prokaryotes or by cell-free expression by a synthetic method, it is more uniform than when it is obtained from eukaryotes. Most preferably, D is obtained by microbial recombinant expression using Escherichia coli (E. coli) bacteria. In other words, it is preferable that the peptide chain of D is not glycosylated, i.e., that D is a non-glycosylated polypeptide.

[0091] [Mode of operation]

[0091] D is -(D 1 )α-D 2 It is a polypeptide produced by D 1 It is a cleavable peptide linker, D 2It is an immune cell engager polypeptide specific to immune cell receptors, and α is 0 or 1. If L is not cleavable, D 1 There exists. Preferably, α is 1 and D 1 There exists, and moreover, D is D 1 The N-terminus or C-terminus, most preferably via the N-terminus, is Z 2 It is connected to.

[0092]

[0092] D 2 It is specific to immune cell receptors and, by activating these receptors, triggers subsequent cellular signaling. 2 The ability of it to bind to at least one of its associated receptors is reduced or completely lost by its binding to the antibody. Therefore, D 2 D 1 Alternatively, it is activated by cleavage of linker L. Since the cleavable site is likely to be cleaved at a specific target, cellular signaling primarily occurs at the target. The antibody is activated against at least one of the associated receptors. 2 In order to interfere with its ability in three dimensions, D 2 The distance between and AB is preferably as short as possible. More preferably, D 2 The shortest atomic chain between A and B consists of 10 to 100 atoms, more preferably selected from B, C, O, N, S, and P. In a preferred embodiment, the atomic chain consists of 15 to 50 atoms selected from C, O, N, S, and P, more preferably from C, O, N, and S.

[0093] [Cuttable Linker D] 1 ]

[0093] D 1 This is a cleavable peptide linker known in the art. 1The molecule contains a cleavable site for a protease, preferably a mammalian protease. In therapeutic applications, the protease cleavage site can be cleaved by a protease typically overexpressed in the vicinity of or target cells, such as cancer cells, infected cells, or pathogens. These proteases may be extracellular enzymes produced by the target cell or intracellular enzymes leaking outside 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 target cell microenvironment, typically in the tumor microenvironment. Such proteases are usually overexpressed in the target microenvironment.

[0094]

[0094] In a preferred embodiment, D 1 It contains a cleavable site that can be recognized by one of the following: serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, metalloproteinase, gelatinase, and asparagine peptide lyase.

[0095]

[0095] In another preferred embodiment, D 1Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K, Cathepsin L, Kallikrein, hKl, hKlO, hKT5, Plasmin, Collagenase, Type IV Collagenase, Stromelicin, Factor Xa, Chymotrypsin-like protease, Trypsin-like protease, Elastase-like protease, Subtilisin-like protease, Actinidine, Bromelain, Calpain, Caspase, Caspase 3, Mirl-CP, Papain, HIV-1 protease, HSV protease, CMV protease, Chymosin, Renin, Pepsin, Matryptase, Membrane-type serine protease 1 (MT-SP1), Regmine, Plasmepsin, Nepenthesin, Metalloexopeptidase, Metalloendopeptidase It contains cleavable sites that are recognized and cleaved by tidase, matrix metalloproteinases (MMPs), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP1O, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM12, urokinase plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), type II transmembrane serine protease (TTSP), neutrophil elastase, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and one of dipeptidyl peptidase IV (DPPIV / CD26). Preferably, the cleavable sites are recognized and cleaved by one of MMP, legmine, matryptase, urokinase, and thrombin, more preferably by at least one of legmine, matryptase, and urokinase.

[0096]

[0096] Any protease-specific cleavable peptide linker known in the art is D 1It can be used as such. Such peptide linkers are known, for example, from U.S. Patent Application Publication No. 2016 / 0194399, which is incorporated herein by reference. Suitable cleavable peptide linkers include 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), 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), and ISSGLLSS (SEQ ID NO: 64). It contains peptide sequences selected from 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), GPSHLLVLT (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).

[0097]

[0097] Peptide sequences having SEQ ID NOs. 43 to 63 can be cleaved by at least one of proteases, legmine, matryptase, and urokinase. Peptide sequences having SEQ ID NOs. 64 to 84 can be cleaved by at least one of proteases MMP. Peptide sequences having SEQ ID NOs. 85 and 86 can be cleaved by thrombin. A preferred cleavable peptide linker includes a peptide sequence selected from SEQ ID NOs. 43 to 63, more preferably a peptide sequence SEQ ID NO. 43 or SEQ ID NO. 44, and most preferably a peptide sequence SEQ ID NO. 43.

[0098]

[0098] The peptide sequence of the cleavable site is D 1 It can be incorporated into longer peptide sequences. The cleavable sites typically contain 3 to 12 amino acids, preferably 4 to 10 amino acids, and more preferably 6 to 8 amino acids. The full length of the peptide D 1 The cleavable site is, as defined herein, a maximum of 50 amino acids longer than the cleavable site, preferably 3 to 30 amino acids longer, and more preferably 8 to 25 amino acids longer. Furthermore, peptide D 1 The total length may be, for example, 4 to 60 amino acids, preferably 10 to 50 amino acids, and more preferably 15 to 40 amino acids. Preferred cleavable peptide linkers are cleavable peptide linkers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2).

[0099]

[0099] Particularly preferably, the cleavable peptide linker comprises the peptide sequence RASRAN (SEQ ID NO: 43), and most preferably comprises cleavable peptide linker 2 (SEQ ID NO: 2). These peptide linkers are recognized by at least three different tumor-specific proteases (regmine, matryptase, and urokinase) and are beneficial for site-specific cleavage of immune cell engager polypeptides at the target site.

[0100] [Immune cell-engaging polypeptide D 2 ]

[0100] Immune cell-engaging polypeptide D 2A cytokine is specific to an immune cell receptor, and typically, to a cytokine receptor on the surface of an immune cell. It should be understood that references to cytokines include mutated variants and cytokines bound to cofactors. For example, IL-15, unless otherwise specified, refers to normal IL-15, but can also refer to IL15α, sushi-IL15, IL15 with point mutations, etc. Preferably, cytokines do not contain cofactors. D 2 If the mutant immune cell-engaging polypeptide is 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(IFN-α1 / 13, IFN The immune cell-engaging polypeptide is selected from the group consisting of IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α14, IFN-α16, IFN-α17, and IFN-α21, and includes mutated variants, and more preferably the immune cell-engaging polypeptide is IL-2 or IL-15. In the most preferred embodiment, the immune cell-engaging polypeptide is IL-15. In an alternative embodiment, the immune cell-engaging polypeptide is not IL-15.

[0101]

[0101] Therefore, in one embodiment, D 2The derivative is IL-15, preferably mutated to enhance or decrease its binding affinity to the receptor, and more preferably mutated to decrease its binding affinity to IL15Ra. IL15Ra binding results in rapid clearance, and therefore mutants with reduced IL15Ra binding affinity result in better PK and higher potency. More preferably, IL15 according to SEQ ID NO: 3 includes amino acid substitutions on E46 and V49, even more preferably the amino acid substitutions are E46G, V49R, and most preferably, there are no other substitutions. Advantageously, IL15Rβ and γ c The binding affinity to is reduced due to steric hindrance of the antibody. However, after cleavage, IL15Rβ and γ c The binding site becomes available, and the immune response is activated.

[0102] [Linking group Z 1 and Z 2 ]

[0102] Z 1 and Z 2 This is a linking group that covalently links the antibody to the payload of the conjugate according to the present invention. The term “linking group” as used herein refers to a structural element resulting from a reaction between Q and F, which links a portion of a conjugate to another portion of the same conjugate. 1 Q 1 and F 1 It is formed by cycloaddition or nucleophilic substitution between Z. Preferably, Z 1 It is formed by cycloaddition. Similarly, Z 2 Q 2 and F 2 It is formed by cycloaddition or nucleophilic substitution between Z. Preferably, Z 2 It is formed by cycloaddition. In this specification, Z is Z 1 and Z 2 Q refers to Q 1 and Q 2 F refers to F 1 and F 2 It refers to.

[0103]

[0103] As will be understood by those skilled in the art, the exact properties of the linking group depend on the exact structure of the reactive parts Q and F. Those skilled in the art will understand that Q reacts with each other and is a suitable reaction partner. 1 / F 1 and Q 2 / F 2 It recognizes the complementary click probe that forms the complementary group. For example, if F contains or is an alkynyl group, the complementary group Q contains an azide group. For example, if F contains or is an azide group, the complementary group Q contains an alkynyl group. For example, if F contains or is a cyclopropenyl group, a trans-cyclooctene group, a cycloheptine or cyclooctin group, the complementary group Q contains a tetradinyl group. In these particular cases, as shown in Figure 1, Z is merely an intermediate structure that releases N2, thereby producing dihydropyridazine (from reaction with an alkene) or pyridazine (from reaction with an alkyne).

[0104]

[0104] In a preferred embodiment, the linking group Z is obtained by a cycloaddition reaction, preferably the cycloaddition is a [4+2] cycloaddition or a 1,3-dipole cycloaddition. Conjugation reactions via cycloaddition are known to those skilled in the art, and those skilled in the art will be able to select appropriate reaction partners F and Q and understand the properties of the resulting linking group Z. Preferred cycloadditions are [4+2]-cycloaddition (e.g., Diels-Alder reaction) or [3+2]-cycloaddition (e.g., 1,3-dipole cycloaddition). Preferably the cycloaddition is a Diels-Alder reaction or a 1,3-dipole cycloaddition. A preferred Diels-Alder reaction is an inverse electron-demanding Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipole cycloaddition, more preferably an alkyne-azide cycloaddition, is used. Cycloadditions such as the Diels-Alder reaction and 1,3-dipolar cycloaddition are known in the art, and those skilled in the art know how to carry them out.

[0105]

[0105] Preferably, Z contains a moiety selected from the group consisting of triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, piperazine, thioether, amide, or imide group. The triazole moiety is particularly preferred to be present in Z. In one embodiment, Z is formed from Q containing a (hetero)cycloalkene moiety, i.e., a (hetero)cycloalkyne moiety. In an alternative embodiment, Z is formed from Q containing a (hetero)cycloalkane moiety, i.e., a (hetero)cycloalkene moiety. In this specification, aromatic rings such as triazole rings are considered heterocycloalkane rings because they are formed by the reaction of an alkyne moiety with an azide moiety.

[0106]

[0106] In a preferred embodiment, Z is structure (Z1): [ka] It has. Here, [ka] The bonds shown as are single or double bonds. Furthermore, - Ring Z is obtained by cycloaddition, preferably ring Z is selected from (Za) to (Zm) as defined below, and the carbon atoms labeled with ** are (Z1) to which ring Z is condensed. [ka] Corresponding to the two carbon atoms of the bond shown as, - R 15 These are, independently, hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)arene substituents, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, - Y 2 C(R 31 )2, O, S, S (+) R 31 S(O)R 31 , S(O)=NR 31 or NR 31 S (+) B (-) It is a cationic sulfur atom that is canceled out by B (-) is an anion, and each R 31 R 15 It is either a connection with D connected via L, - u is 0, 1, 2, 3, 4 or 5, - u' is 0, 1, 2, 3, 4, or 5, and u + u' = 0, 1, 2, 3, 4, 5, 6, 7, or 8, - v is an integer in the range of 8 to 16. Ring Z is formed by cycloaddition and is preferably selected from (Za) to (Zm).

[0107]

[0107] In a preferred embodiment, u+u'=0, 4, 5, 6, 7, or 8, and more preferably 0, 4, or 5. [ka] When the bond shown is a double bond, it is preferable that u+u'=4, 5, 6, 7, or 8, and more preferably that u+u'=4 or 5. [ka] When the bond shown as is a single bond, it is preferable that u+u'=0 or 5. Preferably, the bond marked with an asterisk (*) is an L bond. 6 The connections between AB and the wavy lines marked with ** are connected to L.

[0108]

[0108] Z includes a (hetero)cycloalkene moiety, i.e., [ka] It is particularly preferable that the bond shown as is a double bond. In preferred embodiments, Z is selected from the structures (Z2) to (Z20c) shown below. [ka]

[0109]

[0109] Here, the connection to L is indicated by a wavy line. B (-) is an anion, preferably a pharmaceutically acceptable anion. (+) R is a cation, preferably a pharmaceutically acceptable cation. 36 is a halogen selected from fluorine, chlorine, bromine, and iodine, preferably R 36 Y is fluorine. 4 is a heteroatom, preferably Y 4 R is either O or NH. 35 is hydrogen, C1~C 24 Alkyl alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 (hetero)aryl group and C3~C 24 (hetero)arylalkyl groups, C1-C24 Alkyl 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 with O, Si, S, and NR 14 Optionally interposed by one or more heteroatoms selected from R 14 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably R 35 H, C5H 11 Selected from CH3, CH2CH3, CH2OH, or CH2OTBS.

[0110]

[0110] Ring Z is formed by a cycloaddition reaction and is preferably a triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, oxazolidine, pyrazoline, or piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have a structure selected from (Za) to (Zm) shown below, where the carbon atoms labeled with ** correspond to two carbon atoms of the (hetero)cycloalkane rings (Z2) to (Z20) to which ring Z is condensed. Since the linking group Z is formed by a reaction with a (hetero)cycloalkyne in relation to this embodiment, [ka] The bond shown above is a double bond. [ka]

[0111]

[0111] Here, R 29 is 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)-NR33 -C 1~6 Alkyl and C(O)-NR 33 - Selected from the alphabet, R 33 H or C 1~4 It is alkyl. Preferably, R 29 R is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl. 29 It was found that the presence of hydrogen yields optimal results for the reactivity of the cycloaddition reaction, especially when a ring (Zl) is formed. Therefore, in a preferred embodiment, the ring Z is (Zl), and R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl, more preferably R 29 It is hydrogen.

[0112]

[0112] The following applies to isoxazoline (Zh) and its rearrangement product (Zh'): - R 1 L 10 XR 4 And, - L 10 is structure (C(R 3 )2) z The linker is such that z is 2 or 3, and each R 3 H and C 1~4 Individually selected from alkyl groups, with two R groups 3 The appearance of is combined with C 3~6 (hetero)cycloalkyl groups can be formed, - R 4 H and C 1~4 Selected from alkyl groups, - X is S, O, or NH, - R 2 H and C 1~4 Selected from alkyl groups.

[0113]

[0113] Preferred embodiments are defined for the following nitrone reactive group (F3a), which are equally applicable to isoxazoline (Zh) and rearrangement product (Zh').

[0114]

[0114] The inventors of the present invention have found a substituent R 1 We found that this improves the stability of isoxazoline conjugates. Typically, isoxazolines can be rearranged according to the following scheme. [ka]

[0115]

[0115] The 4-isoxazoline ring may undergo disintegration due to this rearrangement reaction. The isoxazoline ring (a) is rearranged to the aziridine ring (b), from which the ring strain is released in the form of an amphoteric species (c). Addition of water yields a hydroxylamine (e), from which the aldehyde (f) is separated to obtain a ketoneamine (g). This not only destroys the isoxazoline ring but also destroys the covalent bond between the R* substituent and the R** substituent. Therefore, if the isoxazoline ring is part of a bioconjugate, the payload is removed from the biomolecule, thus effectively eliminating its use and potentially leading to serious side effects.

[0116]

[0116] The solution to the above problem developed by the present inventors is a specific substituent R on the nitrogen atom of nitrone. 1 The heteroatom X can capture the imine intermediate (d) by rearrangement through reaction with the imine carbon atom. The inventors have found that this does not result in the separation of the aldehyde (f), but instead an oxazolidine ring (h) is formed, as shown in the following scheme (R 1 We found that this results in a rearrangement of an exemplary scheme having (CH2)2OH. This structure is stable and does not collapse. Therefore, the covalent bond between the substituent at R* and the substituent at R** remains intact. Thus, the bioconjugate remains intact and effective when the payload is bound to a biomolecule. Therefore, even if the rearrangement of the 4-isoxazoline ring formed by the SPANCE reaction is disrupted, the covalent bond is not broken, and thus the bioconjugate according to the present invention represents a great improvement in terms of the stability of payload binding to biomolecules. [ka]

[0117]

[0117] If Z contains a (hetero)cycloalkene moiety, the ring Z is preferably selected from (Za), (Zh), (Zj), (Zk), or (Zl), and the ring Z is more preferably structured as (Za) or (Zl).

[0118]

[0118] In a further preferred embodiment, Z is selected from the structures (Z21) to (Z38d) shown below. [ka]

[0119]

[0119] Here, the connection to L is indicated by a wavy line connection. Structure (Z29) can be an end configuration or an exo configuration, preferably an end configuration. In structure (Z38), B (-) is an anion, preferably a pharmaceutically acceptable anion. (+) is a cation, preferably a pharmaceutically acceptable cation. Ring Z is selected from the structures (Za) to (Zm) defined above.

[0120]

[0120] In preferred embodiments, Z preferably comprises a (hetero)cyclooctene moiety or (hetero)cycloheptene moiety of the structure (Z8), (Z26), (Z27), (Z28), (Z37), or (Z38a), which are optionally substituted. Each of these preferred options for Z is further defined herein below.

[0121]

[0121] Therefore, in a preferred embodiment, Z includes a heterocycloheptene moiety described in structure (Z37) or (Z38a) which is optionally substituted. Preferably, the heterocycloheptene moiety described in structure (Z37) or (Z38a) is not substituted.

[0122]

[0122] In a preferred embodiment, Z includes a (hetero)cyclooctene moiety according to the optionally substituted structure (Z8), more preferably (Z29). Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is not substituted. In relation to this embodiment, Z is preferably a (hetero)cyclooctene moiety according to the structure (Z39) shown below (wherein V is (CH2) l The range is such that 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, more preferably l is 0, 1 or 2, and most preferably l is 0 or 1. In relation to base (Z39), l is most preferably 1. Most preferably, Z is by a structure (Z42) as further defined below.

[0123]

[0123] In alternative preferred embodiments, Z comprises a (hetero)cyclooctene moiety of the structure (Z26), (Z27), or (Z28), which are optionally substituted. In relation to this embodiment, Z is preferably the structure (Z40) or (Z41) shown below (wherein Y 1 is O or NR 11 And R 11 These are, independently, hydrogen, linear or branched C1-C12 12 Alkyl alkyl group or C4-C 12 The structure includes a (hetero)cyclooctene moiety (selected from the group consisting of (hetero)aryl groups). The aromatic ring of (Z40) is optionally O-sulfated at one or more positions, while the ring of (Z41) can be halogenated at one or more positions. Preferably, the (hetero)cyclooctene moiety of structure (Z40) or (Z41) is not further substituted. Most preferably, Z is of structure (Z43), which is further defined below.

[0124]

[0124] In an alternative preferred embodiment, Z comprises a heterocycloheptenyl group, as shown in structure (Z37). [ka]

[0125]

[0125] In a particularly preferred embodiment, Z comprises a cyclooctenyl group, and the structure (Z42): [ka] This is due to the following: - Bonds marked with * are connected to AB, and bonds marked with ** are connected to L. - R 15 These are, independently, hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)arene substituents, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, - R 18 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, - R 19 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24(hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group is optionally interposed by one or more heteroatoms selected from the group consisting of O, N, and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are independently optionally substituted or R 19 This is a second appearance of Z (or Q) or D connected via a spacer portion, and - l is an integer in the range of 0 to 10.

[0126]

[0126] In a preferred embodiment of the base by structure (Z42), R 15 is hydrogen, halogen, -OR 16 , independently selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, R 16 is hydrogen or a C1-C6 alkyl group, more preferably R 15 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R 15 is H. In a preferred embodiment of the base according to structure (Z42), R 18 R is independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably both R 18 is H. In a preferred embodiment of the base according to structure (Z42), R 19 is H. In a preferred embodiment of the base according to structure (Z42), l is 0 or 1, and more preferably l is 1.

[0127]

[0127] In a particularly preferred embodiment, Z comprises a (hetero)cyclooctenyl group, and structure (Z43): [ka] This is due to the following: - Bonds marked with * are connected to AB, and bonds marked with ** are connected to L. - R 15 These are, independently, hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)arene substituents, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, - Y is N or CR 15 And, - The carbon atoms in the condensed aromatic ring may be replaced by nitrogen atoms, as in (Z6a)~(Z6d), preferably Y is CR 15 That is the case.

[0128]

[0128] In a preferred embodiment of the base by structure (Z43), R 15 is hydrogen, halogen, -OR 16 -S(O)3 (-) , independently selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, R 16 is hydrogen or a C1-C6 alkyl group, more preferably R 15 It consists of hydrogen and -S(O)3 (-) It is independently selected from the group consisting of the following. In a preferred embodiment of the group according to structure (Z43), Y is N or CH, and more preferably Y=N.

[0129]

[0129] In a particularly preferred embodiment, Z comprises a heterocycloheptenyl group and is structured as (Z37) or (Z38a) (wherein the ring Z is a triazole). [ka]

[0130]

[0130] In an alternative preferred embodiment, the linking group Z includes a (hetero)cycloalkane moiety, i.e., [ka] The bond shown as is a single bond. The (hetero)cycloalkane group may also be called a heterocycloalkyl group or cycloalkyl group, preferably a cycloalkyl group, and 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 can be optionally substituted. Particularly preferred are a (hetero)cyclopropyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group, and the (hetero)cyclopropyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group is optionally substituted. Preferably, Z includes a cyclopropyl moiety by structure (Z44), a heterocyclobutane moiety by structure (Z45), a norbornane or norbornene group by structure (Z46), a (hetero)cycloheptyl moiety by structure (Z47), or a (hetero)cyclooctyl moiety by structure (Z48). In this specification, Y 3 C(R 23 )2, NR 23 Or selected from O, each R 23 These are, individually, hydrogen, C1-C6 alkyl, or optionally linked to L via a spacer and labeled. [ka] The bonds are single or double bonds. In a further preferred embodiment, the cyclopropyl group is structured as (Z49). In another preferred embodiment, the (hetero)cycloheptane group is structured as (Z50) or (Z51). In yet another preferred embodiment, the (hetero)cyclooctane group is structured as (Z52), (Z53), (Z54), (Z55) or (Z56). [ka]

[0131]

[0131] Here, the R groups on Si in (Z50) and (Z51) are typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is formed by a cycloaddition reaction and is typically selected from structures (Zn)-(Zu), where the carbon atoms labeled with ** correspond to the two carbon atoms of the (hetero)cycloalkane rings (Z44)-(Z56) to which ring Z is condensed, and the carbon atoms labeled with * are linked to AB. Since the linking group Z is formed by a reaction with a (hetero)cycloalkene in relation to this embodiment, [ka] The bond shown above is a single bond. [ka]

[0132]

[0132] Here, R 29 is 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 - Selected from the alphabet, R 33 H or C 1~4 It is alkyl. Preferably, R 29R is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl. 29 It was found that the presence of hydrogen yields optimal results for the reactivity of the cycloaddition reaction, especially when a ring (Zu) is formed. Therefore, in a preferred embodiment, the ring Z is (Zu), and R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl, more preferably R 29 It is hydrogen.

[0133]

[0133] When Z contains a (hetero)cycloalkane moiety, the ring Z is preferably selected from (Zn), (Zs), (Zt), or (Zu), and most preferably the ring Z is structured as (Zu).

[0134]

[0134] In a preferred embodiment, the linking group Z includes a portion selected from (Z1) to (Z56), and the ring Z is selected from (Za) to (Zu).

[0135]

[0135] In the present invention, Z 1 and Z 2 The exact structure is Z 1 Q 1 and F 1 It is formed by the reaction with Z 2 is Q 2 and F 2 Because it is formed by a reaction with, it is typically different. Preferably, as specified herein, F 2 Q 2 Q 1 Q does not respond to this, 1 and Q 2 They should be different.

[0136]

[0136] In a preferred embodiment, F 1 Azid, Q 1 F is a benzocyclized or tetramethylated (hetero)cycloalkyne, while F 2 It is tetrazine, and Q 2 This is bicyclononine. Here, F 1 and Q 1The reaction is preferably due to structure (Z5), (Z6), (Z7), (Z11), (Z17), (Z18), (Z19), or (Z19a), and ring Z is due to structure (Za), preferably due to structure (Z26), (Z27), (Z28), (Z32), (Z37), (Z38), or (Z38a), and more preferably due to linking group Z of structure (Z40), (Z41), or (Z43). 1 It forms F 2 and Q 2 The reaction is preferably represented by structure (Z8) (wherein ring Z is due to structure (Zl)), preferably by structure (Z29), and more preferably by structure (Z42), with a linking group Z 2 It forms.

[0137]

[0137] In yet another preferred embodiment, F 1 is Azid, and also Q 1 Q is a cycloalkyne, while Q 2 is nitrone, ortho-quinone or tetrazine, and F 2 F is a cycloalkene, preferably trans-cyclooctene. Here, F 1 and Q 1 The reaction is preferably due to structures (Z2) to (Z20), and ring Z is due to structure Za, preferably due to linking group Z (Z8). 1 It forms F 2 and Q 2 The reaction is preferably by (Z44)~(Z56), and the ring Z is by (Zo), (Zp), (Zq), (Zr), (Zq), preferably by structure (Zo) or (Zu), more preferably by structure (Zo) of the linking group Z 2 It forms.

[0138]

[0138] In another preferred embodiment, Z 1 While Z can be obtained by a click reaction between cycloalkynes and azides, 2 This can be obtained by a click reaction between cycloalkyne and nitrone.

[0139]

[0139] In a second preferred embodiment, Z 1 The group is formed by a nucleophilic reaction, preferably nucleophilic substitution or Michael addition, preferably Michael addition. A preferred Michael reaction is thiol-maleimide rigation, most preferably where Q is maleimide, F is a thiol group, and the thiol may be part of a disulfide bridge. Preferably, the thiol is located in the side chain of the cysteine ​​residue. Such a conjugation reaction with a thiol may also be called thiol alkylation or thiol arylation. In a preferred embodiment, the linking group Z 1 This comprises a succinimidyl ring or a ring-opened succinic acid amide derivative thereof, which can be formed by hydrolysis of the succinimidyl ring. When a nucleophilic reaction is used for conjugation, the structural part Q-(L 1 ) a -BM-(L 2 bQ is selected from bromomaleimide, bis-bromomaleimide, bis(phenylthiol)maleimide, bis-bromopyridazinedione, bis(halomethyl)benzene, bis(halomethyl)pyridazine, bis(halomethyl)pyridine, or bis(halomethyl)triazole.

[0140]

[0140] Instead, Z 1 The thiol is formed by a nucleophilic reaction at the amino group in the side chain of the lysine residue (F), which can react with an amino-reactive group Q. Such a conjugation reaction with a thiol may also be called amide bond formation or carbamate bond formation. Typical amino-reactive groups Q include N-hydroxysuccinimidyl (NHS) esters, p-nitrophenyl carbonate, pentafluorophenyl carbonate, isocyanates, isothiocyanates, and halogenated benzoyls.

[0141]

[0141] Linking group Z 1 Preferred options for include portions selected from (Z57) to (Z71) as shown below in this specification. [ka]

[0142]

[0142] Here, the connections of one or more slashes marked with * in (Z57) to (Z66) are linked to AB, and the connections of unmarked slashes are linked to the payload via linker L. In addition, R 29 C 1~12 Alkyl, preferably C 1~4 Alkyl, most preferably ethyl, X 1 is O or S, preferably X 1 =O. The nitrogen atoms labeled with ** in (Z67)~(Z71) correspond to the nitrogen atoms of the side chains of the lysine residues of the antibody, and the unlabeled wavy bond is linked to the payload via linker L. The carbon atoms of the phenyl groups in (Z69) and (Z70) are optionally substituted and preferably optionally fluorinated.

[0143] [Linker L]

[0143] Linker L connects payload D to linking group Z 2 Linking group Z (in the conjugate according to the present invention) via 1 Linking to or linking payload D to reactive group Q (in the linker-cytokine construct), or linking an antibody to a reactive group (in the linker-cytokine construct). Linkers are known in the art and may be cleavable or incleavable. Linker L is referred to as cleavable if the linker contains a cleavable site. Typically, the cleavable site is L 2 and L 3 It is formed by D. Therefore, in a preferred embodiment, D 1 It does not exist (i.e., α=0), and L 2 and L 3 Either 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 L2 and L 3 It does not exist.

[0144]

[0144] In a preferred embodiment, the linker L is structure -(L 1 )-(L 2 ) o -(L 3 ) p -(L 4 ) q - It is expressed by, in the formula, - L 1 is, Z 1 Connected to, and L 4 If it exists, Z 2 It is connected to, - L 1 , L 2 , L 3 and L 4 Each of them individually, Z 1 -Z 2 It is a linker that connects them together. - o, p, and q are each individually 0 or 1, preferably o=p=q=0.

[0145] [Linker L 1 ]

[0145] L 1 For example, linear or branched C1-C 200 Alkylene group, C2~C 200 Alkenylene group, C2~C 200 Alkynylene group, C3~C 200 Cycloalkylene group, C5~C 200 Cycloalkenylene group, C8~C 200 Cycloalkylene group, C7~C 200 Alkyl arylene group, C7~C 200 Arylalkylene group, C8~C 200 Arylalkenylene group, C9~C 200The group can be selected from the group consisting of arylalkylene groups.Optionally, alkylene groups, alkenylene groups, alkylylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkylylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkylene groups may be substituted, optionally, the group may be interposed by one or more heteroatoms, preferably 1 to 100 heteroatoms, the heteroatoms preferably O, S(O) y’ and NR 21 Selected from the group consisting of, y' is 0, 1, or 2, preferably y'=2, R 21 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 The substituent is selected from the group consisting of (hetero)arylalkyl groups. In one embodiment, any substituent may be selected from polar groups, such as oxo groups, (poly)ethylene glycol diamines, (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), phosphonic acids or their esters, phosphonic acids or esters, phosphinic acids or esters, sulfoxides, sulfones, sulfonic acids or esters, sulfenic acids or sulfenic acids.

[0146]

[0146] In a preferred embodiment, linker L 1 It contains a polar group, and this is L 1Such polar groups may also be present in the chain. These polar groups include (poly)ethylene glycol diamines (e.g., equivalents including 1,8-diamino-3,6-dioxaoctane or longer ethylene glycol chains), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains and 1,x'-diaminoalkanes (wherein x' is the number of carbon atoms in the alkane, preferably x'=1 to 10), -(O) a -C(O)-NH-S(O)2-NR 13 -(See structure (23) below for further definitions), -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 can be selected. [ka]

[0147]

[0147] Here, with respect to the polar group defined above, any terminal is Z 1 It is connected to (L 2 ) o Whether or not it is linked is irrelevant.

[0148]

[0148] The polar group may also include amino acids, preferably non-natural amino acids selected from Arg, Glu, Asp, Ser, Thr, or cysteic acid. Here, R 13 This is further defined in structure (23) below. t is an integer in the range of integers 30 H and C are individually 1~12 Alkyl, C 1~12 Ariel, C 1~12 Alkalyl or C 1~12 It is Aralkir. Linker L 1It may contain two or more such polar groups, such as at least two polar groups. The polar groups branch off from the branched portion as defined elsewhere, linker L 1 It can also exist during branching. L 1 In relation to this, preferably, nitrogen or carbon atoms are used as the branching portion. -O(CH2CH2O) t - The presence of polar groups is particularly preferable.

[0149]

[0149] In a preferred embodiment, linker L 1 This is a sulfamide group, preferably a sulfamide group in the form of structure (23), or contains the same. [ka]

[0150]

[0150] The wavy line represents the remainder of the compound, typically Q 2 or Z 2 and L 2 , L 3 , L 4 Alternatively, it represents a connection to D. Preferably, (O) a The C(O) part is Q 2 or Z 2 It is connected to NR 13 The part is L 2 , L 3 , L 4 Or D, preferably L 2 It is connected to.

[0151]

[0151] In structure (23), a=0 or 1, preferably a=1, R 13 is hydrogen, C1~C 24 Alkyl alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl groups and C3-C 24 Selected from the group consisting of (hetero)arylalkyl groups, C1~C 24 Alkyl 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 O, S and NR 14 Optionally substituted and optionally interposed by one or more heteroatoms selected from R 14 R is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Alternatively, 13 The spacer portion is optionally selected, preferably defined as Sp 2 D is connected to N via, and in one embodiment, D is -(K 2 ) e -(K 1 ) f -(K 2 ) g It is connected via -C(O)-. Alternatively, R 13 It is optionally connected to other parts of the linker via spacer portions to form an annular structure. For example, R 13 It can be linked to the linker via the CH2CH2 spacer portion to form a piperazinyl ring, and the linkage to D is via the second nitrogen of the piperazinyl ring.

[0152]

[0152] In a preferred embodiment, R 13 is hydrogen, C1~C 20 Alkyl alkyl groups, preferably C1-C 16 Alkyl alkyl groups, more, C1-C 10 It is either an alkyl group, or optionally linked to further appearances of D or other parts of the linker via a spacer portion. Here, the alkyl group is O, S and NR 14 Optionally substituted and interposed by one or more heteroatoms selected from, preferably O, and R 14 R is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. In another preferred embodiment, R 13 C1~C 20 Alkyl groups, fuaC1~C 16 Alkyl alkyl groups, more preferably C1-C 10The alkyl group is optionally interposed by one or more oxygen atoms, and the alkyl group is optionally substituted with an -OH group, preferably a terminal -OH group. In this embodiment, R 13 It is more preferably a (poly)ethylene glycol chain containing terminal -OH groups. In another preferred embodiment, R 13 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl, or is linked to other parts of the linker, more preferably selected from the group consisting of hydrogen, methyl, ethyl, n-propyl and i-propyl, or optionally linked to further appearances of D or other parts of the linker via a spacer portion, even more preferably selected from the group consisting of hydrogen, methyl and ethyl, or optionally linked to further appearances of D or other parts of the linker via a spacer portion. Even more preferably, R 13 is hydrogen, or optionally connected to further appearances of D or other locations of the linker via a spacer portion, most preferably R 13 It is hydrogen.

[0153]

[0153] In a preferred embodiment, L 1 Structure (24): [ka] This is due to...

[0154]

[0154] Here, a and R 13 As defined above, Sp 1 and Sp 2 The spacer portion is independent of the other, 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 These are independently linear or branched C1-C 200 Alkylene group, C2~C 200 Alkenylene group, C2~C200 Alkynylene group, C3~C 200 Cycloalkylene group, C5~C 200 Cycloalkenylene group, C8~C 200 Cycloalkylene group, C7~C 200 Alkyl arylene group, C7~C 200 Arylalkylene group, C8~C 200 Arylalkenylene group and C9~C 200 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkylene group are O, S and NR 16 It is optionally substituted and optionally interposed by one or more heteroatoms selected from the group consisting of R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group and C3-C 24 The group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted. The alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkylynylene group are interposed by one or more heteroatoms as defined above, and it is preferable that the group is interposed by one or more O atoms and / or one or more SS groups.

[0155]

[0155] More preferably, the spacer portion Sp 1 and Sp 2 If present, they are independently linear or branched C1-C 100 Alkylene group, C2~C 100 Alkenylene group, C2~C 100 Alkynylene group, C3~C 100 Cycloalkylene group, C5~C 100 Cycloalkenylene group, C8~C100 Cycloalkylene group, C7~C 100 Alkyl arylene group, C7~C 100 Arylalkylene group, C8~C 100 Arylalkenylene group and C9~C 100 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkylene group are O, S and NR 16 It is optionally substituted and optionally interposed by one or more heteroatoms selected from the group consisting of R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group and C3-C 24 A group consisting of cycloalkyl groups is selected, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted.

[0156]

[0156] More preferably, the spacer portion Sp 1 and Sp 2 If present, they are independently linear or branched C1-C 50 Alkylene group, C2~C 50 Alkenylene group, C2~C 50 Alkynylene group, C3~C 50 Cycloalkylene group, C5~C 50 Cycloalkenylene group, C8~C 50 Cycloalkylene group, C7~C 50 Alkyl arylene group, C7~C 50 Arylalkylene group, C8~C 50 Arylalkenylene group and C9~C 50Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkylene group are O, S and NR 16 It is optionally substituted and optionally interposed by one or more heteroatoms selected from the group consisting of R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group and C3-C 24 A group consisting of cycloalkyl groups is selected, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted.

[0157]

[0157] More preferably, the spacer portion Sp 1 and Sp 2 If present, they are independently linear or branched C1-C 20 Alkylene group, C2~C 20 Alkenylene group, C2~C 20 Alkynylene group, C3~C 20 Cycloalkylene group, C5~C 20 Cycloalkenylene group, C8~C 20 Cycloalkylene group, C7~C 20 Alkyl arylene group, C7~C 20 Arylalkylene group, C8~C 20 Arylalkenylene group and C9~C 20 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkylene group are O, S and NR 16 It is optionally substituted and optionally interposed by one or more heteroatoms selected from the group consisting of R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C24 Alkenyl group, C2~C 24 Alkynyl group and C3-C 24 A group consisting of cycloalkyl groups is selected, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted.

[0158]

[0158] In these preferred embodiments, the alkylene group, alkenylene group, alkylynylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkylynylene group are unsubstituted, and O, S and NR 16 One or more heteroatoms selected from the group, preferably O, are optionally interposed, and R 16 The element is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, and is more preferably hydrogen or methyl.

[0159]

[0159] Most preferably, the spacer portion Sp 1 and Sp 2 If present, they are independently linear or branched C1-C 20 Selected from the group consisting of alkylene groups, the alkylene groups are O, S and NR 16 It is optionally substituted and optionally interposed by one or more heteroatoms selected from the group, R 16 These are, independently, hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group and C3-C 24 The alkyl group is selected from the group consisting of cycloalkyl groups, and the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted. In this embodiment, the alkylene group is unsubstituted and consists of O, S, and NR 16 One or more heteroatoms selected from the group, preferably O and / or SS, are optionally interposed, and R 16 The element is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, and is more preferably hydrogen or methyl.

[0160]

[0160] Therefore, preferred spacer portion Sp 1 and Sp 2 As for, -(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 - is an integer in the range of 1 to 50, preferably 1 to 40, more preferably 1 to 30, even more preferably 1 to 20, and even more preferably 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.

[0161]

[0161] Alternatively, preferred linker L 1 is, -(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 -(K 1 ) f -]2BM-(C(O)) g -(K 1 ) d’ -(K 2 ) e’ -(K1 ) f’ -(C(O)) g’ - can be represented by, - d and d' are 0 or 1 respectively. - e and e' are integers in the range of 1 to 10, respectively. - f and f' are 0 or 1, respectively. - g and g' are integers in the range of 0 to 10, respectively. - k=0 or 1, but if k=1, then d=0. - K 1 Structure (23) [ka] It is a sulfamide group, - K 2 is either the -CH2-CH2-O- or -O-CH2-CH2- part, or (K 2 ) e is -(CH2-CH2-O) e1 -CH2-CH2- or -(CH2-CH2-O) e1 -CH2- is the part where e1 is defined in the same way as e, - K 3 -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 It is C(O)NH-, preferably K 3 -OC(O)NH-, -C(O)(CH2) m It is C(O)NH- or -C(O)NH-, where m is an integer in the range of 0 to 10, preferably m=0, 1, 2, 3, 4, 5, or 6, most preferably m=2 or 3. - Preferably, L 1 is, (K 3 ) k Q via (C(O)) gvia, preferably via C(O), L 2 , L 3 Or D, preferably L 2 It is connected to, - BM is preferably a branched portion selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)ring, or a polycyclic moiety, and more preferably BM is a nitrogen atom.

[0162]

[0162] In relation to this embodiment, the wavy line in structure (23) is (K 3 ) k , (K 2 ) e and (C(O)) g This represents a connection to adjacent bases such as K. 1 This is due to structure (23), where a=1 and R 13 =H or C1~C 20 It is an alkyl group, more preferably R 13 =H or methyl, most preferably R 13 It is preferable that =H

[0163]

[0163] Preferred Linker L 1 is structure-(K 3 ) k -(K 1 ) d -(K 2 ) e -(K 1 ) f -(C(O)) g - has, (a) k=0, d=1, g=1, f=0, K 2 =-CH2-CH2-O-, e=1, 2, 3 or 4, preferably e=2. (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, (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. (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, (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, (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, (g) k=0, d=0, g=1, f=0, K 2 =-CH2-CH2-O-, e=1, 2, 3 or 4, preferably e=2. (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.

[0164]

[0164] Here, if d and / or f = 1, then a = 1 and R 13 It is preferable that =H. Most preferably, the linker is structure (a).

[0165]

[0165] In a preferred embodiment, linker L 1 Q 1 or Z 1 and (L2 ) o Located in the skeleton between, and further part Z as substituent. 1 Or Q 1 It contains a branched nitrogen atom, 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 And R 13 This is linked to the second appearance of D via the spacer portion. Instead, the branched nitrogen atom is structure-(K 3 ) k -(K 1 ) d -(K 2 ) e -(K 1 ) f -(C(O)) g - by L 1 It may be located inside. In one embodiment, L 1 is, [(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 - is represented by K 1 , K 2 , K 3 d, e, f, g and k are as defined above and are individually selected for each occurrence, BM is a branched portion, preferably a branched nitrogen atom, and -(K 1 ) d -(K 2 ) e -(K 1 ) f -(C(O)) g - The two examples are linked. Here, both (C(O)) g The part is -(L 2 ) o -(L 3 ) p -(L4 ) q -Connected to D, L 2 , L 3 , L 4 o, p, q, and D are as defined above and are selected individually. In a preferred embodiment, L 2 , L 3 , L 4 Each of o, p, q, and D is (C(O)) g The same applies to both parts that are connected to it.

[0166]

[0166] Preferred linker L containing branched subatoms 1 is 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’ - has, (i) k=d=g=e'=1, f=d'=g'=0, K 3 =-C(O)-, K 2 =-CH2-CH2-O- and K 1 This is the case for a=0 and R 13 The structure (23) where =H, e=1, 2, 3, or 4, preferably e=2, (j) k=d=g=e'=g'=1, f=d'=0, K 3 =-C(O)-, K 2 =-CH2-CH2-O- and K 1 This is the case for a=0 and R 13 The structure (23) is such that =H, e=1, 2, 3, or 4, preferably e=2.

[0167] [Linker L 2 ]

[0167] Linker L 2 This is a peptide spacer. Linker L2 It is either nonexistent (o=0) or present (o=1). Peptide Spacer L 2 and a severable linker L 3 The combination is known in the art. Linker L 2 The peptide functions as a recognition and cleavage site for the cleavage enzyme, and more preferably, the peptide is recognized by a specific cleavage enzyme. This allows cleavage in specific environments where these cleavage enzymes are expressed, such as in certain tumors. Since various peptide sequences are cleaved by various enzymes, L 2 The base also makes it possible to customize the conjugate for specific treatments.

[0168]

[0168] Peptide spacer is (NH-CR 17 -CO) n It can also be defined by R 17 R represents an amino acid side chain known in this art. 17 Proline, which forms a cyclic portion by bonding with a nitrogen atom, is also included within this definition. Here, the amino acids may be natural or synthetic amino acids. Preferably, all amino acids are in the L configuration. n is an integer in the range of 1 to 5, preferably 2 to 4. Thus, the peptide spacer contains 1 to 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. 17 This preferably represents an amino acid side chain selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolicine, 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, and more preferably those derived from the side chains of Val, Cit, Ala, Glu, and Lys. In other words, R 17Preferably, it is 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(1H-indole-3-yl)(Trp). 17 Particularly preferred embodiments include CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu), and CH(CH3)2(Val). Most preferably, R 17 These are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0169]

[0169] Any peptide spacer can be used, but preferably the peptide spacer is 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-Al a, Phe-Phe, Gly, Gly-Gly, Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO: 19), Leu-Gly, Tyr-Gly, Ala-Gly, Pro-Gly, Phe-Gly, Ph e-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: 26), and Selected from Lys, more preferably Val-Cit, Val-Ala, Glu-Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably Glu-Gly-Cit, Val-Cit, Val-Ala, Asn-Asn, Ala-Ala-Asn, Asn-Ala, most preferably Glu-Gly-Cit, Val-Cit, Val-Ala, or Asn-Ala. Here, 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

[0170]

[0170] R 17 This preferably represents an amino acid side chain selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolicine, 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, and more preferably those derived from the side chains of Val, Cit, Ala, Glu, and Lys. In other words, R 17 Preferably, it is 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(1H-indole-3-yl)(Trp). 17 Particularly preferred embodiments include CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu), and CH(CH3)2(Val). Most preferably, R 17 These are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0171]

[0171] In one embodiment, the R of the amino acid side chain 17Preferably, the group is substituted with a polar group selected from oxo groups, (poly)ethylene glycol diamines, (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), phosphates or their esters, phosphonic acids or esters, phosphinic acids or esters, sulfoxides, sulfones, sulfonic acids or esters, sulfinic acids or sulfenic acids.

[0172]

[0172] In a particularly preferred embodiment, L 2 This includes a peptide spacer according to the general structure (25), preferably L 2 General structure (25): [ka] It is represented by [this].

[0173]

[0173] Here, R 17 This is as defined above, and preferably R 17 This is CH3(Ala) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line is (L 1 ) n and (L 3 ) p This indicates a connection to L by structure (25), preferably by structure (25). 2 (L 1 ) n It is connected to (L via C(O) 3 ) p It is connected to.

[0174]

[0174] L 2These may contain specific peptide sequences that can be cleaved by specific enzymes. When these enzymes are exclusively expressed or overexpressed in the tumor microenvironment or endosomal / lysosomal compartments and leak into the tumor microenvironment of cancer cells, there is an increased probability of targeted release of cytokines in tumors and a decrease in release in healthy tissues.

[0175] [Linker L 3 ]

[0175] Linker L 3 This is a self-cutting spacer, also known as a self-sacrificing spacer. Linker L 3 It either does not exist (p=0) or it exists (p=1). 2 The cutting of linker L 3 This leads to 1,6-β elimination, decarboxylation, and release of the payload, D. This is advantageous because L 2 The enzyme that can cleave the target receptor is overexpressed in the region, increasing the probability of payload release. Furthermore, payload release induces bystander death, which is advantageous for tumors where not all cancer cells overexpress the target receptor. Therefore, Linker L 2 and L 3 It is preferable that both conditions are present (o=p=1) or that neither condition is present (o=p=0).

[0176]

[0176] Preferably, L 3 This is a para-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (L3a). [ka]

[0177]

[0177] Here, the wavy line is L 1 or L 2 and L 4 Or indicates linkage to D. Parentheses indicate an optional carbonyl group. Preferably, a carbonyl group is present. Typically, PABC derivatives are linked to L via NH.1 or L 2 Preferably L 2 And via OC(O) L 4 Alternatively, it is linked to D.

[0178]

[0178] Ring A is a 5-membered or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Preferred 5-membered rings are oxazole, thiazole, and furan. Preferred 6-membered rings are phenyl and pyridyl. Ring A is a halogen, X 2 R 4 , N(R 4 )2, C 1~4 It can be substituted with substituents selected from alkyl and NO2. Here, X 2 and R 4 These, including their preferred embodiments, are as defined above. In preferred embodiments, any substituent may be F, Cl, Br, OH, OR 4 A is selected from SH, NH2, Et, Me, and NO2. In a particularly preferred embodiment, ring A contains 0 to 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.

[0179]

[0179] R 21 H, R 26 , C(O)OH and C(O)R 26 Selected from, R 26 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 These are (hetero)arylalkyl groups, which are O, S, and NR 28 Optionally substituted and optionally interposed by one or more heteroatoms selected from R 28is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Preferably, R 26 C3~C 10 It is a (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H is the most preferred polyalkylene glycol, preferably polyethylene glycol, preferably -(CH2CH2O) s H is an integer in the range of 1 to 10, preferably 1 to 5, most preferably s = 1, 2, 3, or 4. More preferably R 21 is H or C(O)R 26 And R 26 =4-methylpiperazine or morpholine. Most preferably, R 21 H is H.

[0180]

[0180] In an alternative embodiment, linker L 3 It exists (p=1), linker L 2 It does not exist (o=0), L 3 This is a para-glucuronide-meth-amide-benzyloxycarbonyl derivative, preferably with structure (L3b): [ka] It is a glucuronide derivative.

[0181]

[0181] Here, the wavy line is L 1 and L 4 Or indicates linkage to D. Parentheses indicate an optional carbonyl group. Preferably, a carbonyl group is present. Typically, the glucuronide derivative is linked to L via NH. 1 It is connected to L via (O)CO. 4 Alternatively, it is connected to D. Rings A and R 21R is defined similarly to the PABC derivatives by structure (L3a). Preferably, ring A is a six-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. More preferably, R 21 is H or C(O)R 26 And R 26 =4-methylpiperazine or morpholine. Most preferably, R 21 H is H.

[0182]

[0182] Linker L by structure (L3b) 3 Similar to the mechanism in PABC, it can be cleaved by β-glucuronidase, which results in the self-sacrifice of the para-hydroxybenzyloxy group, decarboxylation, and release of the payload. ADCs containing the glucuronide derivative according to structure (L3b) are particularly useful for treating cancers that overexpress β-glucuronidase. The concentration of β-glucuronidase in many solid tumors, including lung cancer, breast cancer, and gastrointestinal cancer, and in the tumor microenvironment has been reported to be higher than in normal tissue, and this enzyme is not found in systemic circulation. Therefore, the conjugate according to the present invention, containing the glucuronide derivative according to structure (L3b), is preferably used for the treatment of patients suffering from lung cancer, breast cancer, and gastrointestinal cancer.

[0183] [Linker L 4 ]

[0183] Linker L 4 It either does not exist (q=0) or it exists (q=1). Linker L 4 teeth, - Structure-NR 22 -(C x -alkylene)-C(O)-(wherein x is an integer in the range of 1 to 20, and R 22 (is H or C1-C4 alkyl) aminoalkanoic acid spacer, - Structure-NR 22 -(CH2-CH2-O) e6 -(CH2)e7 -C(O)-(where e6 is an integer in the range of 1 to 10, e7 is an integer in the range of 1 to 3, and R 22 (These are ethylene glycol spacers which are H or C1-C4 alkyl, and - Structure NR 22 -(SO2-NH-) j (C(O)) h -(In the formula, R 22 Amine spacer (where 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) Selected from.

[0184]

[0184] Linker L 4 This is an aminoalkanoic acid spacer, i.e., -NR 22 -(C x -Alkylene)-C(O)- (wherein x is an integer in the range of 1 to 20, preferably 1 to 10, most preferably 1 to 6). Here, the aminoalkanoic acid spacer is typically L via a nitrogen atom. 3 It is connected to and connected to D via the carbonyl portion. Preferred linker L 4 is selected from 6-aminohexanoic acid (Ahx, x=5), β-alanine (x=2), and glycine (Gly, x=1), and more preferably 6-aminohexanoic acid or glycine. In one embodiment, L 4 =6-aminohexanoic acid. 4 = Glycine. Here, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 H is H.

[0185]

[0185] Instead, Linker L 4 is structure-NR 22 -(CH2-CH2-O) e6 -(CH2) e7-(C(O)-(wherein e6 is an integer in the range of 1 to 10, preferably in the range of 2 to 6, and e7 is an integer in the range of 1 to 3, preferably e7 is 2) may be an ethylene glycol spacer. Here, R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 H is H.

[0186]

[0186] Instead, Linker L 4 is structure-NR 22 -(C x -Alkilen)-NR 22 -(C(O)) h - (wherein h is 0 or 1, x is an integer in the range of 1 to 20, preferably an integer in the range of 2 to 6, more preferably x=2 or 5, most preferably x=2) may be a diamine spacer. 22 R is H or C1-C4 alkyl. Here, 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 is methyl. Here, h is preferably 1, in which case linker L 4 This is particularly suitable for conjugation via phenolic hydroxyl groups present on payload D.

[0187] [Preferred linker]

[0187] Linker L is particularly preferably selected from one of the following structures. [ka]

[0188]

[0188] Here, x is preferably in the range of 0 to 100, preferably in the range of 0 to 50, more preferably in the range of 1 to 30, and most preferably in the range of 1 to 18. Here, parentheses "()" indicate the presence of any oxygen atom.

[0189]

[0189] In another preferred embodiment, α is preferably 0 and the linker L follows one of the following structures.

Chemical formula

[0190]

[0190] Here, L 2 is a peptide sequence that is a recognition site for a protease.

[0191] [Process for synthesizing a conjugate according to general structure (1) or (2)]

[0191] In a further aspect, the present invention is a process for preparing a conjugate according to the present invention, comprising - reacting AB(F 1 ) x with Q 1 -L-Q 2 or (Q 1 )2-L-Q 2 ; - reacting the resulting compound with F 2 -D or the following steps: - reacting D-F 2 with Q 1 -L-Q 2 or (Q 1 )2-L-Q 2 ; - reacting the resulting compound with AB(F 1 ) x ; where AB(F 1 ) x is a functional antibody containing x reactive moieties F 1 , and x is an integer in the range of 1 to 10, Q 1 and F 1 are mutually reactive with each other, and Q 2 and F 2 are mutually reactive with each other, and between Q 1 and F 1 and between Q2 and F 2 The reaction between the reactants forms a covalent bond between them.

[0192]

[0192] In a preferred embodiment, x = 2. The process according to this embodiment can be represented according to schemes 1 to 4, and preferably the process follows scheme 2 or 4.

[0193]

[0193] Scheme 1 [ka]

[0194]

[0194] Here, the immune cell-engaging polypeptide is represented by D. Reactive part Q 1 and F 1 Through a conjugation reaction between them, the linking group Z 1 A reactive part Q is obtained. 2 and F 2 Through a conjugation reaction between them, the linking group Z 2 You can obtain this.

[0195]

[0195] Alternatively, x=2 and the antibody-cytokine conjugate are obtained according to scheme 2.

[0196]

[0196] Scheme 2 [ka]

[0197]

[0197] In yet another embodiment, x=2, and the antibody-cytokine conjugate is obtained according to scheme 3.

[0198]

[0198] Scheme 3 [ka]

[0199]

[0199] Instead, x=2, and the antibody-cytokine conjugate is obtained according to scheme 4.

[0200]

[0200] Scheme 4 [ka]

[0201] [Reactive part Q]

[0201] Reactive part Q 1 and Q 2 These are F 1 and F 2 It is reactive to. In relation to the present invention, Q is Q 1 and Q 2 This refers to the reactive moiety. In relation to the present invention, the term "reactive moiety" may refer to a chemical moiety containing a reactive group, but may also refer to the reactive group itself. For example, the cyclooctinyl group is a reactive group, i.e., a reactive group containing a CC triple bond. However, a reactive group, such as an azide reactive group, may be referred to as a reactive moiety in this specification.

[0202]

[0202] Q is reactive and complementary to F. Here, a reactive group is indicated as "complementary" to a reactive group if the reactive group optionally reacts selectively with the reactive group in the presence of other functional groups. Complementary reactive moieties are known to those skilled in the art and will be described in more detail below. The exact properties of Q and F depend on the type of reaction used. The reaction may be a nucleophilic reaction such as Michael addition or nucleophilic substitution, or it may be a cyclization addition such as a click reaction. Click probes are reactive in cyclization addition (click reaction) and are preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, cydonones, alkene moieties, and alkyne moieties. Preferably, the click probe Q includes or is an alkene or alkyne portion, and more preferably the alkene is a (hetero)cycloalkene and / or the alkyne is a terminal alkyne or (hetero)cycloalkyne.

[0203]

[0203] Therefore, in a particularly preferred embodiment, Q includes a cyclic (hetero)alkyne moiety. The alkynyl group may also be called a (hetero)cycloalkynyl group, i.e., a heterocycloalkynyl group or a cycloalkynyl group, and the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptynyl group, a (hetero)cyclooctinyl group, a (hetero)cyclononinyl group or a (hetero)cyclodecynyl group. Here, the (hetero)cycloalkyne may be optionally substituted. Preferably, the (hetero)cycloalkynyl group is optionally substituted with a (hetero)cycloheptynyl group or optionally substituted with a (hetero)cyclooctinyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctinyl group, and the (hetero)cyclooctinyl group is optionally substituted.

[0204]

[0204] In a particularly preferred embodiment, Q comprises a (hetero)cycloalkynyl group or a (hetero)cycloalkenyl group, and structure (Q1): [ka] This is due to the following: - [ka] The bond shown as is a double bond or a triple bond. - R 15 These are, independently, hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)arene substituents, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, - Y 2 C(R 31 )2, O, S, S (+) R 31 S(O)R 31 , S(O)=NR 31 or NR 31 S (+) B (-) It is a cationic sulfur atom that is canceled out by B (-) is an anion, and each R 31 R 15It is either a connection with D connected via L, - u is 0, 1, 2, 3, 4 or 5, - u' is 0, 1, 2, 3, 4, or 5, and u + u' = 0, 1, 2, 3, 4, 5, 6, 7, or 8, v is an integer in the range of 0 to 16.

[0205]

[0205] Typically, v = (u + u') × 2 (the connection to L shown by the wavy line is Y) 2 (when via ) or [(u+u')×2]-1 (when the bond to L, indicated by the wavy bond, is via one of the carbon atoms of u and u').

[0206]

[0206] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cycloalkynyl group, and structure (Q1a): [ka] This is due to the following: - R 15 and Y 2 This is as defined above, - u is 0, 1, 2, 3, 4 or 5, - u' is 0, 1, 2, 3, 4, or 5, and u+u'=4, 5, 6, 7, or 8, v is an integer in the range of 8 to 16.

[0207]

[0207] In a preferred embodiment, u+u'=4, 5, or 6, and more preferably, u+u'=5.

[0208]

[0208] In a preferred embodiment, v = 8, 9, or 10, more preferably v = 9 or 10, and most preferably v = 10.

[0209]

[0209] In preferred embodiments, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2) to (Q20c) shown below herein. [Chemical]

[0210]

[0210] Here, the linkage to L shown by the wavy line connection can be any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14), and (Q15) can have a linkage to L, or contain a hydrogen atom or be optionally functionalized. B (-) is preferably (-) OTf, Cl (-) , Br (-) or I (-) and is an anion selected from, and most preferably, B (-) is (-) OTf. B (+) is a cation, preferably a pharmaceutically acceptable cation. In the conjugation reaction, since B (-) is exchanged with the anion present in the reaction mixture anyway, B (-) does not need to be a pharmaceutically acceptable anion. When (Q19) is used for Q, the negatively charged counterion is preferably pharmaceutically acceptable when the conjugate according to the present invention is isolated so that the conjugate can be easily used as a drug. R 36 is a halogen selected from fluorine, chlorine, bromine, and iodine, and preferably, R 36 is fluorine. Y 4 is a heteroatom, and preferably, Y 4 is O or NH. R 35 is hydrogen, a C1-C 24 alkyl group, a C3-C 24 cycloalkyl group, a C2-C 24 (hetero)aryl group, a C3-C 24 (hetero)aryl group, and a C3-C 24 (hetero)arylalkyl group, a C1-C 24 alkyl group, a C3-C 24 cycloalkyl group, a C2-C 24 (hetero)aryl group, a C3-C 24 alkyl(hetero)aryl group, and a C3-C24 (Hetero)arylalkyl groups are optionally substituted with O, Si, S, and NR 14 Optionally interposed by one or more heteroatoms selected from R 14 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably R 35 H, C5H 11 Selected from CH3, CH2CH3, CH2OH, or CH2OTBS.

[0211]

[0211] In a further preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21) to (Q38a) shown below herein. [ka]

[0212]

[0212] In the structure (Q38), B (-) Preferably, (-) OTf, Cl (-) , Br (-) or I (-) An anion selected from, most preferably B (-) teeth, (-) It is OTf. In the structure (Q28), B (-) is an anion, preferably a pharmaceutically acceptable anion. The group R on (Q38b), (Q38c), and (Q38d) 35 and R 36 This is defined elsewhere and applies equally to this embodiment.

[0213]

[0213] In preferred embodiments, Q preferably comprises a (hetero)cyclooctin moiety or (hetero)cycloheptine moiety of the structure (Q8), (Q26), (Q27), (Q28), (Q37), or (Q38a), which are optionally substituted. Each of these preferred options for Q is further defined herein below.

[0214]

[0214] Therefore, in a preferred embodiment, Q includes a heterocycloheptine moiety by structure (Q37), also known as TMTHSI, which is optionally substituted. Preferably, the heterocycloheptine moiety by structure (Q37) is not substituted.

[0215]

[0215] In alternative preferred embodiments, Q comprises a cyclooctin moiety of structure (Q8), more preferably (Q29), also known as a bicyclo[6.1.0]nona-4-in-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctin moiety of structure (Q8) or (Q29) is not substituted. In relation to this embodiment, Q is preferably a (hetero)cyclooctin moiety of structure (Q39) shown below (wherein V is (CH2) l Q 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, more preferably l is 0, 1 or 2, and most preferably l is 0 or 1. In relation to base (Q39), l is most preferably 1. Most preferably, Q is by a structure (Q42) as further defined below.

[0216]

[0216] In alternative preferred embodiments, Q comprises a (hetero)cyclooctin moiety of the structure (Q26), (Q27), or (Q28), also referred to as a DIBO, DIBAC, DBCO, or ADIBO group, which are optionally substituted. In relation to this embodiment, Q is preferably the structure (Q40) or (Q41) shown below (wherein Y 1 is O or NR 11 And R 11 These are, independently, hydrogen, linear or branched C1-C12 12 Alkyl alkyl group or C4-C 12The (hetero)cyclooctin moiety is selected from the group consisting of (hetero)aryl groups. The aromatic ring in (Q40) is optionally O-sulfonylated at one or more positions, while the ring in (Q41) can be halogenated at one or more positions. Preferably, the (hetero)cyclooctin moiety of structure (Q40) or (Q41) is not further substituted. Most preferably, Q is of structure (Q43), which is further defined below.

[0217]

[0217] In an alternative preferred embodiment, Q comprises a heterocycloheptynyl group, as shown in structure (Q37). [ka]

[0218]

[0218] In a particularly preferred embodiment, Q comprises a cyclooctinyl group, and structure (Q42): [ka] This is due to the following: - R 15 These are, independently, hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)arene substituents, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, - R 18 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, - R 19 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group is optionally interposed by one or more heteroatoms selected from the group consisting of O, N, and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are independently optionally substituted or R 19 This is a second appearance of Q or D connected via a spacer portion, and - l is an integer in the range of 0 to 10.

[0219]

[0219] In a preferred embodiment of the reactive group by structure (Q42), R 15 is hydrogen, halogen, -OR 16 , independently selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, R 16 is hydrogen or a C1-C6 alkyl group, more preferably R 15 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R 15 is H. In a preferred embodiment of the reactive group according to structure (Q42), R 18 R is independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably both R 18is H. In a preferred embodiment of the reactive group according to structure (Q42), R 19 is H. In a preferred embodiment of the reactive group according to structure (Q42), l is 0 or 1, and more preferably l is 1.

[0220]

[0220] In a particularly preferred embodiment, Q comprises a (hetero)cyclooctinyl group, and structure (Q43): [ka] This is due to the following: - R 15 These are, independently, hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These can be linked together to form optionally substituted cyclic cycloalkyl or optionally substituted cyclic (hetero)arene substituents, R 16 These are independently hydrogen, halogens, and C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, - Y is N or CR 15 And, - The carbon atoms in the fused aromatic ring may be replaced by nitrogen atoms as in (Q6a)~(Q6d), preferably Y is CR 15 That is the case.

[0221]

[0221] In a preferred embodiment of the reactive group by structure (Q43), R 15 is hydrogen, halogen, -OR 16 -S(O)3 (-) , independently selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, R 16 is hydrogen or a C1-C6 alkyl group, more preferably R 15 It consists of hydrogen and -S(O)3 (-) It is independently selected from the group consisting of the following. In a preferred embodiment of the reactive group according to structure (Q43), Y is N or CH, and more preferably Y=N.

[0222]

[0222] In alternative preferred embodiments, Q includes a cyclic alkene moiety. The alkenyl group Q may also be called a (hetero)cycloalkenyl group, i.e., a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, and the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a norbornene group, a norbornadiene group, a trans-(hetero)cycloheptenyl group, a trans-(hetero)cyclooctenyl group, a trans-(hetero)cyclononenyl group, or a trans-(hetero)cyclodecenyl group, all of which may be optionally substituted. Particularly preferred are a (hetero)cyclopropenyl group, a trans-(hetero)cycloheptenyl group, or a trans-(hetero)cyclooctenyl group, and the (hetero)cyclopropenyl group, trans-(hetero)cycloheptenyl group, or trans-(hetero)cyclooctenyl group may be optionally substituted. Preferably, Q includes a cyclopropenyl moiety according to structure (Q44), a heterocyclobutene moiety according to structure (Q45), a norbornene or norbornadiene group according to structure (Q46), a trans-(hetero)cycloheptenyl moiety according to structure (Q47), or a trans-(hetero)cyclooctenyl moiety according to structure (Q48). In this specification, Y 3 C(R 23 )2, NR 23 Or selected from O, each R 23These are, individually, hydrogen, C1-C6 alkyl, or optionally linked to L via a spacer and labeled. [ka] The bond is either a single bond or a double bond. In a further preferred embodiment, the cyclopropenyl group is structured as (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group is structured as (Q50) or (Q51). In yet another preferred embodiment, the trans-(hetero)cyclooctene group is structured as (Q52), (Q53), (Q54), (Q55) or (Q56). [ka]

[0223]

[0223] Here, the R group on Si in (Q50) and (Q51) is typically alkyl or aryl, preferably C1-C6 alkyl.

[0224]

[0224] In an alternative preferred embodiment, Q is a thiol-reactive probe. In this embodiment, Q is a reactive group suitable for cysteine ​​conjugation. Such probes are known in the art and may be selected from the group consisting of maleimide moieties, haloacetamide moieties, allenamide moieties, phosphoamidite moieties, cyanoethynyl moieties, vinylsulfone, vinylpyridine moieties, or methylsulfonylphenyloxadiazole moieties. Most preferably, Q comprises a maleimide moiety. The reagent may be monoalkylated or may be a crosslinking agent for reaction with two cysteine ​​side chains.

[0225]

[0225] In a preferred embodiment, the click probe QZ includes a portion selected from (Q1) to (Q56), and more preferably a portion selected from (Q1) to (Q56).

[0226] [Reactive part F]

[0226] Reactive part F1 and F 2 These are Q 1 and Q 2 It is reactive to. In relation to the present invention, F is F 1 and F 2 This refers to F, which is reactive and complementary to Q. Here, a reactive group is indicated as "complementary" to a reactive group if the reactive group optionally reacts selectively with the reactive group in the presence of other functional groups. Complementary reactive click probes are known to those skilled in the art and will be described in more detail below. The exact properties of Q and F depend on the type of click reaction used. Click probes are reactive in cycloaddition (click reaction) and are preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, cydonones, alkene moieties, and alkyne moieties. Preferably, click probe F contains an azide moiety, a nitrone moiety, or a tetrazine moiety, or is an azide moiety, a nitrone moiety, or a tetrazine moiety.

[0227]

[0227] F is reactive with Q in a conjugation reaction as defined below, preferably the conjugation reaction is a cycloaddition reaction or a nucleophilic reaction. As those skilled in the art will understand, the choices for F are the same as the choices for Q, except that F and Q are reactive with each other. Therefore, F preferably comprises a click probe, a thiol, a thiol-reactive moiety, an amine, or an amine-reactive 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 cycloaddition (click reaction) and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, cydonones, alkene moieties, and alkyne moieties. Preferably, the click probe contains or includes azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, ortho-quinone, dioxothiophene, or cydonone, most preferably azide. Typical thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, allenamide moieties, phosphoamidite moieties, cyanoethynyl moieties, ortho-quinone moieties, vinylsulfone, vinylpyridine moieties, or methylsulfonylphenyloxadiazole moieties. Most preferably, the thiol-reactive moiety contains or is a maleimide moiety. Typical amine-reactive moieties are selected from N-hydroxysuccinimidyl ester, p-nitrophenyl carbonate, pentafluorophenyl carbonate, isocyanate, isothiocyanate, and halogenated benzoyl. 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.

[0228]

[0228] The reactive group F on the antibody is typically introduced by a specific technique, e.g., (bio)chemical or genetic technique. The reactive group placed in the antibody is prepared by chemical synthesis, e.g., by azides or terminal alkynes. Methods for preparing modified antibodies are known in the art from, for example, International Publication Nos. 2014 / 065661, International Publication Nos. 2016 / 170186 and International Publication Nos. 2016 / 053107, which are incorporated herein by reference. From the same literature, the conjugation reaction between the modified antibody and the linker-toxin-construct is known to those skilled in the art.

[0229]

[0229] Preferably, F is a click probe that is reactive to (hetero)cycloalkenes and / or (hetero)cycloalkynes, and is typically selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes and cydonones. Preferred structures of the reactive group are the structures (F1) to (F10) shown below herein. [ka]

[0230]

[0230] Here, the wavy line connection represents a connection to AB or D. For (F3), (F4), (F8) and (F9), the payload can be connected to any one of the wavy line connections. Then, the other wavy line connection is hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Acyl group, C3~C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl group, C3-C 24 (hetero)arylalkyl groups and C1-C 24 They can be linked to R groups selected from sulfonyl groups, each of which (except hydrogen) is O, S, and NR. 32 Optionally substituted and optionally interposed by one or more heteroatoms selected from R32 The R group is independently selected from hydrogen and C1-C4 alkyl groups. Those skilled in the art will understand which R group can be applied to each of the groups F. For example, the R group linked to the nitrogen atom of (F3) may be selected from alkyl and aryl groups, and the R group linked to the carbon atom of (F3) may be selected from hydrogen, alkyl, aryl, acyl, and sulfonyl groups. Preferably, the reactive moiety F is selected from azide, nitrone, or tetrazine.

[0231]

[0231] In a particularly preferred embodiment, F is structure (F8a): [ka] It is tetrazine.

[0232]

[0232] Here, R 29 is 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 - Selected from the alphabet, R 33 H or C 1~4 It is alkyl. Preferably, R 29 R is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl. 29 It was found that having hydrogen yields optimal results in the reactivity of the cycloaddition reaction. Therefore, in preferred embodiments, the ring F, and especially F, are used. 2 is (F8a), and R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl, more preferably R 29 It is hydrogen.

[0233]

[0233] In a particularly preferred embodiment, F is structure (F3a) [ka] This is nitron.

[0234]

[0234] R 1 L 10 XR 4 Here, X is a heteroatom with a lone pair of electrons that can capture the imine intermediate (d) by reacting with the imine carbon atom (see Scheme 3). If this reaction forms a 5 or 6-membered ring, this capture of the imine intermediate (d) is efficient and halts the rearrangement reaction in Scheme 2. Therefore, L 10 It should be a linker of two or three carbon atoms.

[0235]

[0235] More specifically, L 10 is structure (C(R 3 )2) z The linker is (where z is 2 or 3). Each R 3 H and C 1~4 Individually selected from alkyl groups. Instead, R 3 The two appearances are bonded together, forming an oxo group or C 3~6 (Hetero)cycloalkyl groups may be formed. Preferably, L 10 The structure is CH2-C(R 3 )2 or CH2-CH2-C(R 3 )2 linker, more preferably L 10 =CH2-C(R 3 )2. R 3 When the two occurrences of these elements combine to form a spirobonded ring, such a ring is preferably a C3-C6 ring, more preferably a C4 or C5 ring.

[0236]

[0236] L 10 A preferred embodiment is (L 10 A)-(L 10 P): [ka] That is the case.

[0237]

[0237] Here, the wavy line bonds indicated by * are linked to the nitrogen atom of the nitron group, and the wavy line bonds indicated by * are linked to the XR 4 It is connected to. The ring (L) is L 10 It is a spiro linked to the skeletal atom of . The ring (L) is preferably a cyclobutyl ring or a cyclopentyl ring, most preferably a cyclobutyl ring. If ring L is a cyclobutyl ring, (L 10 A), (L 10 B), (L 10 C) and (L 10 G) is particularly preferred.

[0238]

[0238] X is S, O, or NH. Most preferably, X is O. 4 H, C 1~4 Selected from alkyl groups. Typically, R 4 If X is O or NH, then H and R 4 If X is S, then H or C 1~4 It is alkyl. Therefore, XR 4 These are typically OH, NH2, SH, and SC 1~4 Selected from alkyl groups. In preferred embodiments, XR 4 These are SH, OH, and NH2, most preferably XR 4 It is OH.

[0239]

[0239] R 2a and R 2b The properties of are not important to the present invention, and any suitable substituent for the nitrone compound can be used. 2a and R 2b They may be the same or different; typically, they are different. 2a and R 2b If different, the arrangement of the double bond between the nitrogen atom and carbon atom of the nitron group can be either the E configuration or the Z configuration. The exact configuration does not affect the operation of the present invention. Here, the linkage to AB or D is typically R 2a or R 2b It is mediated through.

[0240]

[0240] Typically, R 2a R is selected from H and C1-C6 (cyclo)alkyl. In a preferred embodiment, 2a is selected from H and C1-C5 (cyclo)alkyl, more preferably R 2a is H, Me, or Et, most preferably R 2a H is H.

[0241]

[0241] Typically, R 2b is hydrogen, C1~C 24 Alkyl alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl groups and C3-C 24 Selected from the group consisting of (hetero)arylalkyl groups, C1~C 24 Alkyl 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 O, S and NR 14 Optionally substituted and optionally interposed by one or more heteroatoms selected from R 14 R is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, and instead, 2a and R 2b These combine to form a (complex) ring part. In an alternative embodiment, R 2b is L(D) r (wherein r is an integer in the range of 1 to 10, and L is a linker that covalently bonds D to the nitron group). In further alternative embodiments, R 2b L 6 AB and L 6 This is a linker that covalently bonds AB to the nitrone group. Antibody AB, payload D, linker L and L 6 Furthermore, preferred embodiments of the integer r are defined elsewhere.

[0242]

[0242] In a preferred embodiment, R 2b is hydrogen, C1~C 20 Alkyl alkyl groups, preferably C1-C 16 Alkyl groups, fuaC1~C 10 Alkyl group, L(D) r or L 6 It is AB. Here, the alkyl group is O, S and NR 14 Optionally substituted and interposed by one or more heteroatoms selected from, preferably O, and R 14 R is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. In another preferred embodiment, R 2b This includes hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, and L(D) r or L 6 Selected from the group consisting of A and B, more preferably hydrogen, methyl, ethyl, n-propyl, i-propyl, L(D) r or L 6 Selected from the group consisting of A and B, and more preferably hydrogen, methyl, ethyl, L(D) r or L 6 The group is selected from the group consisting of A and B.

[0243]

[0243] It is particularly preferable that the nitrone compound according to the present invention is used in the preparation of the bioconjugate, and that antibody AB is covalently linked to payload D. Therefore, R 2b L(D) r or L 6 It is particularly preferable that it be AB. In one embodiment, R 2b is L(D) r The nitrone compound is coupled with a (hetero)cycloalkyne compound containing antibody AB. In one embodiment, R 2b L 6 B is the nitrone compound, which is coupled with the (hetero)cycloalkyne compound containing payload D. The nitrone is coupled to the payload, and therefore R 2b L(D) r It is particularly preferable that this be the case.

[0244]

[0244] In a preferred embodiment, the click probe F 1 and F 2 The group is selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, dioxothiophenes, cydonones, iminosidenones, and catechols. Note that catechols are oxidized in situ to an ortho-quinone group and react as click probes. Similarly, the term "tetrazine" also includes "hydrotetrazine," a known precursor that forms tetrazine upon oxidation in situ. Such precursors of click probes that form reactive groups in situ are also included in the present invention. Figure 12 shows known examples of click probes formed by oxidation in situ. In preferred embodiments, F 1 is Azid, F 2 is an iminosideonone, a catechol that forms an ortho-quinone group in situ, or a tetrazine. More preferably, F 2 This is an iminosideonone with structure (F7), a catechol that forms structure (F10) in situ, or a tetrazine with structure (F8). Most preferably, F 1 This is an azide due to structure (F1), and F 2 This is tetrazine according to structure (F8a).

[0245]

[0245] In the present invention, Q 1 and Q 2 The exact structure is different, and as a result, Q 1 is, F 2 There was no response to Q, on the other hand. 2 is F 2 It is responsive to that.

[0246]

[0246] In a preferred embodiment, F 1 is Azid, Q 1 F is a benzocyclized or tetramethylated (hetero)cycloalkyne, while F 2 It is tetrazine, and Q 2 This is bicyclononine. Here, Q1 Preferably, the structure is (Q5), (Q6), (Q7), (Q11), (Q17), (Q18), (Q19), or (Q19a), more preferably (Q26), (Q27), (Q28), (Q32), (Q37), (Q38), or (Q38a), and most preferably (Q40), (Q41), or (Q43). Here, Q 2 Preferably, this is due to structure (Q8), more preferably to structure (Q29), and most preferably to structure (Q42).

[0247]

[0247] In another preferred embodiment, F 1 is Azid, Q 1 Q is a cycloalkyne, 2 is trans-cyclooctene, F 2 It is nitrone or tetrazine.

[0248]

[0248] In a preferred embodiment, DF 2 The product is obtained by a ligase-mediated step, preferably the ligase being tubulintyrosine ligase, transglutaminase, lipoic acid ligase, farnesyltransferase, glycosyltransferase, formyl-glycine-producing enzyme (FGE), or tripsyligase / subtillol ligase, and more preferably the ligase being saltase. Preferably the saltase-mediated step is as follows. *D-LPX 1 TG·X 2 n +(G) n’ -F 2 →*D-LPX 2 T · (G) n’ -F 2 , or F 2 -LPX 1 TG·X 2 n +(G) n’ -D*→F 2 -LPX 2 T · (G)n’ -D

[0249]

[0249] Here, D* is the remainder of D, L is leucine, P is proline, T is threonine, and G is glycine. X 1 is any amino acid, X 2 is any hydrophobic amino acid, n is an integer in the range of 1 to 20, preferably n is 3 to 15 (SEQ ID NOs: 27 to 30). 2 This may include PEG chains. More preferably, LPX 1 TGX 2 n LPETGGH 10 (Sequence ID 31), where n' is 3. In a particularly preferred embodiment, F 2 It is an azide, and conjugation proceeds either via a SPAAC reaction or F 2 It is tetrazine, and the conjugation proceeds via a reverse-requested Diels-Alder reaction.

[0250]

[0250] In another preferred embodiment, F is nitron and DF 2 This is obtained by converting the terminal serine or threonine to a nitrone. The conversion to a nitrone can be achieved by oxidizing the serine or threonine side chain and then converting it to a nitrone, as shown below. [ka]

[0251]

[0251] Such conversion of serine or threonine to nitrones can be carried out by conventional chemical conversion. Here, [ox] is an oxidizing agent such as NaIO4. R is R as defined above with respect to (F3a). 1 Corresponds to R. Its exact properties are irrelevant to the present invention, but preferably R as described above. 1It is defined as follows. The conjugate obtained in this way has improved stability because it can displace into a stable conjugate as explained above.

[0252]

[0252] The oxidation of terminal amino acids, conversion to nitrones, and conjugation to linkers are particularly preferably carried out in a one-pot synthesis. Preferably, Q 2 is trans-cyclooctene, or Q 2 It is a cycloalkyne, and the conjugation reaction is strain-enhanced alkyne-nitrone cycloaddition (SPANC).

[0253] [Application]

[0253] The conjugate of the present invention is suitable for medical procedures, and is particularly suitable for the treatment of cancer or autoimmune diseases. Alternatively or additionally, the conjugate of the present invention can be used to treat or reduce hematological disorders such as neutropenia, anemia and thrombocytopenia. Such hematological disorders are common side effects of treatment with immune cell engagers such as IL-15. The conjugate according to the present invention avoids serious side effects typically associated with treatment with immune cell engagers. Common side effects include nausea, vomiting, abdominal pain, fatigue, leukopenia, neutropenia and thrombocytopenia, which occur when immune cell engagers are shielded by the stereobulk of the antibody and activated only upon cleavage at the target site.

[0254]

[0254] Accordingly, in a preferred embodiment, the present invention further relates to a method for treating cancer, comprising administering a conjugate according to the present invention to a subject in need thereof. The subject in need thereof 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 present invention is particularly suitable in this respect. The method described is typically suitable for the treatment of cancer. In the method according to this embodiment, the antibody conjugate is typically administered in a therapeutically effective dose. A preferred dose for administration of the conjugate according to the present invention is in the range of 3 to 20 mg per kg of body weight, every 3 weeks, or every 2 weeks, or every week, preferably every 3 weeks. This embodiment of the present invention may also be expressed as a conjugate according to the present invention for use in the treatment of cancer. In other words, this embodiment relates to the use of a conjugate according to the present invention for preparing a drug or pharmaceutical composition for use in the treatment of cancer. In this regard, the treatment of cancer is assumed to encompass treating tumors, imaging them, diagnosing them, preventing tumor growth, suppressing tumors, and reducing tumors.

[0255]

[0255] This aspect of the present invention is a method for targeting tumor cells expressing a specific extracellular receptor, comprising contacting a conjugate according to the present invention with cells that may express the extracellular receptor, wherein the antibody specifically targets the extracellular receptor, and the method may also be expressed as a method. 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 present invention to a subject that requires it. Alternatively, the method may be carried out ex vivo or in vitro. In a preferred embodiment, cells that may optionally express the extracellular receptor are cells that express the extracellular receptor. Targeting tumor cells preferably includes one or more of treating tumor cells, preventing their proliferation, inhibiting and reducing their growth.

[0256]

[0256] In relation to diagnosis, it is typically unclear whether the cells being contacted actually express the specific extracellular receptor being examined. For example, in the diagnosis of HER2-positive breast cancer, a conjugate containing an antibody that targets HER2, such as trastuzumab, may be contacted with cells. If the tumor cells actually express HER2, the conjugate will target the cells; however, if the tumor cells do not express HER2, the conjugate will not target the cells. Similarly, in the treatment of cancer cells that specifically express an extracellular receptor, those skilled in the art will understand that a cell-binding agent, such as an antibody that targets that specific extracellular receptor, should be used.

[0257]

[0257] In the method of the present invention, the extracellular receptors are 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, Brevican, c-KIT (CD117), c-Met, C4.4a (LYPD3), CA-IX (CA9) / MN, Cadherin-6, CanAg, CCR7, CD11 7(c-KIT), CD123(IL-3Rα), CD13, CD133, CD138 / syndecan-1, CD166(ALCAM), CD19, CD20, CD203C, CD205, Ly75, CD21, CD22, CD228(P79, SEMF), CD25(IL- C D70, CD71(TF-R), CD72, CD74, CD79a, CD79b, CDH6, Cadherin-6, CEACAM5(CD66e), Claudine, CLDN18.2, CLDN6(Claudine-6, Skullin), CLEC12A, CLL-1 / CLEC12A, Crypt, CS1(SLAMF7, CD319), CXCR5, DKL-1, DLL3(Delta-like 3), DPEP3, E16, EGFR, EGFRvIII, ENPP3, CD203c(AGS-16), EpCAM, EphA2, EphB2R, Ephrin-A4(E FNA4), ETBR, FAP, FGFR2, FGFR3, Fibronectin EDB, FLT3, FOLR1 (FR-a), Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, GloboH, GPC3 (Glypican-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, Nectin-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 The antibody is selected from the group consisting of 5b, SEZ6, SLITRK6 (SLC44A4), STEAP1, STEAP2, STn, TAG72, TENB2, TF (CD142, thromboplastin), TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, TNF-α, TROP-2 (TACSTD2), TWEAKR, receptor tyrosine kinase (RTK), and tenascin, or the antibody is specific to extracellular proteins and / or tumor-associated glycogen antigens (TACAs) resulting from viral infection. Similarly, it is preferable 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-binding agent capable of targeting that extracellular receptor. In preferred embodiments, the extracellular receptor is selected from PD-L1, HER2, and nectin-4, more preferably from PD-L1 and HER2.

[0258]

[0258] The conjugates of the present invention are particularly suitable as antibiotics, antiviral agents, anti-inflammatory agents, and anti-autoimmune agents. Therefore, in alternative embodiments, the present invention relates to a method for treating inflammation of infectious diseases and autoimmune diseases, comprising administering the conjugates according to the present invention to a subject in need thereof. In this embodiment, the antibody conjugate is typically administered in a therapeutically effective dose. This embodiment of the present invention may also be expressed as the conjugates according to the present invention for use in the treatment of inflammation of infectious diseases and autoimmune diseases. In other words, this embodiment relates to the use of the conjugates according to the present invention for preparing agents or pharmaceutical compositions for use in the treatment of inflammation of infectious diseases and autoimmune diseases, where the infection may be bacterial or viral.

[0259]

[0259] In this embodiment, the antibody is preferably specific to an extracellular protein resulting from a viral infection and / or tumor-associated glycosylation antigen. Here, the extracellular protein resulting from a viral infection may be human poliovirus (HPV), human cytomegalovirus (HCMV), or human papillomavirus (HPV). Tumor-associated glycosylation antigens (TACAs) include Tn, STn, T-antigen, LDN, Lewis c (Le c ), Ciaril-Lewis c (SLe c ), 6-Sialil-Lewis c (6SLe c ), LN, alpha-Gal, 3SLN, 6SLN, H antigen, A antigen, B antigen, Lewis a (Le a ), Ciaril-Lewis a (SLe a ), 6-Sialil-Lewis a (6SLe a ), Lewis b (Le b ), Ciaril-Lewis b (SLe b ), 6-Sialil-Lewis b (6SLe b ), Lewis x (Le x ), Ciaril-Lewis x (SLex ), 6-Sialil-Lewis x (6SLe x ), Lewis y (Le y ), Ciaril-Lewis y (SLe y ), 6-Sialil-Lewis y (6SLe y ) and / or combinations thereof may be selected. The antibody may also be specific to both extracellular proteins and TACA simultaneously.

[0260]

[0260] In preferred embodiments, the present invention relates to conjugates for use in the treatment of autoimmune diseases. Those skilled in the art will understand which combinations of target and payload are most suitable for a particular autoimmune disease. Cytokines may have inflammatory activity, such as IL-17 and interferon-γ, or they may have modulating effects in inflammation, such as IL-10 and TGF-β. In the treatment of autoimmune diseases, the conjugate of the present invention preferably contains a modulating cytokine, more preferably the cytokine is selected from the group consisting of IL-4, IL-6, IL-9, IL-10, IL-11 and IL-13, IFN-α and TGF-β. Alternatively, the conjugate contains an antibody that specifically targets abnormal cells causing the autoimmune disease, wherein the cytokine is preferably pro-inflammatory, and wherein 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.

[0261]

[0261] From this viewpoint, the present invention also relates to a pharmaceutical composition comprising a conjugate according to the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition typically contains the conjugate according to the present invention in a pharmaceutically effective dose. [Brief explanation of the drawing]

[0262] [Figure 1]This shows a representative (but not exhaustive) set of functional groups (F) in biomolecules, which are either naturally occurring or introduced by genetic engineering, and which generate linking groups Z upon reaction with reactive groups. Functional groups F can be artificially introduced (manipulated) into biomolecules at any selected site. The pyridazine linking group (underline) is formed during the reaction of tetrazine with alkynes and is a product of rearrangement of a tetrazabicyclo[2.2.2]octane linking group, with N2 lost. Linking groups Z in structures (10a)-(10j) are preferred linking groups used in the present invention. [Figure 2] Preferred embodiments relating to cyclooctyne and reactive moiety Q suitable for metal-free click chemistry are shown. This list is not exhaustive, and for example, alkynes may be further activated by fluorination, substitution of aromatic rings, or introduction of heteroatoms in aromatic rings. [Figure 3] The following are some structures of UDP sugar derivatives of galactosamine that can be modified, for example, with a 3-mercaptopropionyl group at position 2 (11a), an azidoacetyl group (11b), or an azidodifluoroacetyl group (11c), or with an azide group at position 6 of N-acetylgalactosamine (11d), or with a thiol group at position 6 of N-acetylgalactosamine (11e). The monosaccharide (i.e., with UDP removed) is the preferred partial Su used in the present invention. [Figure 4] This shows a general process for the non-genetic conversion of a monoclonal antibody to an antibody containing probes for click conjugation (F). The click probes can be located at various positions within the antibody, depending on the technique used. For example, the antibody may be converted to an antibody containing two click probes for click conjugation (left structure), four click probes (bottom structure), or eight probes (right structure). [Figure 5]This paper demonstrates 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 conjugate (Q) during the reaction, with the polypeptide being selected from any polypeptide capable of binding to immune cells, thereby forming a bispecific antibody. Polypeptide modification using a single click probe Q can be achieved by any selective genetic or non-genetic method. Probes for click conjugation can be selected from any suitable combination shown in Figure 1. The stoichiometry of the resulting bispecific antibody depends on the number of click probes F placed in the first modification of the antibody. Symmetrical bivalent IgG can be used (CDR1=CDR2), thus resulting in a bispecific antibody with a 2:2 molecular format (2× polypeptide conjugation). Asymmetric antibodies can also be used (CDR1≠CDR2), thus yielding a triplicate antibody with a 1:1:2 molecular format. If more than two click probes F are placed, the molecular format may change further, resulting in, for example, a 2:4 molecular format (4×F placed on a symmetric antibody) or a 1:1:8 molecular format (8×F placed on an asymmetric antibody). [Figure 6] Three alternative methods for placing a single immune cell-engaging polypeptide on a full-length antibody are shown (2:1 molecular format). Thus, the full-length antibody is initially modified with two click probes F. In one method (arrow below), IgG(F2) is subjected to a polypeptide modified with two complementary click probes Q linked via a suitable spacer, both of which react with the appearance of one F on the antibody. In a second method (arrow to the right), IgG(F2) is subjected to a trivalent construct containing three complementary probes Q, two of which react with IgG(F2), leaving one unit of Q free for subsequent reaction with the F-modified polypeptide. In a third method (arrow diagonal), IgG(F2) is subjected to a trivalent construct containing two complementary probes Q and one unreactive click probe F2 (also different from F). The two click probes Q react with IgG(F2), leaving F2 aside for a subsequent reaction with the Q2-modified polypeptide. [Figure 7] This paper presents a concrete example of forming a bispecific antibody in a 2:2 molecular format based on glycan remodeling of full-length IgG and azido-cyclooctin click chemistry. IgG is first enzymatically remodeled by endoglycosidase-mediated trimming of all different glycoforms, followed by glycosyltransferase-mediated transfer of azido sugars onto a core GlcNAc released by endoglycosidase. In the next step, the azido-modeled IgG is subjected to an immune cell-engaging polypeptide modified with single cyclooctin for metal-free click chemistry (SPAAC), resulting in a bispecific antibody in a 2:2 molecular format. The cyclooctin-polypeptide construct also exhibits a specific spacer between cyclooctin and the polypeptide, allowing for adjustment of the IgG-polypeptide distance or conferring other properties to the resulting bispecific antibody. [Figure 8] This figure illustrates how an azide-glyco-remodeled antibody can be converted to bispecificity in a 2:1 molecular format by first subjecting it to a trivalent cyclooctin construct suitable for clipping onto a bisazide antibody, releasing one cyclooctin for subsequent SPAAC using an azide-modified polypeptide, and effectively introducing only one polypeptide onto IgG. The latter polypeptide may also be modified with other complementary click probes for reaction with cyclooctin, such as a tetrazine moiety for reverse electron-demanded Diels-Alder cycloaddition. Any combination of F and Q (Figure 1) can be assumed here. [Figure 9]This paper presents various options for trivalent constructs for reactions with bis-azido sugar-modified mAbs. Trivalent constructs can be homotrivalent or heterotrivalent (2+1 format). A homotrivalent construct (X=Y) may consist of 3×cyclooctin, or 3×acetylene, or 3×maleimide, or 3×other thiol-reactive groups. A heterotrivalent construct (X≠Y) may consist, for example, two cyclooctin groups and one maleimide group or two maleimide groups and one trans-cyclooctene group. Heterotrivalent constructs can exist in any combination of X and Y, as long as X and Y do not react with each other (e.g., maleimide + thiol). [Figure 10] Various divalent BCN reagents (105, 107, 118, 125, 129, 134), trivalent BCN reagents (143, 145, 150), and monovalent BCN reagents for soltagging (154, 157, 161, 163, 168) are shown. [Figure 11] Various divalent or trivalent crosslinkers (XL01-XL13) are shown. [Figure 12] This shows various antibody variants as starting materials for subsequent conversion into antibody conjugates. [Figure 13] The structures of various variants of IL-15(PF18) or IL-15R-IL-15 fusion proteins (207, 208, PF26, and PF57-PF59, the Sushi domain of the IL-15 receptor) are shown, 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). Derivatives of these proteins, each equipped with a suitable click probe (BCN, tetrazine, or azide) or maleimide, are also shown, modified at their N-terminus to enable site-specific modification in each case. 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)). [Figure 14]The structure of an IL-15 variant with a non-cleavable spacer is shown. The peptide sequences correspond to SEQ ID NO: 5 (structure(PF18)), SEQ ID NO: 10 (structure(PF54)), and SEQ ID NO: 11 (structure(PF55)). Peptide sequence YR(G4S)3 (in structure(PF19)) corresponds to SEQ ID NO: 39, and peptide sequence R(G4S)3 (in structure(PF21)) corresponds to SEQ ID NO: 41. [Figure 15] The structure of hydroxylamine HO1-HO4 is shown. [Figure 16] The structures of IL-15 variants and IL-15 mutants (PF53 and PF56) with different N-terminal sequence tags (PF50-52) are shown. The peptide sequences correspond to sequence number 6 (structure (PF50)), sequence number 7 (structure (PF51)), sequence number 8 (structure (PF52)), sequence number 9 (structure (PF53)), and sequence number 12 (structure (PF56)). [Figure 17A] The structures of various nitrone variants of the IL-15R-IL-15 fusion protein (PF57) obtained by introducing hydroxylamine HO1-HO4 are shown. Peptide sequence YR(G4S)8 corresponds to SEQ ID NO: 40 and peptide sequence YR(G4S)3 corresponds to SEQ ID NO: 39. [Figure 17B] The structures of various nitrone variants of different IL-15 mutants (PF18, PF50, PF51, PF53, and PF56) obtained by introducing hydroxylamine HO1 and HO2 are shown. Peptide sequence YR(G4S)8 corresponds to SEQ ID NO: 40 and peptide sequence YR(G4S3) corresponds to SEQ ID NO: 39. [Figure 18] This paper presents SDS-page analysis of various antibody-cytokine conjugates based on mPD-L1 and IL-15 variants PF51-PF54 and FPF56 under reducing conditions. [Figure 19] This shows RP-UPLC analysis under non-reducing conditions, demonstrating the formation of a 2:1 format between trastuzumab and IL-15 variants with different N-terminal sequences. [Figure 20]This shows RP-UPLC analysis under non-reducing conditions demonstrating the formation of a 2:1 format for IL-15 variants modified with trastuzumab and hydroxylamine HO2-HO4. [Figure 21] This shows the aggregation of antibody-cytokine conjugates after 0-5 freeze-thaw cycles, as measured by SE-HPLC. [Figure 22] This shows the cleavage of mPD-L1-v1a-218-NHO2-PF52(2:2) by matryptase. [Figure 23] This demonstrates the cleavage of various antibody-cytokine conjugates by matryptase. [Figure 24] This paper presents in vitro IL-15 activity assays of various intact antibody-cytokine conjugates. Matryptase-treated mPD-L1-v1a-218-NHO2-PF52(2:2) is included as a reference. [Figure 25] This paper presents in vitro IL-15 activity assays of various matriptase-treated antibody-cytokine conjugates. [Figure 26] The in vitro IL-15 activity of mPD-L1-v1a-218-NHO2-PF52(2:2) and mPD-L1-v1a-218-NHO2-PF56(2:2) with and without pretreatment with matryptase MT-SP1 is shown. Free PF52 is included for reference. [Figure 27] This 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. [Figure 28] This study demonstrates 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. Mice were subcutaneously administered single doses of 6 mg / kg (top graph), 12 mg / kg (middle graph), and 18 mg / kg (bottom graph). [Figure 29]This study demonstrates the in vivo tolerability of antibody-cytokine conjugates in female BALB / c mice. The conjugates consist of different IL-15 variants and different molecular formats (2:2 and 2:1). Mice were administered subcutaneously as a single dose at the indicated dose levels. [Figure 30] This study demonstrates in vivo efficacy in a mouse CT26 syngeneic colon cancer model. The 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 was monitored in mice. Data are plotted as mean + / - standard error (upper graph) and median tumor volume (lower graph). [Figure 31] This study demonstrates in vivo efficacy in a mouse CT26 syngeneic colon cancer model compared to antibody-cytokine conjugates based on different IL-15 variants and different molecular formats (2:2 and 2:1). In all cases, immune cytokines were administered at either 3 mg / kg or 6 mg / kg dose levels, as shown, on days 0 and 7. Tumor volume in mice was monitored. Data are plotted as mean + / - standard error (upper graph) and median tumor volume (lower graph). [Figure 32] This study demonstrates the in vivo efficacy of a mouse MC38 syngeneic colorectal cancer model, comparing vehicle-treated mice with mice treated with 3 mg / kg and 6 mg / kg mPD-L1-v1a-218-NHO2-PF52. Mice were administered at either 3 mg / kg or 6 mg / kg dose levels, as indicated, on days 0, 7, and 14 in all cases. Tumor volume was monitored in the mice. Data are plotted as mean + / - standard error of the mean.

[0263] [Examples]

[0294] The present invention will be explained by the following examples.

[0264] [Synthesis of hydroxylamine compounds] [Example 1. Synthesis of compound HO2]

[0295] 2-oxoacetic acid hydrate (1.24 g, 1 equivalent, 13.5 mmol) and hydroxylamine hydrochloride (936 mg, 1 equivalent, 13.5 mmol) were stirred in water (60 mL) at room temperature. 1 M NaOH was added to raise the pH to 5, and then sodium borohydride cyanohydride (2.12 g, 2.5 equivalents, 33.7 mmol) was added. The reaction mixture was stirred for 3 hours. The mixture was concentrated under vacuum, and the resulting crude was purified by crystallization with minimal water to obtain HO2 (754 mg, 8.28 mmol, 62%) as a white solid. ¹H NMR (400 MHz, D₂O) δ 3.77 (s, ¹H). ³C NMR (101 MHz, DMSO) δ 172.40, 55.10. [ka]

[0265] [Example 2. Synthesis of compound HO3] [ka]

[0296] To a solution of ammonium chloride in 3 mL of water (793 mg, 2.7 equivalents, 14.8 mmol), 2-nitroethane-1-ol (500 mg, 394 μL, 1 equivalent, 5.49 mmol) and methanol (12 mL) were added. The resulting solution was stirred, and zinc (729 mg, 2.03 equivalents, 11.1 mmol) was added in small amounts while maintaining a temperature below 65°C. The mixture was stirred at room temperature for 30 minutes and then filtered. The precipitate was washed with methanol and H2O. Hydrogen chloride in 1,4-dioxane (400 mg, 2.75 mL, 4 moles, 2 equivalents, 11.0 mmol) was added to the filtrate, and the solution was concentrated under vacuum. The resulting crude was triturated with IPA to remove insoluble NH4Cl crystals. The filtrate was concentrated under vacuum, and the resulting crude was dissolved in H2O. The aqueous phase was extracted with Et2O and siRNA to remove residual 2-nitroethane-1-ol. The aqueous phase was concentrated under vacuum to obtain HO3 (444 mg, 3.91 mmol, 71%) as a yellow oily substance. 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.

[0266] [Example 3. Synthesis of compound HO4] [ka]

[0297] To a solution of ammonium chloride in 3 mL of water (551 mg, 2.7 equivalents, 10.3 mmol), 1-(nitromethyl)cyclobutan-1-ol (500 mg, 1 equivalent, 3.81 mmol) and methanol (12 mL) were added. The resulting solution was stirred, and zinc (506 mg, 2.03 equivalents, 7.74 mmol) was added in small amounts while maintaining a temperature below 65°C. The mixture was stirred at room temperature for 15 minutes and then filtered. The precipitate was washed with methanol and H2O. Hydrogen chloride in dioxane (278 mg, 1.91 mL, 4 mol, 2 equivalents, 7.63 mmol) was added to the filtrate, and the solution was concentrated under vacuum. The resulting crude was triturated with IPA to remove insoluble NH4Cl crystals. The filtrate was concentrated under vacuum, and the resulting crude was dissolved in H2O. The aqueous phase was extracted with Et2O and ELISA to remove residual 1-(nitromethyl)cyclobutan-1-ol. The aqueous phase was concentrated under vacuum to obtain HO4 (495 mg, 3.22 mmol, 85%) as a yellow oily substance. 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.

[0267] [BCN-compound synthesis] [Example 4. Synthesis of Compound 218] [ka]

[0298] To a solution of 2-[2-(2-aminoethoxy)ethoxy]ethane-1-amine (71 mg, 0.48 mmol, 1 equivalent) in anhydrous DCM (1 mL), BCN-OPNP (339.2 mg, 1.07 mmol, 2.2 equivalents) and triethylamine (100 μL, 0.71 mmol, 1.5 equivalents) were added. After stirring at room temperature for 3 hours, the reaction mixture was purified by flash column chromatography on silica gel (0% → 30% siRNA in DCM (to remove p-nitrophenol), followed by 0% → 20% MeOH in DCM) to obtain 218 as a colorless oil (191.5 mg, 0.36 mmol, 74%). LCMS (ESI+) calculated value for C48H41N2O6S+(M+H+) 501.63, actual value 501.56. 1H NMR(400MHz,CDCl3)δ(ppm)4.16(d,J=8.0Hz,4H),3.62(s,4H),3.57(dd,J=5.6,4.7Hz,4H),3. 40(q,J=5.4Hz,4H),2.36-2.17(m,12H),1.61(m,4H),1.37(p,J=8.6Hz,2H),1.01-0.90(m,4H).

[0268] [Example 5. Synthesis of Compounds 127 and 128] [ka]

[0299] To a solution of diethylene glycol 126 (446 μL, 0.50 g, 4.71 mmol) in DCM (20 mL), 4-nitrophenol chloroformate (1.4 g, 7.07 mmol) and Et3N (3.3 mL, 2.4 g, 23.6 mmol) were added. The mixture was stirred, filtered, and concentrated under vacuum (55°C). The residue was purified by silica gel chromatography (15% → 75% RINKAN in heptane) and two products were isolated. Product 127 was obtained as a white solid (511 mg, 1.17 mmol, 25%). 1H NMR (400MHz, 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 was obtained as a colorless oil (321 mg, 1.18 mmol, 25%). 1H NMR(400MHz,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).

[0269] [Example 6. Synthesis of Compound 145] [ka]

[0300] To a solution of 128 (200 mg, 0.45 mmol) in DCM (1 mL), triethylamine (41.6 μL, 0.30 mmol) and tris(2-aminoethyl)amine 144 (14.9 μL, 0.10 mmol) were added. The mixture was stirred for 150 minutes and then concentrated under vacuum. The residue was purified by silica gel column chromatography (25% → 100% HCl in DCM, then 0% → 10% MeOH in DCM) to obtain 145 (45.4 mg, 42.5 μmol) as a yellow oil in a 43% yield. 1H NMR (400MHz, CDCl3): δ(ppm)5.68-5.18(m,6H),4.32-4.18(m,6H),4.18-4.11(d,J=7.9Hz,6H),3.74-3.61(m,6H),3.61-3.51(m,6H),3.4 3-3.29(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=8.9Hz,3H),1.03-0.87(m,6H).

[0270] [Examples 7-10: Synthesis of Compound XL14] [ka] [Example 7. Synthesis of Compound 219]

[0301] To a solution of sulfo-D-alanine hydrate (554 mg, 1 equivalent, 2.96 mmol) and triethylamine (599 mg, 825 μL, 2 equivalents, 5.92 mmol) in DMF (15 mL), di-tert-butyl dicarbonate (775 mg, 1.2 equivalents, 3.55 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. The solvent was evaporated under vacuum. The residue was redissolved in 10 mL of CH2Cl2 and treated with Et2O (30 mL) with vigorous stirring. The organic solvent was decanted and discarded. The oily residue was washed with Et2O (20 mL) and dried under vacuum to obtain compound 219 (1134 mg, 2.9 mmol, 100%, purity 70%) as a colorless oil. The LCMS(ESI-) calculated value for C8H14NO7S-(MH-) was 268.1, and the measured value was 268.3.

[0271] [Example 8. Synthesis of Compound 220]

[0302] To a solution of compound 219 (100 mg, 1 equivalent, 371 μmol) in DMF (1 mL), 2,2'-(ethane-1,2-diylbis(oxy))bis(ethane-1-amine) (550 mg, 542 μL, 10 equivalents, 3.71 mmol), HATU (155 mg, 1.1 equivalents, 409 μmol), and DIPEA (144 mg, 194 μL, 3 equivalents, 1.11 mmol) were added. The reaction mixture was stirred for 18 hours, and then purified by reverse-phase chromatography (10% in H2O → 100% MeCN + 1% AcOH) to obtain compound 220 (42 mg, 0.11 mmol, 28%) as a colorless oil. The LCMS(ESI+) value for C14H30N3O8S+(M+H+) was calculated at 400.2, and the measured value was 400.5.

[0272] [Example 9. Synthesis of Compound 221]

[0303] To a solution of compound 220 (50 mg, 1 equivalent, 0.13 mmol) in DMF (1000 μL), TMTHSI-OSu (64 mg, 1.5 equivalents, 0.19 mmol) and triethylamine (51 mg, 70 μL, 4 equivalents, 0.5 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by reverse-phase chromatography (10% in H2O → 100% MeCN + 1% AcOH) to obtain a colorless oily substance, the Boc-intermediate (41 mg, 66 μmol, 52%). The calculated LCMS (ESI+) value for C25H45N4O10S2+(M+H+) was 625.3, and the measured value was 625.5.

[0273]

[0304] To a solution of Boc-intermediate (41.0 mg, 1 equivalent, 65.6 μmol) in DCM (1000 μL), TFA (150 mg, 101 μL, 20 equivalents, 1.31 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum to obtain compound 221 (41.9 mg, 65.6 μmol, 100%) as a colorless oil. The calculated LCMS(ESI+) value for C20H37N4O8S2+(M+H+) was 525.2, and the measured value was 525.5.

[0274] [Example 10. Synthesis of Compound XL14]

[0305] To a solution of compound 221 (41.9 mg, 1 equivalent, 65.6 μmol) in DMF (1000 μL), ((1R,8S,9s)-bicyclo[6.1.0]non-4-in-9-yl)methyl(2,5-dioxopyrrolidine-1-yl)carbonate (28.7 mg, 1.5 equivalents, 98.4 μmol) and triethylamine (26.6 mg, 36.6 μL, 4 equivalents, 262 μmol) were added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by silica gel flash column chromatography (0 → 30% MeOH / DCM) to obtain XL14 (11.9 mg, 17.0 μmol, 25.9%) as a white solid. The calculated LCMS (ESI+) value for C31H49N4O10S2+(M+H+) was 701.3, and the measured value was 701.5.

[0275] [Cytokine expression and isolation] [General Procedures for Mass Spectral Analysis of Cytokines, Antibodies, and Antibody-Cytokine Fusions]

[0306] IgG was treated with IdeS before mass spectrometry analysis. This enabled the analysis of Fc / 2 fragments. For Fc / 2 fragment analysis, a solution of 20 μg (modified) IgG was incubated with IdeS / Fabricator® (1.25 U / μL) in PBS (pH 7.4) in a total volume of 10 μL at 37°C for 1 hour. The sample was diluted to 80 μL using PBS (pH 7.4). For cytokine analysis, a solution of 4 μg (modified) cytokines was diluted to 80 μL using PBS (pH 7.4). MS analysis was performed using a JEOL AccuTOF LC-plus JMS-T100LP system (ESI-TOF) combined with an HPLC system (Agilent 1100 series, Hewlett Packard). The HPLC system was fitted with a MassPREP® online desalting cartridge (Waters P / N 186002785). For cytokine analysis, a 4 μg (modified) cytokine solution was diluted to 80 μL using PBS, followed by analytical electrospray ionization time-of-flight (ESI-TOF) on a JEOL AccuTOF. Deconvolution spectra were obtained using Magtran software.

[0276] [General Procedures for Cytokine Expression and Inclusion Body Isolation]

[0307] Cytokine expression was initiated by transformation into corresponding BL21(DE3) cells (Novagen). Transformed cells were seeded on LB agar containing ampicillin and incubated overnight at 37°C. Single colonies were harvested and used to inoculate into small cultures in TB medium + ampicillin, followed by incubation overnight at 37°C. The overnight cultures were then used to inoculate larger volumes by performing a 20-fold dilution in TB medium + ampicillin. The cultures were incubated at 37°C, 160 RPM, and induced with 1 mM IPTG (addition of 1 mL of 1 M stock solution) when the OD600 reached 2.5–4.0. After induction for over 16 hours at 37°C, 160 RPM, the cultures were pelletized by centrifugation (5000xg for 5 minutes). The cultured cell pellets were dissolved in BugBuster® containing benzoase and incubated on a roller bank at room temperature for 30 minutes. After dissolution, the insoluble fraction was separated from the soluble fraction by centrifugation (15 minutes, 15000xg). Half of the insoluble fraction was dissolved in BugBuster® (final concentration 200 pg / mL) containing lysozyme and incubated on a roller bank for 10 minutes. The solution was then diluted with 10 volumes of miliQ and centrifuged at 15000xg for 15 minutes. The pellet was washed with a 1:10 diluted BugBuster® solution and centrifuged at 12000xg for 10 minutes. This washing round was repeated 1 to 3 times until a very white inclusion pellet was obtained.

[0277] [General procedure for cytokine refolding]

[0308] The purified inclusion bodies were denatured by dissolving them in 5M guanidine (pH 8.0) containing 40 mM cysteamine and 20 mM Tris, to a final concentration of 1 mg / mL. The solution was then incubated on a roller bank at room temperature for 2 hours. Subsequently, 10 volumes of refolding buffer (pH 8.0, 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) were added dropwise in a 4°C cold room, stirring as needed. The solution was left to stand at 4°C for at least 24 hours. The solution was dialyzed using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO to 10 mM NaCl and 20 mM Tris (pH 8.0) for one overnight period and two 4-hour periods. The refolded product was loaded onto an equilibrated Q-trap anion exchange column (GE Healthcare) of 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) with a 60 mL gradient from buffer A to buffer B. Mass spectrometry was performed for product analysis.

[0278] [Example 11. Design of the IL-15 variant]

[0309] We designed cleavable IL-15 variants consisting of a chemically modifiable N-terminal sequence comprising either (M)SYR-, (M)ST-, or (M)S- (amino acid sequences identified by SEQ ID NOs: 1 and 2) fused to a protease-cleavable linker, and either wild-type or mutant human IL-15 (the amino acid sequence of wild-type IL-15 is identified by SEQ ID NO: 3). The N-terminal methionine is cleaved after expression, leaving the N-terminal serine required for chemical modification. Both cleavable linkers are designed for recognition by several tumor-specific proteases, including matryptase (MT-SP1), regmine, and urokinase (UpA). Non-cleavable variants were designed by replacing the cleavable linker with either the -(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). Mutant IL-15 includes the N72D mutation, which enhances IL-15Rβ-binding (amino acid sequence identified by SEQ ID NO: 8), and the E46G and V49R mutations, which lack IL-15Rα-binding (amino acid sequence identified by SEQ ID NO: 11).

[0279] [Example 12. Gene synthesis and cloning into an expression vector]

[0310] The codon-optimized DNA 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 expression of the protein in Escherichia coli (E. coli).

[0280] [Example 13. Expression and refolding of cytokines PF18 and PF50-PF59]

[0311] Expression and inclusion body isolation for each cytokine listed in Table 1 were performed according to general procedures for cytokine expression and inclusion body isolation. Cytokine refolding was performed according to general procedures for cytokine refolding with minor adaptations. Refolding was successfully performed for cytokines PF51, PF52, PF53, PF54, and PF56. For cytokines PF18, PF50, and PF55, the cytokines were denatured by dissolving them in 5M guanidine (pH 8.0) containing 40mM cysteamine and 20mM tris, resulting in a final concentration of 2 mg / mL instead of 1 mg / mL. For cytokines PF57 and PF59, the cytokines were denatured by dissolving them in 5M guanidine (pH 8.0) containing 40mM cysteamine and 20mM tris, resulting in a final concentration of 3 mg / mL instead of 1 mg / mL. For cytokine PF58, the cytokine was denatured by dissolving it in 5M guanidine (pH 8.0) containing 40 mM cysteamine and 20 mM tris, and the final concentration was listed as unknown instead of 1 mg / mL.

[0281] [Table 1]

[0282] [Preparation of Modified Cytokines] [Example 14. N-terminal nitrone functionalization of PF18 to obtain NHO1-PF18]

[0312] IL15 PF18 (8070 μL, 50 μM in PBS) was mixed with 2 equivalents of NaIO4 (16.4 μL of 50 mM stock solution in PBS). The reaction mixture was incubated at 4°C for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 14109 Da). The reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and eluted using PBS. To the eluate (11500 μL, 32 μM in PBS), 160 equivalents of NHO1 (558 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (558 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at 25°C for 3 hours. Mass spectrometry showed a single peak corresponding to NHO1-PF18 (measured mass 14119 Da). The reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva), and eluted using PBS.

[0283] [Example 15. N-terminal nitrone functionalization of PF50 to obtain NHO1-PF50]

[0313] PF50 (2500 μL, 80 μM in PBS) was mixed with 4 equivalents of NaIO4 (7.8 μL of 100 mM stock solution in PBS). The reaction mixture was incubated at 4°C for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 15294 Da). The reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and eluted using PBS. To the eluate (3500 μL, 50 μM in PBS), 160 equivalents of NHO1 (308 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (308 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated overnight at 25°C. Mass spectrometry showed a single peak corresponding to NHO1-PF50 (measured mass 15306 Da). The reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva), and eluted using PBS.

[0284] [Example 16. N-terminal nitrone functionalization of PF51 to obtain NHO1-PF51]

[0314] Two equivalents of NaIO4 (68.5 μL of 100 mM stock solution in PBS) were added to PF51 (56100 μL, 61 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 14594 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (2260 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO1 (5500 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (5500 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO1-PF51 (measured mass 14604 Da). The reaction mixture was dialyzed to PBS once overnight and twice for 3 hours using Spectrum®Spectra / Por®2 RC Dialysis Membrane Tubing 12-14 kDa MWCO.

[0285] [Example 17. N-terminal nitrone functionalization of PF55 to obtain NHO1-PF55]

[0315] Two equivalents of NaIO4 (16.5 μL of 100 mM stock solution in PBS) were added to PF55 (12807 μL, 61 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 15683 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (546 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO1 (1366 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (1366 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO1-PF55 (measured mass 15694 Da). The reaction mixture was loaded directly onto a desalting column (HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase in an AKTA explorer (GE Healthcare).

[0286] [Example 18. N-terminal nitrone functionalization of PF57 to obtain NHO1-PF57]

[0316] Two equivalents of NaIO4 (11 μL of 100 mM stock solution in PBS) were added to PF57 (8000 μL, 69 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 25707 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (364 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO1 (870 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (870 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO1-PF57 (measured mass 25720 Da). The reaction mixture was loaded directly onto a desalting column (HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase in an AKTA explorer (GE Healthcare).

[0287] [Example 19. N-terminal nitrone functionalization of PF58 to obtain NHO1-PF58]

[0317] Two equivalents of NaIO4 (18.8 μL of 10 mM stock solution in PBS) were added to PF58 (1443 μL, 65 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 25388 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (62 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO1 (150 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (150 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO1-PF58 (measured mass 25401 Da). The reaction mixture was loaded directly into a Superdex® 75 Increase (Cytiva) using PBS (pH 7.5) as the mobile phase in an AKTA explorer (GE Healthcare).

[0288] [Example 20. N-terminal nitrone functionalization of PF59 to obtain NHO1-PF59]

[0318] Two equivalents of NaIO4 (6 μL of 10 mM stock solution in PBS) were added to PF59 (1000 μL, 30 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 25494 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (19.8 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO1 (49 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (49 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO1-PF59 (measured mass 25503 Da). The reaction mixture was purified by performing five rounds of buffer exchange to PBS (pH 7.5) using an Amicon Ultra-0.5 centrifugation filter unit (10 kDa MWCO).

[0289] [Example 21. N-terminal nitrone functionalization of PF52 to obtain NHO2-PF52]

[0319] Two equivalents of NaIO4 (10 mM stock solution in PBS, 1113 μL) were added to PF52 (88679 μL, 61 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 14375 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (3672 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO2 (100 mM stock solution in PBS, 8901 μL) and 160 equivalents of p-anisidine (100 mM stock solution in PBS, 8901 μL) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO2-PF52 (measured mass 14429 Da). The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 7.5) for 1 hour twice using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO. For purification, the dialyzed product was directly loaded onto an AEX column (Cytiva 2x5 mL, HiTrap® Q HP) using an AKTA explorer (GE Healthcare). After loading the product, the column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 7.5). The retained protein was eluted with a linear gradient to buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). The eluate was directly loaded onto a desalting column (Cytiva, HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase with an AKTA explorer (GE Healthcare).

[0290] [Example 22. N-terminal nitrone functionalization of PF53 to obtain NHO2-PF53]

[0320] Two equivalents of NaIO4 (278 μL of 10 mM stock solution in PBS) were added to PF53 (22170 μL, 61 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 14376 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (918 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO2 (2225 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (2225 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO2-PF53 (measured mass 14430 Da). The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 7.5) for 1 hour twice using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO. For purification, the dialyzed product was directly loaded onto an AEX column (Cytiva 2x5 mL, HiTrap® Q HP) using an AKTA explorer (GE Healthcare). After loading the product, the column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 7.5). The retained protein was eluted with a linear gradient to buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). The eluate was directly loaded onto a desalting column (Cytiva, HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase with an AKTA explorer (GE Healthcare).

[0291] [Example 23. N-terminal nitrone functionalization of PF54 to obtain NHO2-PF54]

[0321] Two equivalents of NaIO4 (85 μL of 100 mM stock solution in PBS) were added to PF54 (69660 μL, 61 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 14090 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (2807 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO2 (6800 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (6800 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO2-PF54 (measured mass 14146 Da). The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 7.5) for 1 hour twice using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO. For purification, the dialyzed product was directly loaded onto an AEX column (Cytiva 2x5 mL, HiTrap® Q HP) using an AKTA explorer (GE Healthcare). After loading the product, the column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 7.5). The retained protein was eluted with a linear gradient to buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). The eluate was directly loaded onto a desalting column (Cytiva, HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase with an AKTA explorer (GE Healthcare).

[0292] [Example 24. N-terminal nitrone functionalization of PF56 to obtain NHO2-PF56]

[0322] Two equivalents of NaIO4 (55.7 μL of 100 mM stock solution in PBS) were added to PF56 (45629 μL, 61 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 14359 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (1837 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO2 (4453 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (4453 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO2-PF56 (measured mass 143414 Da). The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 7.5) for 1 hour twice using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO. For purification, the dialyzed product was directly loaded onto an AEX column (Cytiva 2x5 mL, HiTrap® Q HP) using an AKTA explorer (GE Healthcare). After loading the product, the column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 7.5). The retained protein was eluted with a linear gradient to buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). The eluate was directly loaded onto a desalting column (Cytiva, HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase with an AKTA explorer (GE Healthcare).

[0293] [Example 25. N-terminal nitrone functionalization of PF57 to obtain NHO2-PF57]

[0323] Two equivalents of NaIO4 (16.3 μL of 10 mM stock solution in PBS) were added to PF57 (1350 μL, 61 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 25708 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (54 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO2 (133 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (133 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO2-PF57 (measured mass 25763 Da). The reaction mixture was purified by performing five rounds of buffer exchange to PBS (pH 7.5) using an Amicon Ultra-0.5 centrifugation filter unit (10 kDa MWCO).

[0294] [Example 26. N-terminal nitrone functionalization of PF57 to obtain NHO3-PF57]

[0324] Two equivalents of NaIO4 (16.3 μL of 10 mM stock solution in PBS) were added to PF57 (1350 μL, 61 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 25708 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (54 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO3 (133 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (133 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO3-PF57 (measured mass 25749 Da). The reaction mixture was purified by performing five rounds of buffer exchange to PBS (pH 7.5) using an Amicon Ultra-0.5 centrifugation filter unit (10 kDa MWCO).

[0295] [Example 27. N-terminal nitrone functionalization of PF57 to obtain NHO4-PF57]

[0325] Two equivalents of NaIO4 (16.3 μL of 10 mM stock solution in PBS) were added to PF57 (1350 μL, 61 μM in PBS). The reaction mixture was incubated at room temperature for 5 minutes. Mass spectrometry showed oxidation of serine to the corresponding hydrate (measured mass 25708 Da). To quench the remaining NaIO4 during the reaction, 6.6 equivalents of p-methoxybenzenethiol were added (54 μL, 10 mM in DMF). To the quenched reaction mixture, 160 equivalents of NHO4 (133 μL of 100 mM stock solution in PBS) and 160 equivalents of p-anisidine (133 μL of 100 mM stock solution in PBS) were added. The reaction mixture was incubated at room temperature overnight. Mass spectrometry showed a single peak corresponding to NHO4-PF57 (measured mass 25789 Da). The reaction mixture was purified by performing five rounds of buffer exchange to PBS pH 7.5 using an Amicon Ultra-0.5 centrifugation filter unit (10kDa MWCO).

[0296] [Example 28. Functionalization of N-terminal BCN of NHO1-PF51 by SPANC to obtain 218-NHO1-PF51]

[0326] 10 equivalents of BCN-PEG2-BCN218 (8562 μL, 4 mM in DMF) were added to NHO1-PF51 (77055 μL, 44 μM in PBS). The reaction mixture was incubated overnight at room temperature. The solution was dialyzed to 10 mM NaCl and 20 mM Tris (pH 8.0) for 1 hour twice using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO. For purification, the dialyzed product was directly loaded onto an AEX column (Cytiva 2x5 mL, HiTrap® Q HP) using an AKTA explorer (GE Healthcare). 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 via a linear gradient to buffer B (20 mM Tris buffer, 1 M NaCl, pH 8.0). The eluate was dialyzed to PBS using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO, once overnight and twice for 3 hours. Mass spectrometry revealed the exact product corresponding to 218-NHO1-PF51 (measured mass 15105 Da).

[0297] [Example 29. Functionalization of N-terminal BCN of NHO2-PF52 by SPANC to obtain 218-NHO2-PF52]

[0327] To NHO2-PF52 (173349 μL, 22 μM in PBS), 10 equivalents of BCN-PEG2-BCN218 (17335 μL, 2 mM in DMF) were added. The reaction mixture was incubated overnight at room temperature with stirring. The solution was dialyzed to 100 mM NaCl and 20 mM Tris (pH 7.5) for 1 hour twice using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO. For purification, the dialyzed product was directly loaded onto an AEX column (Cytiva 2x5 mL, HiTrap® Q HP) using an AKTA explorer (GE Healthcare). After loading the product, the column was first washed with buffer A (20 mM Tris, 100 mM NaCl, pH 7.5). The retained protein was eluted via a linear gradient to buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). The eluate was directly loaded onto a desalting column (Cytiva, HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase in an AKTA explorer (GE Healthcare). Mass spectrometry revealed the exact product corresponding to 218-NHO2-PF52 (measured mass 14929 Da).

[0298] [Example 30. Functionalization of N-terminal BCN of NHO2-PF53 by SPANC to obtain 218-NHO2-PF53]

[0328] To NHO2-PF3 (62408 μL, 22 μM in PBS), 10 equivalents of bisBCN-PEG2218 (6934 μL, 2 mM in DMF) were added. The reaction mixture was incubated overnight at room temperature with stirring. The solution was dialyzed to 100 mM NaCl and 20 mM Tris (pH 7.5) for 1 hour twice using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO. For purification, the dialyzed product was directly loaded onto an AEX column (Cytiva 2x5 mL, HiTrap® Q HP) using an AKTA explorer (GE Healthcare). After loading the product, the column was first washed with buffer A (20 mM Tris, 100 mM NaCl, pH 7.5). The retained protein was eluted via a linear gradient to buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). The eluate was directly loaded onto a desalting column (Cytiva, HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase in an AKTA explorer (GE Healthcare). Mass spectrometry revealed the exact product corresponding to 218-NHO2-PF53 (measured mass 14930 Da).

[0299] [Example 31. Functionalization of N-terminal BCN of NHO2-PF54 by SPANC to obtain 218-NHO2-PF54]

[0329] To NHO2-PF54 (160712 μL, 22 μM in PBS), 10 equivalents of bisBCN-PEG2218 (17857 μL, 2 mM in DMF) were added. The reaction mixture was incubated overnight at room temperature with stirring. The solution was dialyzed to 100 mM NaCl and 20 mM Tris (pH 7.5) for 1 hour twice using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO. For purification, the dialyzed product was directly loaded onto an AEX column (Cytiva 2x5 mL, HiTrap® Q HP) using an AKTA explorer (GE Healthcare). After loading the product, the column was first washed with buffer A (20 mM Tris, 100 mM NaCl, pH 7.5). The retained protein was eluted via a linear gradient to buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). The eluate was directly loaded onto a desalting column (Cytiva, HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase in an AKTA explorer (GE Healthcare). Mass spectrometry revealed the exact product corresponding to 218-NHO2-PF54 (measured mass 14646 Da).

[0300] [Example 32. Functionalization of N-terminal BCN of NHO1-PF55 by SPANC to obtain 218-NHO1-PF55]

[0330] 10 equivalents of bisBCN-PEG2218 (2000 μL, 5 mM in DMF) were added to NHO1-PF55 (22000 μL, 45 μM in PBS). The reaction mixture was incubated overnight at room temperature. The reaction mixture was loaded directly onto a desalting column (Cytiva, HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase in an AKTA explorer (GE Healthcare). Mass spectrometry revealed the exact product corresponding to 218-NHO1-PF55 (measured mass 16196 Da).

[0301] [Example 33. Functionalization of N-terminal BCN of NHO2-PF56 by SPANC to obtain 218-NHO2-PF56]

[0331] To NHO2-PF56 (93103 μL, 22 μM in PBS), 10 equivalents of bisBCN-PEG2218 (10345 μL, 2 mM in DMF) were added. The reaction mixture was incubated overnight at room temperature with stirring. The solution was dialyzed to 100 mM NaCl and 20 mM Tris (pH 7.5) for 1 hour twice using a Spectrum® Spectra / Por® 2 RC Dialysis Membrane Tubing 12-14 kDa MWCO. For purification, the dialyzed product was directly loaded onto an AEX column (Cytiva 2x5 mL, HiTrap® Q HP) using an AKTA explorer (GE Healthcare). After loading the product, the column was first washed with buffer A (20 mM Tris, 100 mM NaCl, pH 7.5). The retained protein was eluted via a linear gradient to buffer B (20 mM Tris buffer, 1 M NaCl, pH 7.5). The eluate was directly loaded onto a desalting column (Cytiva, HiPrep® 26 / 10 desalting column) using PBS (pH 7.5) as the mobile phase in an AKTA explorer (GE Healthcare). Mass spectrometry revealed the exact product corresponding to 218-NHO2-PF56 (measured mass 14914 Da).

[0302] [Preparation of modified antibodies] [General Procedures for Analytical RP-UPLC of Cytokines, Monoclonal Antibodies, and Antibody-Cytokine Conjugates]

[0332] 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 using a Waters Acquity UPLC-SQD. A sample (5 μL) was injected at 0.4 mL / min into a Bioresolve RP mAb 2.1 × 150 mm 2.7 μm (Waters) column at a column temperature of 70°C. A linear gradient was applied for 9 minutes with 0.1% TFA and 30–54% acetonitrile in water.

[0303] [General Procedure for SE-HPLC Analysis of Cytokines, Monoclonal Antibodies, and Antibody-Cytokine Conjugates]

[0333] HPLC-SEC analysis was performed using an Agilent 1100 series (Hewlett Packard) with an Xbridge BEH200A (3.5 μM, 7.8 x 300 mm, PN 186007640 Waters) column. Samples were diluted to 1 mg / mL in PBS and measured for 16 minutes using the isocratic method at 0.86 mL / min (0.1 M sodium phosphate buffer pH 6.9 (NaHPO4 / Na2PO4) containing 10% isopropanol).

[0304] [Example 34A. Transient expression of mPD-L1 in CHO]

[0334] mPD-L1 is an antibody that targets mouse PD-L1 as described in U.S. Patent Application Publication No. 2016 / 0340429A1, and consists of an LC sequence identified by SEQ ID NO: 16 and an HC sequence identified by SEQ ID NO: 17. mPD-L1 was transiently expressed in CHO Kl cells in 500 mL scale by Evitria (Zurich, Switzerland). The antibody was purified using two HiTrap MabSelect Sure 5 mL columns connected in series. After loading the supernatant, the columns were washed 10 CV with TBS (pH 7.5). 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 dialyze passes with 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), yielding a final yield of 87 mg.

[0305] [Example 34B. Transient reaction of enfortumab in CHO]

[0335] The nectin-4 targeted antibody enfortumab was transiently expressed in CHO Kl cells in 3 L scale 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 10 CV with TBS (pH 7.5). IgG was eluted with 0.1 M sodium acetate (pH 3.0) and neutralized with 2.5 M Tris-HCl (pH 7.2). After dialyzing three times with 20 mM histidine-HCl and 150 mM NaCl (pH 7.5), IgG was concentrated to 19.96 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) to obtain a final yield of 99 mg.

[0306] [Example 34C. Enzymatic remodeling of enfortumab (6-N3-GalNAc)2 (enfo-v1a)]

[0336] Enfortumab (3509 μL, 80 mg, 17.7 mg / mL in 20 mM histidine-HCl, 150 mM NaCl, pH 7.5) was incubated at 30°C for 16 hours in 20 mM histidine-HCl (pH 7.5), 150 mM NaCl (pH 7.5), and 6 mM MnCl2 with EndoSH (1 w / w%), His-TnGalNAcT (3 w / w%), alkaline phosphatase (commercially available from Roche, 0.01 w / w%), and UDP-6-N3-GalNAc (25 equivalents compared to IgG) prepared according to PCT / EP2016 / 059194, as described in PCT / EP2017 / 052792. Next, functionalized IgG was purified using two HiTrap MabSelect Sure 5 mL columns connected in series. After loading the reaction mixture, the columns were washed with TBS + 0.2% Triton for 10 CV, followed by TBS (pH 7.5) for another 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 dialyze passes to TBS (pH 7.5), IgG was concentrated to approximately 25 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (measured mass 24365 Da, approximately 90% of total Fc / 2) corresponding to the 6-N3-GalNAc-GlcNAc(Fuc)-substituted Fc / 2 fragment.

[0307] [Example 35. Enzymatic remodeling of trastuzumab against tras(6-N3-GalNAc)2 (tras-v1a)]

[0337] Trastuzumab (Herceptin) obtained from a pharmacy was reconstituted and buffered using a HiPrep® 26 / 10 desalting column (Cytiva) on an AKTA Purifier-10 (Cytiva) with 20 mM histidine-HCl and 150 mM NaCl (pH 7.5). Trastuzumab (31.3 mL, 1128 mg, 36.0 mg / mL in 20 mM histidine-HCl and 150 mM NaCl, pH 7.5) was incubated at 30°C for 16 hours in 20 mM histidine-HCl (pH 7.5), 150 mM NaCl (pH 7.5), and 6 mM MnCl2 with EndoSH (1 w / w%), His-TnGalNAcT (3 w / w%), alkaline phosphatase (commercially available from Roche, 0.01 w / w%), and UDP-6-N3-GalNAc (10 equivalents compared to IgG) prepared according to PCT / EP2016 / 059194, as described in PCT / EP2017 / 052792. Next, 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 for 10 CVs, followed by TBS (pH 7.5) for another 10 CVs. 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 dialyze passes to TBS (pH 7.5), the IgG was concentrated to approximately 25 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). Mass spectral analysis of the sample after IdeS treatment revealed one major Fc / 2 product corresponding to the 6-N3-GalNAc-GlcNAc(Fuc)-substituted Fc / 2 fragment (measured mass 24366 Da, approximately 90% of total Fc / 2) and one small amount of Fc / 2 product corresponding to the 6-N3-GalNAc-GlcNAc-substituted Fc / 2 fragment (measured mass 24220 Da, approximately 10% of total Fc / 2).

[0308] [Example 36. Intramolecular crosslinking of tras-v1a having trivalent linker 145 to obtain tras-v1a-145]

[0338] To a solution of tras-v1a (1698 μL, 35 mg, 20.61 mg / mL in TBS, pH 7.5), 1802 μL of TBS pH 7.5, propylene glycol (3427 μL), and trivalent linker 145 (23.3 μL, 40 mM solution in DMF, 4 equivalents compared to IgG) were added. The reaction mixture was incubated overnight at room temperature, followed by purification using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 16 / 600 column (Cytiva) with AKTA Purifier-10 (Cytiva). Mass spectral analysis of the IdeS-digested sample showed one major product (calculated mass 49799 Da, measured mass 49799 Da) corresponding to the intramolecularly cross-linked trastuzumab derivative tras-v1a-145.

[0309] [Example 37. Enzymatic remodeling of mPD-L1 for mPD-L1(6-N3-GalNAc)2(mPD-L1-v1a)]

[0339] mPD-L1 (4528 μL, 87 mg, 19.3 mg / mL in 20 mM histidine-HCl, 150 mM NaCl, pH 7.5) was incubated at 30°C for 16 hours in 20 mM histidine-HCl (pH 7.5), 150 mM NaCl (pH 7.5), and 6 mM MnCl2 with EndoSH (1 w / w%), His-TnGalNAcT (4 w / w%), alkaline phosphatase (commercially available from Roche, 0.01 w / w%), and UDP-6-N3-GalNAc (25 equivalents compared to IgG) prepared according to PCT / EP2016 / 059194, as described in PCT / EP2017 / 052792. Next, functionalized IgG was purified using two HiTrap MabSelect Sure 5 mL columns connected in series. After loading the reaction mixture, the columns were washed with TBS + 0.2% Triton for 10 CV, followed by TBS (pH 7.5) for another 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 dialyzing three times with PBS (pH 7.4), the IgG was concentrated to approximately 25 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (measured mass 24368 Da, approximately 90% of total Fc / 2) corresponding to the 6-N3-GalNAc-GlcNAc(Fuc)-substituted Fc / 2 fragment.

[0310] [Example 38. Intramolecular crosslinking of mPD-L1-v1a having trivalent linker 145 to obtain mPD-L1-v1a-145]

[0340] To a solution of mPD-L1-v1a (1569 μL, 50 mg, 31.86 mg / mL in PBS, pH 7.4), 3431 μL of TBS pH 7.5, 4967 μL of propylene glycol, and 33.3 μL of trivalent linker 145 (40 mM solution in DMF, 4 equivalents compared to IgG) were added. The reaction mixture was incubated overnight at room temperature, followed by purification using PBS (pH 7.4) as the mobile phase on an AKTA Purifier-10 (Cytiva) 16 / 600 Increase Superdex200 column (Cytiva). Mass spectral analysis of the IdeS-digested sample showed one major product (calculated mass 49799 Da, measured mass 49799 Da) corresponding to the intramolecularly cross-linked mPD-L1 derivative mPD-L1-v1a-145.

[0311] [Example 39. Conjugation of mPD-L1-v1a having divalent linker XL14 to obtain mPD-L1-v1a-XL14]

[0341] To a solution of mPD-L1-v1a (1883 μL, 60 mg, 31.86 mg / mL in PBS, pH 7.4), 1617 μL of TBS pH 7.5 and divalent linker XL14 (500 μL, 20 mM solution in DMF, 25 equivalents compared to IgG) were added. The reaction mixture was incubated overnight at room temperature, followed by purification using PBS (pH 7.4) as the mobile phase on a 16 / 600 Increase Superdex200 column (Cytiva) with an AKTA Purifier-10 (Cytiva). Mass spectrometry of the IdeS-digested sample showed one major product (measured mass 25063 Da, approximately 90% of total Fc / 2) corresponding to the desired product with XL14 conjugated to a single Fc / 2 fragment, leaving a second reactive handle available for further conjugation. A small amount of product corresponding to the undesirable intramolecular crosslinking derivative was observed (measured mass 49427 Da, approximately 10% of total Fc / 2).

[0312] [Preparation of antibody-cytokine conjugates] [General Procedures for Purifying Antibody-Cytokine Conjugates]

[0342] Unless otherwise specified, antibody-cytokine conjugates were purified according to the following procedure. The reaction was diluted with 2 volumes of 20 mM Tris-HCl (pH 8.0) to reduce the salt concentration to 50 mM NaCl. Next, the sample was loaded onto a 5 mL HiTrap Q HP column (Cytiva) using 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl as the mobile phase. After loading, the column was washed with 20 or more column volumes (CV) of 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 0.2% Triton-X100, followed by washing with 20 or more CV of 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. Next, the product was eluted with 20 mM Tris-HCl (pH 8.0) and 500 mM NaCl using a 20 CV linear gradient. The fraction containing the desired product was collected and concentrated to approximately 5 mL by pooling using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). The sample was then purified using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 16 / 600 column (Cytiva). The final antibody-cytokine conjugate was rapidly frozen and stored at -80°C until use.

[0313] [Example 40. Transient expression of gene fusion IC1 in CHO]

[0343] The gene fusion IC1 consists of the same mPD-L1 antibody targeting mouse PD-L1, with the same cleavable linker and C-terminal fusion to the 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 in 1100 mL scale by Evitria (Zurich, Switzerland). The antibody was purified using two HiTrap MabSelect Sure 5 mL columns connected in series. After loading the supernatant, the columns were washed 10 CV with TBS (pH 7.5). 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 obtain 22 mg of 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 using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 16 / 600 column (Cytiva) on an AKTA Purifier-10 (Cytiva). IC1 was analyzed according to SE-HPLC, yielding a final monomer level of 12 mg and 95%. Isolation of IC1 was confirmed by reduced SDS-PAGE analysis (Figure 18). The broad band measured for the heavy chain (HC) fused to IL-15 indicates heterogeneity and may be due to glycosylation at the IL-15 moiety.

[0314] [Example 41. Conjugation of tras-v1a-145 to NHO1-PF50 to obtain the conjugate tras-v1a-145-NHO1-PF50 (2:1)]

[0344] To a solution of tras-v1a-145 (247 μL, 2 mg, 8.1 mg / mL in PBS, pH 7.4), NHO1-PF50 (150 μL, 3.7 mg / mL in PBS, pH 7.4, 2.6 equivalents compared to IgG) was added. The reaction mixture was incubated overnight at room temperature. The reaction mixture was diluted 20-fold using 20 mM Tris (pH 8.0) and loaded onto a 1 mL HiTrap Q HP column (Cytiva) using 20 mM Tris-HCl (pH 8.0) and 10 mM NaCl as the mobile phase. The product was then eluted using a 50 CV linear gradient in 20 mM Tris-HCl (pH 8.0) and 1 M NaCl. The fraction containing the desired 2:1 format was collected and concentrated to 1 mL using a centrifuge filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Next, the sample was purified using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 10 / 300 GL column (Cytiva) on an AKTA Purifier-10 (Cytiva). Mass spectral analysis of the intact sample showed one major product (measured mass 162697 Da) corresponding to the conjugate tras-v1a-145-NHO1-PF50 (2:1).

[0315] [Example 42. Conjugation of mPD-L1-v1a to 218-NHO1-PF51 to obtain the conjugate mPD-L1-v1a-218-NHO1-PF51(2:2)]

[0345] A solution of mPD-L1-v1a (659 μL, 21 mg, 31.9 mg / mL in PBS (pH 7.4)) was mixed with 218-NHO1-PF51 (1352 μL, 7.8 mg / mL in PBS (pH 7.4), 5 equivalents compared to IgG). The reaction mixture was incubated overnight at 37°C and then purified according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO1-PF51(2:2) (Figure 18).

[0316] [Example 43. Conjugation of mPD-L1-v1a to 218-NHO2-PF52 to obtain the conjugate mPD-L1-v1a-218-NHO2-PF52(2:2)]

[0346] A solution of mPD-L1-v1a (785 μL, 25 mg, 31.9 mg / mL in PBS (pH 7.4)) was mixed with 218-NHO2-PF52 (2077 μL, 6.0 mg / mL in PBS (pH 7.4), 5 equivalents compared to IgG). The reaction mixture was incubated overnight at 37°C and then purified according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO2-PF52(2:2) (Figure 18). In addition, mass spectrometry of the IdeS digestion sample showed one major product (measured mass 39295 Da) corresponding to the conjugated Fc / 2 fragment.

[0317] [Example 44. Conjugation of mPD-L1-v1a to 218-NHO2-PF53 to obtain the conjugate mPD-L1-v1a-218-NHO2-PF53(2:2)]

[0347] A solution of mPD-L1-v1a (606 μL, 19.3 mg, 31.9 mg / mL in PBS (pH 7.4)) was mixed with 218-NHO2-PF53 (2639 μL, 3.64 mg / mL in PBS (pH 7.4), 5 equivalents compared to IgG). The reaction mixture was incubated overnight at 37°C and then purified according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO2-PF53(2:2) (Figure 18). In addition, mass spectrometry of the IdeS digestion sample showed one major product (measured mass 39296 Da) corresponding to the conjugated Fc / 2 fragment.

[0318] [Example 45. Conjugation of mPD-L1-v1a to 218-NHO2-PF54 to obtain the conjugate mPD-L1-v1a-218-NHO2-PF54(2:2)]

[0348] A solution of mPD-L1-v1a (942 μL, 30 mg, 31.9 mg / mL in PBS (pH 7.4)) was mixed with 218-NHO2-PF54 (3261 μL, 5.39 mg / mL in PBS (pH 7.4), 6 equivalents compared to IgG). The reaction mixture was incubated overnight at 37°C and then purified according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO2-PF54(2:2) (Figure 18). In addition, mass spectrometry of the IdeS digestion sample showed one major product (measured mass 39012 Da) corresponding to the conjugated Fc / 2 fragment.

[0319] [Example 46. Conjugation of mPD-L1-v1a to 218-NHO2-PF56 to obtain the conjugate mPD-L1-v1a-218-NHO2-PF56(2:2)]

[0349] A solution of mPD-L1-v1a (691 μL, 22 mg, 31.9 mg / mL in PBS (pH 7.4)) was mixed with 218-NHO2-PF56 (1667 μL, 6.56 mg / mL in PBS (pH 7.4), 5 equivalents compared to IgG). The reaction mixture was incubated overnight at 37°C and then purified according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-218-NHO2-PF56(2:2) (Figure 18). In addition, mass spectrometry of the IdeS digestion sample showed one major product (measured mass 39280 Da) corresponding to the conjugated Fc / 2 fragment.

[0320] [Example 47. Conjugation of mPD-L1-v1a-145 to NHO2-PF52 (2:1) conjugate]

[0350] A solution of mPD-L1-v1a-145 (869 μL, 23.6 mg, 27.1 mg / mL in PBS (pH 7.4)) was mixed with NHO2-PF52 (910 μL, 7.47 mg / mL in PBS (pH 7.4), 3 equivalents compared to IgG). The reaction mixture was incubated overnight at room temperature and then purified according to standard procedures. SDS-PAGE analysis confirmed the formation of mPD-L1-v1a-145-NHO2-PF52(2:1) (Figure 18). In addition, mass spectrometry of the IdeS digestion sample showed one major product (measured mass 64226 Da) corresponding to a crosslinked and conjugated Fc / 2 fragment (Fc / 2-IL-15-Fc / 2).

[0321] [Example 48. Conjugation of mPD-L1-v1a-XL14 to NHO2-PF52 (2:2) conjugate]

[0351] A solution of mPD-L1-v1a-XL14 (1541 μL, 45 mg, 29.21 mg / mL in PBS (pH 7.4)) was mixed with NHO2-PF52 (1739 μL, 7.47 mg / mL in PBS (pH 7.4), 3 equivalents compared to IgG). The reaction mixture was incubated overnight at 37°C and then purified according to standard procedures. Mass spectrometry of the IdeS digestion sample showed one major product (measured mass 39495 Da) corresponding to the conjugated Fc / 2 fragment.

[0322] [Example 49. Conjugation of tras-v1a-145 to NHO1-PF57 to obtain the conjugate tras-v1a-145-NHO1-PF57(2:1)]

[0352] To a solution of tras-v1a-145 (1600 μL, 16 mg, 10 mg / mL in PBS (pH 7.4)), NHO1-PF57 (727 μL, 6.20 mg / mL in PBS (pH 7.4), 1.6 equivalents compared to IgG) was added. The reaction mixture was incubated overnight at room temperature. The reaction mixture was diluted 20-fold using 20 mM Tris (pH 8.0) and loaded onto a 1 mL HiTrap Q HP column (Cytiva) using 20 mM Tris-HCl (pH 8.0) and 10 mM NaCl as the mobile phase. The product was then eluted using a 50 CV linear gradient in 20 mM Tris-HCl (pH 8.0) and 1 M NaCl. The fraction containing the desired 2:1 format was collected and concentrated to 1 mL using a centrifuge filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Next, the sample was purified using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 10 / 300 GL column (Cytiva) on an AKTA Purifier-10 (Cytiva). Mass spectral analysis of the intact sample showed one major product (measured mass 173099 Da) corresponding to the conjugate tras-v1a-145-NHO1-PF57 (2:1).

[0323] [Example 50. Conjugation of tras-v1a-145 to NHO1-PF58 to obtain the conjugate tras-v1a-145-NHO1-PF58(2:1)]

[0353] To a solution of tras-v1a-145 (86 μL, 0.7 mg, 8.1 mg / mL in PBS (pH 7.4)), NHO1-PF58 (42 μL, 7.94 mg / mL in PBS (pH 7.4), 3 equivalents compared to IgG) was added. The reaction mixture was incubated overnight at room temperature. The sample was then purified using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 10 / 300 GL column (Cytiva) on an AKTA Purifier-10 (Cytiva). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO1-PF58 (2:1) (Figure 19).

[0324] [Example 51. Conjugation of tras-v1a-145 to NHO1-PF59 to obtain the conjugate tras-v1a-145-NHO1-PF59 (2:1)]

[0354] To a solution of tras-v1a-145 (85 μL, 0.9 mg, 10 mg / mL in PBS (pH 7.4)), NHO1-PF59 (140 μL, 2.16 mg / mL in PBS (pH 7.4), 2 equivalents compared to IgG) was added. The reaction mixture was incubated overnight at room temperature. The sample was then purified using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 10 / 300 GL column (Cytiva) on an AKTA Purifier-10 (Cytiva). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO1-PF59 (2:1) (Figure 19).

[0325] [Example 52. Conjugation of tras-v1a-145 to NHO2-PF57 to obtain the conjugate tras-v1a-145-NHO2-PF57(2:1)]

[0355] A solution of tras-v1a-145 (95 μL, 1.0 mg, 10.6 mg / mL in PBS (pH 7.4)) was mixed with NHO2-PF57 (69 μL, 7.49 mg / mL in PBS (pH 7.4), 3 equivalents compared to IgG). The reaction mixture was incubated overnight at room temperature. The sample was then purified using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 10 / 300 GL column (Cytiva) on an AKTA Purifier-10 (Cytiva). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO2-PF57 (2:1) (Figure 20).

[0326] [Example 53. Conjugation of tras-v1a-145 to NHO3-PF57 to obtain the conjugate tras-v1a-145-NHO3-PF57(2:1)]

[0356] A solution of tras-v1a-145 (95 μL, 1.0 mg, 10.6 mg / mL in PBS (pH 7.4)) was mixed with NHO3-PF57 (86 μL, 5.96 mg / mL in PBS (pH 7.4), 3 equivalents compared to IgG). The reaction mixture was incubated overnight at room temperature. The sample was then purified using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 10 / 300 GL column (Cytiva) on an AKTA Purifier-10 (Cytiva). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO3-PF57 (2:1) (Figure 20).

[0327] [Example 54A. Conjugation of tras-v1a-145 to NHO4-PF57 (2:1) conjugate]

[0357] A solution of tras-v1a-145 (95 μL, 1.0 mg, 10.6 mg / mL in PBS (pH 7.4)) was mixed with NHO4-PF57 (73 μL, 7.06 mg / mL in PBS (pH 7.4), 3 equivalents compared to IgG). The reaction mixture was incubated overnight at room temperature. The sample was then purified using PBS (pH 7.4) as the mobile phase on a Superdex200 Increase 10 / 300 GL column (Cytiva) on an AKTA Purifier-10 (Cytiva). RP-UPLC analysis of the intact sample confirmed the formation of the conjugate tras-v1a-145-NHO4-PF57 (2:1) (Figure 20).

[0328] [Example 54B. Conjugation of tras-v1a to 218-NHO2-PF52 to obtain the conjugate tras-v1a-218-NHO2-PF52(2:2)]

[0358] A solution of tras-v1a (607 μL, 14.4 mg, 23.72 mg / mL in PBS (pH 7.4)) was mixed with 218-NHO2-PF52 (1285 μL, 5.57 mg / mL in PBS (pH 7.4), 5 equivalents compared to IgG). The reaction mixture was incubated overnight at 37°C and then purified according to standard procedures. Mass spectrometry of the IdeS digestion sample showed one major product (measured mass 39294 Da) corresponding to the conjugated tras-v1a-218-NHO2-PF52(2:2)Fc / 2 fragment.

[0329] [Example 54C. Conjugation of enfo-v1a to 218-NHO2-PF52 to obtain the conjugate enfo-v1a-218-NHO2-PF52(2:2)]

[0359] To a solution of enfo-v1a (470 μL, 10.8 mg, 22.97 mg / mL in PBS (pH 7.4)), 218-NHO2-PF52 (964 μL, 5.57 mg / mL in PBS (pH 7.4), 5 equivalents compared to IgG) was added. The reaction mixture was incubated overnight at 37°C and then purified according to standard procedures. Mass spectrometry of the IdeS digestion sample showed one major product (measured mass 39294 Da) corresponding to the conjugated enfo-v1a-218-NHO2-PF52(2:2)Fc / 2 fragment.

[0330] [Evaluation of Antibody-Cytokine Conjugates] [Example 55: Freeze-thaw stability test]

[0360] 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 using liquid nitrogen and thawed again. This process was repeated five times. The aggregate levels were determined by SE-HPLC after cycles 0, 1, 3, and 5 (Figure 21). 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) tended to aggregate less readily compared to the gene fusion IC1.

[0331] [Example 56: Stability testing of antibody-cytokine conjugates prepared using IL-15 variants with variable N-terminal IL-15 sequences and different hydroxylamines]

[0361] Antibody-cytokine conjugates were diluted to 1 mg / mL in PBS (pH 7.4) and incubated at 37°C. Stability was determined by analyzing the samples by RP-UPLC at the indicated time points (Table 2). Variants prepared with hydroxylamine HO2, HO3, and HO4 showed improved stability compared to the variant prepared with HO1.

[0332] [Table 2]

[0333] [Example 57: Cleavage of antibody-cytokine conjugate by matryptase (MT-SP1)]

[0362] mPD-L1-v1a-218-NHO2-PF52(2:2) was incubated at 37°C for 2 hours at a final concentration of 1 mg / mL using 0.5%, 1.0%, 2.0%, and 4.0 (w / w)% recombinant human matryptase (commercially available from Bio-techne), followed by RP-UPLC analysis. Complete cleavage was observed using 1.0% matryptase (Figure 22).

[0334] [Example 58. Treatment of antibody-cytokine conjugates using MT-SP1 before in vitro evaluation]

[0363] The antibody-cytokine conjugate sets shown in Figure 23 (35 μg each) were incubated at 37°C for 2 hours at a final concentration of 1 mg / mL using 2 (w / w)% recombinant human matryptase (commercially available from Bio-techne), followed by RP-UPLC analysis. Complete cleavage was observed in all cases (Figure 23). The cleaved constructs were used to evaluate IL-15 activity in an in vitro IL-15 bioassay.

[0335] [Example 59: Cleavage of antibody-cytokine conjugates by urokinase (uPA)]

[0364] tras-v1a-145-NHO1-PF50(2:1) was incubated at a final concentration of 1 mg / mL at room temperature for 3 hours with 0.1%, 1.0%, and 10.0 (w / w)% recombinant human urokinase (uPA, commercially available from Bio-techne) at a room temperature. Intact samples were analyzed by MS. Under the 10% (w / w) uPA condition, mass spectrometry of the intact sample showed one major product corresponding to the cleavage antibody without IL-15 (measured mass 149118 Da) and one minor product corresponding to free IL-15 (measured mass 13598 Da). Under the 0.1% and 1.0% (w / w) uPA conditions, mass spectrometry of the intact sample showed uncleaved tras-v1a-145-NHO1-PF50(2:1) (measured mass 162708 Da) as the major product. Samples treated with 10% (w / w) uPA were used to evaluate IL-15 activity in an in vitro IL-15 bioassay.

[0336] [In vitro evaluation] [General procedure for in vitro IL-15 bioassay]

[0365] Monitoring was performed 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 the read. When IL-15 binds to its receptor, luminescence is induced by receptor-mediated signaling, which can be detected upon substrate addition and quantified with a luminometer. The assay was performed according to the manufacturer's instructions. Briefly, cleaved or intact antibody-cytokine conjugates or controls were added to IL-15 cells in Duplo at the square root of a 10-fold dilution series to obtain final concentrations ranging from 0.1 to 1000 pM unless otherwise specified (for cleaved constructs, the concentration refers to the initial antibody-cytokine conjugate). IL-15 cells were incubated at 37°C and in a humidified atmosphere of 5% CO2 for 6 hours. Next, the detection reagent (Bio-Glo® luciferase assay reagent) was added, and the chemiluminescence intensity was recorded using a microplate reader (Envision, PerkinElmer). Data analysis was performed using Graphpad prism software. EC 50 The values ​​were calculated using nonlinear regression, and the maximum induced magnification was calculated by dividing the relative luminescence units (RLU) of the treated cells by the RLU of the untreated cells (both with background subtraction).

[0337] [Example 60. In vitro IL-15 activity of intact and cleaved antibody-cytokine conjugates]

[0366] The activity of five antibody-cytokine conjugates was measured via an in vitro IL-15 bioassay. mPD-L1-v1a-218-NHO2-PF52(2:2) was included both with and without pretreatment using MT-SP1 for comparison. MT-SP1 treatment was performed according to Example 58. All intact antibody-cytokine conjugates showed only minimal activation in the induction factor range of 1.0–2.5, while the cleaved variant of mPD-L1-v1a-218-NHO2-PF52(2:2) showed an induction factor of 7.2 (Figure 24 and Table 3), demonstrating that IL-15 is inactivated or attenuated when linked to the antibody, but becomes activated upon linker cleavage by MT-SP1.

[0338] [Table 3]

[0339] [Example 61. In vitro IL-15 activity assay of cleaved antibody-cytokine conjugates]

[0367] Antibody-cytokine conjugates were pretreated with MT-SP1 according to Example 58 to obtain free cytokines. The activity of five cleaved antibody-cytokine conjugates was measured via an in vitro IL-15 bioassay following a general procedure. Both mPD-L1-v1a-218-NHO1-PF51(2:2) and mPD-L1-v1a-218-NHO2-PF52(2:2) showed the same cleavable linker, IL-15 variant, and stoichiometry as the EC1 compared to the gene fusion IC1. 50 It showed higher efficiency in terms of values ​​and maximum induction ratio (Figure 25 and Table 4). The corresponding 2:1 format mPD-L1-v1a-145-NHO2-PF52(2:1) had approximately twice the EC compared to the 2:2 format, as expected based on the amount of IL-15 that could be emitted. 50The values ​​were shown. The variant enhanced with IL-15Rβ binding mPD-L1-v1a-218-NHO2-PF53(2:2) showed equivalent in vitro potency to mPD-L1-v1a-218-NHO2-PF52(2:2), and this assay was performed using a mouse cell line. This mutation can be explained by the fact that it binds with higher affinity to human IL-15Rβ but does not bind to mouse IL-15Rβ.

[0340] [Table 4]

[0341] [Example 62. In vitro IL-15 activity of intact and cleaved antibody-cytokine conjugates]

[0368] mPD-L1-v1a-218-NHO2-PF52(2:2) and mPD-L1-v1a-218-NHO2-PF56(2:2) were pretreated with MT-SP1 according to Example 58 to obtain free cytokines. The activity of both compounds was measured using an in vitro IL-15 bioassay with and without pretreatment with MT-SP1. Unmodified cytokine PF52 was included as a reference. Cleaved and intact mPD-L1-v1a-218-NHO2-PF56(2:2) was added at higher final concentrations (square root of the 10-fold dilution series) in the range of 32 pM to 316 nM. Both antibody-cytokine conjugates showed activation upon pretreatment with MT-SP1, as demonstrated by the increase in induction factor (Figure 26 and Table 5). mPD-L1-v1a-218-NHO2-PF56(2:2) has a high EC ratio exceeding 1000 times. 50 The values ​​showed that high-affinity binding to IL-15Rα disappeared, but binding to IL-15Rβ / γ was retained.

[0342] [Table 5]

[0343] [Example 63. In vitro IL-15 activity assay using alternative antibody and cleavable linker]

[0369] tras-v1a-145-NHO1-PF50(2:1) was pretreated with urokinase (UpA) according to Example 59 to obtain the free cytokine. The activity of tras-v1a-145-NHO1-PF50(2:1) was measured using an in vitro IL-15 bioassay with and without pretreatment using UpA. Unmodified cytokine PF50 and unmodified antibody trastuzumab were included as controls. Intact tras-v1a-145-NHO1-PF50(2:1) showed minimal activation (Table 6 and Figure 27), while cleaved tras-v1a-145-NHO1-PF50(2:1) showed a similar response to modified cytokine PF50.

[0344] [Table 6]

[0345] [In Vivo Review] [Example 64. In vivo tolerability in female BALB / c mice]

[0370] Female BALB / c mice (a group of three mice, 6–8 weeks old at the start of the study, obtained from Vital River Laboratory Animal Technology Co., Ltd. in China) were treated with either a vehicle (PBS, pH 7.4) or the indicated antibody-cytokine conjugate (at 6 mg / kg) and compared to IC1 (at 6 mg / kg). In all cases, a single dose was administered by sc injection. After administration, the body weight of all animals was measured daily (Figures 28 and 29, upper graph). Based on the results of this first study, a second tolerability study was conducted using the same procedure but with dose concentrations ranging from 3 to 12 mg / kg (Figures 28 and 29, middle graph). Finally, a third study was conducted using the same procedure but with dose concentrations ranging from 4.5 to 18 mg / kg (Figures 28 and 29, lower graph). Antibody-cytokine conjugates with cleavable IL-15mPD-L1-v1a-218-NHO1-PF51(2:2) and mPD-L1-v1a-218-NHO2-PF52(2:2) showed no signs of toxicity at 6 mg / kg and 12 mg / kg doses, but at 18 mg / kg, moderate body weight loss (average body weight loss of less than 10%) was observed, indicating an MTD of approximately 18 mg / kg (Figure 28). Variants lacking IL-15Rα-binding mPD-L1-v1a-218-NHO2-PF56(2:2) showed an MTD of approximately 4.5 mg / kg, and at an initial dose of 6 mg / kg, one out of three mice died on day 5. Compared to mPD-L1-v1a-218-NHO2-PF52(2:2), the variants mPD-L1-v1a-145-NHO2-PF52(2:1) and mPD-L1-v1a-218-NHO2-PF53(2:2) also showed decreased tolerability at estimated MTDs of 9 mg / kg and 15 mg / kg, respectively (Figure 29).

[0346] [Example 65. In vivo efficacy in a CT26 syngeneic colon cancer model]

[0371] Female BALB / c mice (obtained from Vital River Laboratory Animal Technology Co., Ltd. in China, 5-8 weeks old at the start of the study) were given 5 × 10 in 0.1 mL of PBS to induce tumor development. 5 Individual CT26 tumor cells were subcutaneously inoculated into the right posterior flank region. The tumor volume was 70-120 mm². 3 When within the specified range, groups of eight mice were subcutaneously injected with either a vehicle (PBS pH 7.4) or the indicated antibody-cytokine conjugate (either 3 mg / kg or 6 mg / kg as indicated). In all cases, the dose was administered on days 0 and 7. Tumor volume and body weight were measured 2-3 times per week after randomization. mPD-L1-v1a-218-NHO2-PF52(2:2) showed improved antitumor efficacy compared to the corresponding gene fusion IC1 and the cleavable variant mPD-L1-v1a-218-NHO2-PF54(2:2) (Figure 30). Furthermore, the 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 to the vehicle group (Figure 31).

[0347] [Example 66. In vivo efficacy in an MC38 syngeneic colon cancer model]

[0372] Female C57BL / 6 mice (obtained from Vital River Laboratory Animal Technology Co., Ltd. in China, 6-7 weeks old at the start of the study) were given 1 × 10⁶ cells in 0.1 mL of PBS to induce tumor development. 6 Individual MC38 colorectal cancer cells were subcutaneously inoculated into the right posterior flank region. The average tumor volume was 94 mm². 3Upon reaching a certain stage, mice were assigned to groups of 10 and subcutaneously injected with either a vehicle (PBS pH 7.4) or mPD-L1-v1a-218-NHO2-PF52(2:2) (3 mg / kg or 6 mg / kg as indicated). In all cases, the dose was administered on days 0, 7, and 14. Tumor volume and body weight were measured 2-3 times per week after randomization. mPD-L1-v1a-218-NHO2-PF52(2:2) showed a significant reduction in tumor volume compared to the vehicle group (p<0.001 for both dose levels, as determined on day 16 by a one-way analysis of variance (ANOVA) followed by Tukey's HSD test).

[0348] [Sequence List]

[0373] The following list herein provides a selection of peptide sequences relevant to the present invention. A complete list is disclosed in another sequence listing document. Sequence identification of protease-cleavable linker 1 (SEQ ID NO: 1): GSSGGSGGSGGSGLSGRSDNHGSSGS Sequence identification of protease-cleavable linker 2 (SEQ ID NO: 2): GGGGSGGGGSRASRANGS Sequence identification of human IL-15 (SEQ ID NO: 3): NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Sequence identification of the IL-15Rα-IL-15 fusion protein (SEQ ID NO: 4) [ka] Sequence identification of SYR-(G4S)3-IL15(PF18) (Sequence ID 5): SYRGGGGSGGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Sequence identification of SYR-cleavable linker 1-IL15(PF50)(SEQ ID NO: 6): [ka] Sequence identification of SYR-cleavable linker 2-IL15(PF51)(SEQ ID NO: 7): MSYRGGGGSGGGGSRASRANGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Sequence identification of ST-cleavable linker 2-IL15(PF52) (Sequence ID 8): STGGGGSGGGGSRASRANGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Sequence identification of ST-cleavable linker 2-IL15(N72D)(PF53)(SEQ ID NO: 9): STGGGGSGGGGSRASRANGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Sequence identification of ST-(G4S)3GGS-IL15(PF54) (Sequence ID 10): STGGGGSGGGGGSGGGGSGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Sequence identification of SYR-(G4S)8-IL15(PF55)(SEQ ID NO: 11): [ka] Sequence identification of ST-cleavable linker 2-IL15(E46G, V49R)(PF56)(SEQ ID NO: 12): MSTGGGGSGGGGSRASRANGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLGLQRISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Sequence identification of SYR-(G4S)8-IL15Rα-IL-15(PF57)(SEQ ID NO: 13): [ka] Sequence identification of S-(G4S)8-IL15Rα-IL-15(PF58)(SEQ ID NO: 14): [ka] Sequence identification of ST-(G4S)8-IL15Rα-IL-15(PF59)(SEQ ID NO: 15): [ka] Sequence identification of the mPD-L1 light chain (SEQ ID NO: 16): [ka] Sequence identification of the mPD-L1 heavy chain (SEQ ID NO: 17): [ka] Sequence identification of the gene fusion (IC1) heavy chain (SEQ ID NO: 18): [ka]

Claims

1. Structure (1) or Structure (2) 【Chemistry 1】 (In the formula, - AB is an antibody, - L is a linker connecting AB to D, - x is an integer in the range of 1 to 10. - Z １ and Z ２ is a linking group, and at least Z １ This includes products of cycloaddition or nucleophilic reactions. - b is 0 or 1, - L ６ is -(H) ｖ -S-(L ７ ) ｗ’ - and ○H is a monosaccharide, ○v is an integer in the range of 1 to 10. ○S is a sugar or a sugar derivative. ○w' is 0 or 1, and ○L ７ is -N(H)C(O)-(CH ２ -O) ｚ -(CH ２ -CH ２ -O) γ -CH ２ - or CH ２ -(O-CH ２ -CH ２ ) γ - where γ is an integer in the range of 0 to 100, and z is 0 or 1, - D is - (D １ ) α -D ２ It is a polypeptide produced by ○D １ It is a cleavable peptide linker, ○D ２ It is an immune cell engager polypeptide specific to immune cell receptors, ○α is either 0 or 1. (L is a linker that can be cut when α = 0) Antibody-cytokine conjugate containing the antibody.

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

3. The antibody is specific to extracellular receptors on tumor cells, and preferably the extracellular receptors on the tumor cells are 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, Brevican, c-KIT (CD117), c-Met, C4.4a (LYPD3), CA-IX (CA 9) / MN, cadherin-6, CanAg, CCR7, CD117 (c-KIT), CD123 (IL-3Rα), CD13, CD133 , CD138 / syndecan-1, CD166 (ALCAM), CD19, CD20, CD203C, CD205, Ly75, CD21, CD 22, CD228 (P79, SEMF), CD25 (IL-2R-α), CD30 (TNFRSF8), CD324 (CDH1 / E-cad Herin), CD33, CD352 (SLAMF6, NTB-A), CD37, CD38, CD44v6, CD45, CD46, CD47, C D48a (SLAMF2), CD56 (NCAM), CD70, CD71 (TF-R), CD72, CD74, CD79a, CD79b, CDH6, Cadherin-6, CEACAM5 (CD66e), Claudine, CLDN18.2, CLDN6 (Claudine-6, Skullin), CLEC12A, CLL-1 / CLEC12A, Crypt, CS1 (SLAMF7, CD319), CXCR5, DKL-1, DLL3 (Delta-like 3), DPEP3, E16, EGFR, EGFRvIII, ENPP3, CD203c (AGS-16), EpCAM, E phA2, EphB2R, Ephrin-A4 (EFNA4), ETBR, FAP, FGFR2, FGFR3, Fibronectin EDB, FLT3, FOLR1 (FR-a), Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, GloboH, GPC3 (Glypican-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, NaPi 2b (SLC34A2), NCA, Nectin-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 Selected from the group consisting of 5b, SEZ6, SLITRK6 (SLC44A4), STEAP1, STEAP2, STn, TAG72, TENB2, TF (CD142, thromboplastin), TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, TNF-α, TROP-2 (TAACSTD2), TWEAKR, receptor tyrosine kinase (RTK), and tenascin. Or, the antibody is specific to extracellular proteins and / or tumor-associated glycosylation antigens (TACAs) arising from viral infection, and / or the immune cell-engaging polypeptide is specific to 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, IFN- α (IFN-α1 / 13, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α14, IFN-α16, IFN-α17 and The antibody-cytokine conjugate according to claim 1 or 2, wherein the immune cell-engaging polypeptide is selected from the group consisting of IFN-β, IFN-Y, IFN-A, TFN-α, TNF-β, TGF-β1, M-CSF, G-CSF, GM-CSF, and CXL10, and more preferably the immune cell-engaging polypeptide is IL-2 or IL-15, more preferably IL-15.

4. Each Z １ Structure (Z2) to (Z20c): 【Chemistry 2】 (In the formula, the connection to L is represented by a wavy line, B （－） is an anion, and B （＋） is a cation, and the nitrogen atoms of (Z10), (Z13), (Z14), and (Z15) may have a linkage to L, or contain or are substituted with hydrogen atoms. R ３６ is a halogen selected from fluorine, chlorine, bromine, and iodine, Y ４ is a heteroatom, R ３５ is hydrogen, C １ ~C ２４ alkyl group, C ３ ~C ２４ Cycloalkyl groups, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups and C ３ ~C ２４ Selected from the group consisting of (hetero)arylalkyl groups, the C １ ~C ２４ alkyl group, C ３ ~C ２４ Cycloalkyl groups, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups and C ３ ~C ２４ (Hetero)arylalkyl groups are O, Si, S and NR １４ Optionally substituted and optionally interposed by one or more heteroatoms selected from R １４ is hydrogen and C １ ~C ４ Independently selected from the group consisting of alkyl groups, Ring Z is selected from triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, or piperazine. Selected individually from, Preferably, Z １ The structure is selected from (Z21) to (Z38a), and more preferably, Z １ (Z29): 【Transformation 3】 And, More specifically, Z １ This can be obtained by a click reaction between cycloalkyne and azide, and Z ２ The antibody-cytokine conjugate according to any one of claims 1 to 3, which can be obtained by a click reaction between cycloalkyne and nitrone.

5. Linker L is structure - (8) １ )-(L ２ ) ｏ - (8) ３ ) ｐ - (8) ４ ) ｑ - (In the formula, - L １ is, Z １ Connected to, and L ４ If it exists, Z ２ It is connected to, - L １ , L ２ , L ３ and L ４ Each of them individually, Z １ to Z ２ It is a linker that connects together with it. - o, p, and q are each individually 0 or 1, preferably o = p = q = 0. Preferably, a) Linker L １ teeth, -(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 -] ２ BM-(C(O)) ｇ -(K １ ) ｄ’ -(K ２ ) ｅ’ -(K １ ) ｆ’ -(C(O)) ｇ’ - (In the formula, - d and d' are 0 or 1, respectively. - e and e' are integers in the range of 1 to 10, respectively. - f and f' are 0 or 1, respectively. - g and g' are integers in the range of 0 to 10, respectively. - k = 0 or 1, but if k = 1, then d = 0. - K １ Structure (23) 【Chemistry 4】 It is a sulfamide group, a = 0 or 1, and R １３ is hydrogen, C １ ~C ２４ alkyl group, C ３ ~C ２４ Cycloalkyl groups, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups and C ３ ~C ２４ Selected from the group consisting of (hetero)arylalkyl groups, the C １ ~C ２４ alkyl group, C ３ ~C ２４ Cycloalkyl groups, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups and C ３ ~C ２４ (Hetero)arylalkyl groups are O, S and NR １４ Optionally substituted and optionally interposed by one or more heteroatoms selected from R １４ is hydrogen and C １ ~C ４ Independently selected from the group consisting of alkyl groups, - K ３ -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -(O)(CH2) ｍ C(O)-, -C(O)(CH ２ ) ｍ C(O)NH- or -(4-Ph)CH ２ NHC(O)(CH ２ ) ｍ C(O)NH-, where m is an integer in the range of 0 to 10. - K ２ is, -CH ２ -CH ２ -O- or -O-CH ２ -CH ２ - is a part, or (K ２ ) ｅ is, -(CH ２ -CH ２ -O) ｅ１ -CH ２ -CH ２ - or - (CH ２ -CH ２ -O) ｅ１ -CH ２ - It is a part, and e1 is defined in the same way as e, - BM is preferably a branched portion selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)ring, or a polycyclic moiety, and more preferably BM is a nitrogen atom. Represented by and / or b) Linker L ２ This is a peptide spacer, preferably L ２ It has a protease recognition sequence, preferably L ２ It comprises 1 to 5 amino acids, optionally including non-natural amino acids, more preferably a dipeptide, tripeptide, or tetrapeptide spacer, and / or c) Linker L ３ This is a spacer related to the cutting mechanism, preferably L ３ This refers to a para-aminobenzyl-carbonyl derivative or glucuronic acid-functionalized p-hydroxybenzyl-carbonyl (L3b) based on structure (L3a): 【Transformation 5】 (In the formula, parentheses indicate an optional carbonyl group, R ２１ H, R ２６ or C(O)R ２６ And R ２６ C １ ~C ２４ (hetero)alkyl group, C ３ ~C １０ (hetero)cycloalkyl group, C ２ ~C １０ (hetero)aryl group, C ３ ~C １０ Alkyl (hetero)aryl groups and C ３ ~C １０ (Hetero)arylalkyl groups, these are O, S and NR ２８ Optionally substituted and optionally interposed by one or more heteroatoms selected from R ２８ is hydrogen and C １ ~C ４ A group independently selected from the group consisting of alkyl groups, preferably R ２１ is H or C(O)R ２６ And R ２６ = 4-N-methylpiperazine or morpholine, most preferably R ２１ (is H) Selected from and / or d) Linker L ４ This is a connecting spacer, preferably L ４ is, -NR ２２ - (C ｚ -Alkilen)-NR ２２ - (SO ２ -NH-) ｊ (C(O)) ｈ - is a diamine by R ２２ is H or C １ ~C ４ It is an alkyl group, z is an integer in the range of 1 to 10, j is 0 or 1, and h is 0 or 1, or Linker L ４ is structure-NR ２２ - (CH ２ -CH ２ -O) ｅ６ - (CH ２ ) ｅ７ -C(O)- (wherein e6 is an integer in the range of 1 to 10, e7 is an integer in the range of 1 to 3, and R ２２ is H or C １ ~C ４ Ethylene glycol spacers (which are alkyl), or Structure-NR ２２ - (C ｚ -Alkylene)-C(O)-(wherein x is an integer in the range of 1 to 20, and R ２２ is H or C １ ~C ４ (It is an aminoalkanoic acid spacer due to being alkyl.) The antibody-cytokine conjugate according to any one of claims 1 to 4, which is a linker.

6. α = 1, and D is D １ Z via the N-terminus ２ An antibody-cytokine conjugate according to any one of claims 1 to 5, which is linked to the antibody-cytokine conjugate.

7. D １ These are proteases that are overexpressed in the tumor microenvironment, preferably serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, glutamate proteases, metalloproteinases, gelatinases and asparagine peptide lyases, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, kallikrein, hKl, hKlO, hKT5, plasmin, collagen Nase, type IV collagenase, stromericin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidine, bromelain, calpain, caspase, caspase-3, mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, membrane-bound serine protease 1 (MT- SP1), Regmine, Plasmepsin, Nepenthesin, Metalloexopeptidase, Metalloendopeptidase, Matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP1O, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM12, Urokinase plasminogen activator (uPA), Enterokinase, Prostate-specific target (PSA, hK3), Inter An antibody-cytokine conjugate according to any one of claims 1 to 6, comprising a cleavable site that is cleaved by one of the following: leukin-1b converting enzyme, thrombin, FAP (FAP-α), type II transmembrane serine protease (TTSP), neutrophil elastase, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

8. L has the following structure: 【Transformation 6】 (In the formula, x is in the range of 0 to 100, preferably in the range of 0 to 50, more preferably in the range of 1 to 30, and most preferably in the range of 1 to 18.) An antibody-cytokine conjugate according to any one of claims 1 to 7, comprising any one of the following.

9. L-Z ２ The following: 【Transformation 7】 (In the formula, ○L １ The connection of the wavy line connected to is Z １ Representing a connection to, and ring Z is as defined in claim 4, preferably in the definition of D, α is 0, and ○L １ and L ２ (This is as defined in claim 5.) The antibody-cytokine conjugate according to any one of claims 1 to 11.

10. An antibody-cytokine conjugate according to any one of claims 1 to 9, for use in medical procedures.

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

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

13. A process for preparing an antibody-cytokine conjugate according to any one of claims 1 to 9, - AB(F) １ ) ｘ Q １ -L-Q ２ or (Q １ ) ２ -L-Q ２ The process of reacting with, - The obtained compound is F ２ - The process of reacting with D Includes or - D-F ２ Q １ -L-Q ２ or (Q １ ) ２ -L-Q ２ The process of reacting with, - The resulting compound is AB(F １ ) ｘ The process of reacting with Includes AB(F １ ) ｘ This consists of x reactive parts F １ A functional antibody containing, where x is an integer in the range of 1 to 10, and Q １ and F １ They are mutually reactive with each other, and Q ２ and F ２ They are mutually reactive with each other, Q １ and F １ Between and Q ２ and F ２ The reaction between two reactants is a process that forms a covalent bond between the reactants.

14. F １ , is Azid, Q １ It contains cycloalkynes, Q ２ It contains trans-cyclooctene and F ２ The process according to claim 13, comprising nitrone or tetrazine.

15. - F ２ F is nitron ２ - A step in which the terminal serine or threonine of D is converted to nitrone in order to obtain D, - D to F ２ A ligase-mediated ligation step for conversion to -D, wherein the ligase is preferably selected from tubulin tyrosine ligase, transglutaminase, lipoic acid ligase, farnesyltransferase, glycosyltransferase, formyl-glycine-producing enzyme (FGE), and tripsy ligase / subtillase, and more preferably the ligase is saltase. This process is carried out by, preferably, D is produced by microbial recombinant expression, and then D-F ２ The process according to claim 13 or 14, wherein the microorganism is converted to a prokaryote, most preferably Escherichia coli.

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