Antibodies functionalized on both sides via cycloaddition

Abstract

The present invention provides an antibody-payload conjugate having a payload antibody ratio of 1. The antibody-payload conjugate has the structure (1): Formula (1) (wherein a, b, and c are each independently 0 or 1; L 1 , L 2 and L 3 is a linker; D is a payload; BM is a branching moiety; and Z is a connecting group obtainable by a cycloaddition reaction. The present invention further provides methods for preparing antibody-payload conjugates according to the invention, intermediate compounds in the preparation methods, and medical uses of antibody-payload conjugates according to the invention.

Description

[Technical Field]

[0001] The present invention relates to the field of bioconjugation, in particular to antibody conjugates containing a single payload (drug-to-antibody ratio of 1). More particularly, the present invention relates to conjugates, compositions, and methods suitable for attaching a payload to an IgG-type antibody via a cycloaddition reaction. Monofunctionalized antibody conjugates as compounds, compositions, and methods can be useful in providing novel drugs for targeted delivery of payloads, such as highly potent cytotoxic or immunomodulatory agents. [Background technology]

[0002] Antibody-drug conjugates (ADCs), considered a therapeutic magic bullet, consist of an antibody to which a drug is attached. The antibody (also known as a ligand) can be in a small protein format (e.g., scFv, Fab fragment, DARPin, affibody), but is typically a monoclonal antibody (mAb), selected for its high selectivity and affinity for a given antigen, its long circulating half-life, and its minimal to no immunogenicity. Thus, as protein ligands for carefully selected biological receptors, mAbs provide an ideal delivery platform for selective targeting of drugs. For example, monoclonal antibodies known to selectively bind to specific cancer-associated antigens can be used to deliver chemically conjugated cytotoxic agents to tumors via binding, internalization, intracellular processing, and eventual release of activated catabolites. The cytotoxic agent can be a small molecule toxin, a protein toxin, or other formats, such as an oligonucleotide. As a result, tumor cells can be selectively eradicated while sparing normal cells not targeted by the antibody. Similarly, chemical conjugation of antimicrobial drugs (antibiotics) to antibodies can be applied to treat bacterial infections, while conjugates of anti-inflammatory drugs are under investigation for the treatment of autoimmune diseases, and, for example, the attachment of oligonucleotides to antibodies is a potentially promising approach for the treatment of neuromuscular diseases. Thus, the concept of targeted delivery of active pharmaceutical agents to selected specific cellular locations is a powerful approach for treating a wide range of diseases, with many beneficial aspects compared to systemic delivery of the same drugs.

[0003]

[0003] An alternative strategy for employing monoclonal antibodies for targeted delivery of specific protein drugs is by genetic fusion of the former protein to one or more antibody termini, which may be the N- or C-terminus of the light or heavy chain (or both). In this case, a biologically active protein of interest, e.g., a protein toxin such as Pseudomonas exotoxin A (PE38) or an anti-CD3 single-chain variable fragment (scFv), is genetically encoded as a fusion with the antibody, possibly, but not necessarily, via a peptide spacer, such that 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 also be genetically engineered to modify their structure and thereby introduce (or remove) specific properties. For example, mutations can be introduced into antibody Fc fragments to nihilate binding to Fc gamma receptors, modulate binding to FcRn receptors, or to specific cancer targets, or engineer antibodies to lower their pI and control their clearance rate from the circulation. An emerging strategy in cancer treatment involves the use of antibodies, also known as T cell or NK cell redirected antibodies, that can bind to receptors present on upregulated tumor-associated antigens (TAAs, or simply targets) as well as cancer-destroying immune cells (e.g., T cells or NK cells). While the immune cell redirection approach has a history of over 30 years, new technologies are overcoming the limitations of first-generation immune cell redirected antibodies, particularly by extending their half-life to allow for intermittent dosing, reducing immunogenicity, and improving safety profiles. Most commonly, T cell-redirecting bispecific antibodies (TRBAs) are generated by genetically swapping the complement-dependent region (CDR) of one arm of a Fab fragment with an antibody fragment that tightly binds to CD3 or CD137 (4-1BB) on T cells. However, in addition to these traditional T cell-engaging bispecific antibodies, a wide variety of other molecular structures, typically IgG types, have also been developed, as disclosed, for example, in Yu and Wang, J. Cancer Res. Clin. Oncol. 2019, 145, 941-956. Similarly, NK cell recruitment to the tumor microenvironment has been extensively investigated. NK cell engagement is typically based on the insertion of an antibody (fragment) that selectively binds to CD16, CD56, NKp46, or other NK cell-specific receptors into an IgG scaffold.

[0005]

[0005] A common strategy in the field of ADCs as well as in the field of immune cell engagement employs nihilation or removal of antibodies' ability to bind to Fc gamma receptors, which has multiple pharmaceutical implications. The primary consequence of removing Fc gamma receptor binding is, for example, a reduction in Fc gamma receptor-mediated uptake of antibodies by macrophages or megakaryocytes, which can result in dose-limiting toxicities, 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 can be beneficial because high mannose glycoforms are known to impair therapeutic efficacy by leading to nonspecific uptake by endogenous mannose receptors and rapid clearance, as described, 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). Furthermore, Van de Bovenkamp et al., J. Immunol. 2016, 196, 1435-1441 (incorporated by reference) describes how high mannose glycans can affect immunity. Inappropriate glycosylation of monoclonal antibodies can contribute to ineffective production from expressed Ig genes, as described by Reusch and Tejada, Glycobiology 2015, 25, 1325-1334 (incorporated by reference). In the field of immunotherapy, binding of glycosylated antibodies to Fc gamma receptors on immune cells can induce systemic activation of the immune system before the antibody binds to tumor-associated antigens, resulting in cytokine storm (cytokine release syndrome, CRS). Therefore, to reduce the risk of CRS, the majority of immune cell engagers in clinical settings are based on Fc-silencing antibodies that lack the ability to bind to Fc gamma receptors.Furthermore, various companies in the field of bispecific antibodies are adapting molecular structures to have a defined ratio of target binding to immune cell-engaging antibody domains. For example, Roche is developing T cell engagers based on asymmetric monoclonal antibodies that retain bivalent binding to TAA (e.g., CD20 or CEA) through both CDRs, but have an additional anti-CD3 fragment engineered into only one of the two heavy chains (2:1 ratio of target binding to CD3 binding). Similar strategies can be employed for T cell engagement / activation with anti-CD137 (4-1BBB) or NK cell engagement / activation with anti-CD16, CD56, NKp46, or other NK cell-specific receptors.

[0006]

[0006] The abrogation of binding to Fc gamma receptors can be achieved in various ways, for example, by specific mutations of the antibody (specifically, the Fc fragment) or by the addition of Fc fragments (C HGlycan removal can be achieved by genetic modification of the Fc domain, such as the N297Q or T299A mutation, or by enzymatic removal of glycans after recombinant expression of the antibody, using, for example, PNGase F or endoglycosidases. For example, endoglycosidase H is known to trim high-mannose and hybrid glycoforms but not complex-type glycans, whereas endoglycosidase S can trim complex-type glycans and, to some extent, hybrid glycans but cannot trim high-mannose forms. Endoglycosidase F2 can trim complex-type glycans (but not hybrids), and endoglycosidase F3 can only trim complex-type glycans that are also 1,6-fucosylated. Another endoglycosidase, endoglycosidase D, can hydrolyze only Man5 (M5) glycans. A detailed activity summary of various endoglycosidases is disclosed in Freeze et al., Curr. Prot. Mol. Biol., 2010, 89:17.13A.1-17, which is incorporated herein by reference. An additional advantage of deglycosylation of proteins for therapeutic applications is facilitated batch-to-batch consistency and significantly improved homogeneity.

[0007]

[0007] In the field of ADCs, chemical linkers are typically employed to attach pharmaceutical agents to antibodies. This linker must possess several important attributes, including the requirement for stability in plasma for an extended period of time after drug administration. A stable linker allows localization of the ADC to a planned site or cell in the body and prevents premature release of the payload into the circulation, which would indiscriminately induce any kind of undesirable biological response, thereby reducing the therapeutic index of the ADC. Upon internalization, the ADC should be processed so that the payload can be effectively released and bind to its target.

[0008]

[0008] There are two families of linkers: non-cleavable and cleavable. Non-cleavable linkers consist of a chain of atoms between the antibody and the payload that is completely stable under physiological conditions, regardless of the organ or biological compartment in which the antibody-drug conjugate resides. As a result, release of the payload from an ADC containing a non-cleavable linker relies on complete (lysosomal) degradation of the antibody after cellular internalization of the ADC. This degradation results in the release of the linker and the payload, which still retains peptide fragments and / or amino acids derived from the antibody to which the linker was originally attached. Cleavable linkers exploit the unique properties of cells or cell compartments for selective release of the payload from the ADC, generally leaving no trace of the linker after metabolic processing. There are three commonly used mechanisms for cleavable linkers: 1) sensitivity to specific enzymes, 2) pH sensitivity, and 3) sensitivity to the redox state of the cell (or its microenvironment).

[0009] Enzyme-based strategies are generally based on the endogenous presence of specific proteases, esterases, glycosidases, etc. For example, the majority of ADCs used in oncology utilize the predominant proteases found in tumor cell lysosomes to recognize and cleave specific peptide sequences in the linker. Dubowchik et al., Bioconjug Chem. 2002, 13, 855-69, incorporated by reference, pioneered the discovery of specific dipeptides as the intracellular cleavage mechanism by cathepsins. Other enzymes known to be upregulated in tumor lysozyme or the tumor microenvironment include plasmin, matrix metalloproteinases (MMPs), urokinase, etc., all of which can recognize specific peptide sequences in ADCs and induce the release of the payload from the linker by hydrolytic cleavage of one of the peptide bonds. Esterases can also be employed for intracellular release of payloads via hydrolysis of ester bonds; for example, human carboxylesterase 2 (CES2, hiCE) demonstrated in vivo antitumor efficacy of a doxorubicin prodrug against CES2-positive xenografts that was superior to or comparable to the in vivo antitumor efficacy of the payload itself, as demonstrated by Barthel et al., J. Med. Chem. 2012, 55, 6595-6607, which are incorporated by reference. Third, various glycosidases, particularly galactosidases (for removal of galactose) or glucuronidases (for removal of glucuronic acid), as exemplified by Torgov et al., Bioconj. Chem. 2005, 16, 717-721 and Jeffrey et al., J. Med. Chem. 2006, 17, 831-840, respectively, which are incorporated by reference, can be employed for selective cleavage of specific monosaccharides. Other endogenous enzymes that can be employed for tumor-specific hydrolytic cleavage of the bond are, for example, phosphatases or sulfatases.

[0010]

[0010] In addition to the use of endogenous enzymes, local concentration enhancement of any selected enzyme that may not be abundant in nature can be achieved by strategies such as systemic administration by intravenous injection, intratumoral injection, or other methods such as ADEPT (antibody-directed enzyme prodrug therapy).

[0011] Acid-sensitive strategies take advantage of the low pH of the endosomal (pH 5-6) and lysosomal (pH 4.8) compartments of human cells compared to the cytosol (pH 7.4) to trigger hydrolysis of acid-labile groups in linkers, such as hydrazones. See, e.g., Ritchie et al., mAbs 2013, 5, 13-21, incorporated by reference. Alternative acid-sensitive linkers, such as those based on silyl ethers, as disclosed in U.S. Patent Application Publication No. 20180200273, can also be employed.

[0012]

[0012] A third release strategy based on a redox mechanism takes advantage of intracellular glutathione, which is at a higher concentration than in plasma. Thus, a linker containing a disulfide bridge releases a free thiol group upon reduction by glutathione, which can remain part of the payload or can further self-immolate to release the free payload. An alternative reduction mechanism for the release of the free payload can be based on the conversion of an (aromatic) nitro group or an (aromatic) azido group to an aniline, which can be part of the payload or part of a self-immolative assembly unit.

[0013]

[0013] The self-immolative building block of an antibody-drug conjugate links the drug unit to the rest of the conjugate or its drug-linker intermediate. The primary function of the self-immolative building block is to conditionally release the free drug at the site targeted by the ligand unit. The activatable self-immolative moiety comprises an activatable group and a self-immolative spacer unit. Activation of the activatable group, for example by enzymatic conversion of an amide group to an amino group or by reduction of a disulfide to a free thiol group, initiates a self-immolative reaction sequence that results in the release of the free drug by one or more of a variety of mechanisms, which may involve (transient) 1,6-elimination of a p-aminobenzyl group to a p-quinone methide, optionally with the release of carbon dioxide and / or followed by a second cyclization-release mechanism. The self-immolative building block can be part of a chemical spacer (via a functional group) connecting the antibody and payload. Alternatively, the self-immolative group is not an inherent part of the chemical spacer, but is branched from the chemical spacer connecting the antibody and payload.

[0014]

[0014] The majority of antibody-drug conjugates approved for marketing or currently in late-stage clinical trials employ one of the above mechanisms for the release of the active drug. For example, Adcetris® is an ADC used to treat various hematological malignancies. It consists of a CD30-targeting antibody (ligand) connected to the highly potent tubulin inhibitor MMAE (payload) via a linker consisting of a cathepsin-sensitive fragment connected to a self-immolative p-aminobenzyloxycarbonyl group (PAB). The same mechanism for MMAE release is active for polatuzumab-vedotin (Polivy®). Other ADCs in pivotal trials employing protease / peptidase-sensitive linkers are SYD985, ADCT-402, ASG-22CE, and DS-8201a. Protease-mediated release of payloads is also part of the design of RG7861 (DSTA4637S), an ADC being developed in areas outside of oncology, specifically to treat bacterial infections.

[0015] Two ADCs consisting of antibodies linked to a DNA-damaging payload (calicheamicin) via acid-sensitive groups, specifically hydrazone groups, have been approved (Besponsa® and Mylotarg®). Similarly, sacituzumab govitecan, an ADC in Phase III clinical trials, employs payload release via acid hydrolysis of a carbonate group. Glutathione-sensitive disulfide groups are part of the linker in mirvetuximab soravtansine to connect the antibody to the maytansinoid payload DM4, and also in IMGN853. Currently, more than 75 ADCs are in various stages of clinical trials, at least 70% of which contain some form of cleavable linker.

[0016] As mentioned above, the self-immolative unit is at least part of the linker of many ADCs, most often an (acylated) para-aminobenzyl unit connected to a protease-sensitive peptide fragment for enzymatic release of the amino group. In addition to the aminobenzyl group, other aromatic moieties, such as heteroaromatic moieties such as pyridine or thiazole, can also be employed as moieties for the self-immolative unit. See, for example, U.S. Pat. No. 7,754,681 and U.S. Patent Application Publication No. 2005 / 0256030. Substitution of the aminobenzyl group can be at the para or ortho position, and in both cases can result in the same 1,6-elimination mechanism. The benzyl position can be substituted with an alkyl or carbonyl derivative, such as an ester or amide derived from mandelic acid, as disclosed, for example, in WO 2015 / 038426, which is incorporated by reference. The benzyl position of the self-immolative unit is typically connected to a heteroatom leaving group, such as, but not limited to, an oxygen- or nitrogen-based one. There is primarily a benzyl functionality of the carbamate moiety, which releases carbon dioxide via a 1,6-elimination mechanism, and a primary or secondary amino group. The primary or secondary amino group may be part of the toxic payload itself and may be an aromatic or aliphatic amino group. In the latter case, the amino group of the free payload likely has a higher pKa and is therefore protonated primarily under physiological conditions (pH 7-7.5), and particularly in the acidic environment of tumors (pH < 7).

[0017]

[0017] The primary or secondary amino group may be part of another self-immolative group, such as an N,N-dialkylethylenediamine moiety. On the other hand, the N,N-dialkylethylenediamine moiety may be connected to another carbamate group, which upon cyclization can liberate an alcohol group as part of a toxic payload, as demonstrated, for example, by Elgersma et al., Mol. Pharm. 2015, 12, 1813-1835, which is incorporated by reference. The primary or secondary amino group of the carbamate moiety may also form part of an N,O-acetal, a method used in several drug delivery strategies, for example, to release 5-fluorouracil (Madec-Lougerstay et al., J. Chem. Soc. Perkin Trans I, 1999, 1369-1375) and SN-38 (Santi et al., J. Med. Chem. 2014, 57, 2303-2314). More recently, a similar approach was adopted by Kolakowski et al., Angew. Chem. Int. Ed. 2016, 55, 7948-7951, which are incorporated by reference, to design a linker with prolonged serum exposure due to the long circulation time of the ADC, in combination with a beta-glucuronidase-promoted release mechanism to release an aliphatic alcohol. The functional group at the benzylic position of the self-immolative aromatic moiety can also be a phenolic oxygen (see, e.g., Toki et al., J. Org. Chem. 2002, 67, 1866-1872 and U.S. Pat. No. 7,553,816, which are incorporated by reference), but cannot be an aliphatic alcohol, as aliphatic alcohols do not have sufficient leaving group ability (typically pKa 13-15). Another option for the benzyl functionality is a quaternary ammonium group that releases a trialkylamino group or a heteroarylamine upon elimination, as reported by Burke et al., Mol. Cancer Ther. 2016, 15, 938-945 and Staben et al., Nat. Chem. 2016, 8, 1112-1119, which are incorporated by reference.

[0018]

[0018] Currently, payloads utilized in ADCs include microtubule-disrupting agents (e.g., monomethyl auristatin E (MMAE) and maytansinoid-derived DM1 and DM4), DNA-damaging agents (e.g., calicheamicin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine dimers, duocarmycins, anthracyclines), topoisomerase inhibitors (e.g., SN-38, exatecan and its derivatives, simitecan), or RNA polymerase II inhibitors (e.g., amanitin). While ADCs have demonstrated clinical and preclinical activity, it remains unclear which factors, in addition to antigen expression on targeted tumor cells, determine such efficacy. For example, drug:antibody ratio (DAR), ADC binding affinity, payload potency, receptor expression level, internalization rate, transport, multidrug resistance (MDR) status, and other factors have all been implicated in affecting the outcome of ADC treatment in vitro. In addition to directly killing antigen-positive tumor cells, ADCs also have the ability to kill neighboring antigen-negative tumor cells—the so-called “bystander killing” effect—as first reported by Sahin et al., Cancer Res. 1990, 50, 6944–6948 and studied, for example, by Li et al., Cancer Res. 2016, 76, 2710–2719. Generally speaking, neutral cytotoxic payloads exhibit bystander killing, whereas ionic (charged) payloads do not, as a result of the fact that ionic species do not readily cross cell membranes by passive diffusion. For example, as disclosed by Ogitani et al., Cancer Sci. 2016, 107, 1039–1046, incorporated by reference, evaluation of various exatecan derivatives showed that acylation of the primary amine with hydroxyacetic acid provided a derivative (DXd) with substantially enhanced bystander killing compared to various aminoacylated exatecan derivatives.

[0019] A drawback of most clinical trials and marketed ADCs in this field is that the toxic payload can induce dose-limiting off-target toxicity, as outlined by Donaghy et al., MAbs 2016, 8, 659-71, which is incorporated by reference. For example, it was demonstrated by Thon et al., Blood 2012, 120, 1975-84, which is incorporated by reference, that ADCs can be taken up by differentiating hematopoietic stem cells, resulting in the release of the toxic payload, inhibition of megakaryocyte proliferation and differentiation, and thus preventing platelet production and ultimately leading to thrombocytopenia. Similarly, the instability of the hydrazone linker is believed to have contributed to safety issues with Mylotarg®, which was withdrawn from the market in 2010 (but later reintroduced). It has been shown that linkers designed for proteolytic cleavage by cathepsins can also be cleaved by other enzymes, such as the esterase Ces1c (reported by Dorywalska et al., Mol. Cancer Ther. 2016, 15, 958-970, incorporated by reference). Indeed, even in the absence of cathepsin B, peptide-based cleavable linkers readily undergo cellular processing to release free payloads, as demonstrated by Caculitan et al., Cancer Res. 2017, 7027-7037, incorporated by reference. Furthermore, Zhao et al. (Mol. Cancer Ther. 2017, 16, 1866-1876, incorporated by reference) demonstrated that excretion of elastase by differentiating neutrophils can cause premature release of toxic payloads and is one of the causes of neutropenia, a common adverse event in cancer patients treated with MMAE-based ADCs.

[0020]

[0020] Antibody conjugates known in the art can suffer from several drawbacks. For antibody-drug conjugates, a measure of the toxin loading on the antibody is given by the drug-antibody ratio (DAR), which gives the average number of active substance molecules per antibody. In general, two general approaches can be identified for the production of ADCs: one via random (stochastic) conjugation to endogenous amino acids, and one involving conjugation to one or more specific sites in the antibody, which can be natural sites on the antibody or sites engineered into the antibody for such purposes.

[0021]

[0021] Methods for preparing ADCs by stochastic conjugation typically yield products with DARs between 2.5 and 4; however, in practice, such ADCs contain mixtures of antibody conjugates with numbers of target molecules varying from 0 to 8 or more. In other words, antibody conjugates formed by stochastic conjugation typically have DARs with high standard deviations. For example, gemtuzumab ozogamicin is a heterogeneous mixture of 50% conjugated antibody (0 to 8 calicheamicin moieties per IgG molecule, with an average of 2 or 3 randomly linked to solvent-exposed lysine residues of the antibody) and 50% unconjugated antibody (Bross et al., Clin. Cancer Res. 2001, 7, 1490; Labrijn et al., Nat. Biotechnol. 2009, 27, 767, both incorporated by reference). For brentuximab vedotin (Adcetris®), Kadcyla® (T-DM1), and other ADCs in the clinic, exactly how many drugs are attached to any given antibody remains uncontrollable, and thus the ADC results as a statistical distribution of conjugates, with the majority having a DAR of 3 to 4. One approach to achieving a higher DAR is by reducing all (four) interchain disulfide bonds in the monoclonal antibody, thereby liberating a total of eight cysteine ​​side chains as free thiols, followed by overall conjugation with a maleimide-functionalized payload to reach a final DAR of between 6 and 8. This methodology has been applied to a variety of clinical-stage ADCs, including, for example, IMMU-132, IMMU-110, DS-8201a, U3-1402, SGN-CD48a, and SGN-CD228A, and can be applied to a variety of payloads, but is less suitable for antibodies other than IgG1 due to fragment scrambling during the reduction step.

[0022] Many techniques for bioconjugation are known, as summarized in GT Hermanson, "Bioconjugate Techniques," Elsevier, 3rd Edition 2013, which is incorporated by reference. Two main techniques can be recognized for the preparation of ADCs by random conjugation, based on acylation of the lysine side chain or based on alkylation of the cysteine ​​side chain. Acylation of the ε-amino group of the lysine side chain is typically achieved by subjecting the protein to a reagent based on an activated ester or activated carbonate derivative, for example, SMCC is applied in the production of Kadcyla®. The main chemistry for alkylating the thiol group of the cysteine ​​side chain is based on the use of maleimide reagents, as applied, for example, in the production of Adcetris®. In addition to standard maleimide derivatives, a range of maleimide variants are also applicable for more stable cysteine ​​conjugation, 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 of which are incorporated by reference. Another important technique for conjugation to cysteine ​​side chains is via disulfide bonds, a bioactivatable linkage that has been utilized to reversibly connect protein toxins, chemotherapeutic drugs, and probes to carrier molecules (see, e.g., Pillow et al., Chem. Sci. 2017, 8, 366-370).Other approaches for cysteine ​​alkylation include nucleophilic substitution of haloacetamides (typically bromoacetamide or iodoacetamide) (see, e.g., Alley et al., Bioconj. Chem. 2008, 19, 759-765, both of which are incorporated by reference), or reaction 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 of which are incorporated by reference), phosphonamidates (p phosphonamidates (see, e.g., Kasper et al., Angew. Chem. Int. Ed. 2019, 58, 11625-11630, which are incorporated by reference), reactions with allenamides (see, e.g., Abbas et al., Angew. Chem. Int. Ed. 2014, 53, 7491-7494, which are incorporated by reference), reactions with cyanoethynyl reagents (see, e.g., Kolodych et al., Bioconj. Chem. 2015, 26, 197-200, which are incorporated by reference), reactions with vinyl sulfones (see, e.g., Gil These involve various approaches based on nucleophilic addition to unsaturated bonds, such as reaction with vinylpyridine (see, e.g., de Montes et al., Chem. Sci. 2019, 10, 4515-4522) or with vinylpyridine (see, e.g., https: / / iksuda.com / science / permalink / (accessed January 7, 2020)). 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.

[0023]

[0023] While the majority of clinical ADCs (approximately 65%) are based on random payload attachment, there is a clear trend toward site-specific conjugated ADCs, based on the finding that site-specific ADCs offer improved therapeutic indices. To this end, several methods have been developed that enable the creation of antibody-drug conjugates with defined DARs by site-specific conjugation to one or more predetermined site(s) on an antibody. Site-specific conjugation is typically achieved by engineering specific amino acids (or sequences) into the antibody to serve as anchor points for payload attachment (see, e.g., Aggerwal and Bertozzi, Bioconj. Chem. 2014, 53, 176-192, incorporated by reference), most typically cysteines. Moreover, over the past decade, a range of other site-specific conjugation techniques have been explored, most notably the genetic coding of unnatural amino acids, such as p-acetophenylalanine, suitable for oxime ligation, or p-azidomethylphenylalanine, suitable for click chemistry conjugation. Most approaches based on genetic redesign of antibodies result in ADCs with a DAR of approximately 2.An alternative approach to antibody conjugation that does not involve antibody redesign involves reduction of interchain disulfide bridges followed by the use of cysteine ​​bridging reagents, such as bis-sulfone reagents (see, e.g., Balan et al., Bioconj. Chem. 2007, 18, 61-76 and Bryant et al., Mol. Pharmaceuticals, 2007, 18, 61-76, both incorporated by reference). 2015, 12, 1872-1879), mono- or bis-bromomaleimides (see, e.g., Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269, both of which are incorporated by reference), bis-maleimide reagents (see, e.g., WO 2014114207), bis(phenylthio)maleimides (see, e.g., Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269 and Aubrey et al., Bioconj. Chem. 2018, 29, 3516-3521, both of which are incorporated by reference), bis-bromopyridazinediones (see, e.g., Robinson et al., RSC Advances 2017, 7, 9073-9077), bis(halomethyl)benzenes (see, e.g., Ramos-Tomillero et al., Bioconj. Chem. 2018, 29, 1199-1208, incorporated by reference), or other bis(halomethyl)aromatics (see, e.g., WO 2013173391). Typically, ADCs prepared by crosslinking of cysteines have a drug-antibody loading ratio (DAR4) of about 4.

[0024] Based on enzymatic remodeling of native antibody glycans at N297 (trimming with endoglycosidases and introduction of azide-modified GalNAc derivatives under the action of glycosyltransferases), followed by attachment of a cytotoxic payload using click chemistry, homogeneous ADCs can be prepared and selectively tailored to DAR2 or DAR4, as shown in WO 2014065661; van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242; and Verkade et al., Antibodies 2018, 7, 12, all of which are incorporated by reference. ADCs prepared by this technique were found to exhibit significantly increased therapeutic indices compared to a range of other conjugation techniques, including the glycan remodeling conjugation technique currently being applied clinically in ADCT-601 (ADC Therapeutics).

[0025] A similar enzymatic approach to converting antibodies into azide-modified antibodies, reported by Lhospice et al., Mol. Pharmaceut. 2015, 12, 1863-1871, incorporated by reference, employs the bacterial enzyme transglutaminase (BTG or TGase). Deglycosylation of the native glycosylation site N297 with PNGase F liberates the adjacent N295 to become a substrate for TGase-mediated introduction, which was shown to convert the deglycosylated antibody into a bis-azido antibody when subjected to an azide-bearing molecule in the presence of TGase. Subsequent reaction of the bis-azido antibody with a DBCO-modified cytotoxin yielded an ADC bearing DAR2. A genetic method based on C-terminal TGase-mediated azide introduction followed by conversion of the ADC via metal-free click chemistry was reported by Cheng et al., Mol. Cancer Therap. 2018, 17, 2665-2675, incorporated by reference.

[0026] Other methods for introducing azides into antibodies have been reported, based on prior genetic modification of the antibody followed by the introduction of unnatural amino acids using the genetic code based on AMBER suppression codons, as demonstrated, for example, by Axup et al., Proc. Nat. Acad. Sci. 2012, 109, 16101-16106, which is incorporated by reference. Similarly, Zimmerman et al., Bioconj. Chem. 2014, 25, 351-361, which is incorporated by reference, employ cell-free protein synthesis to introduce azidomethylphenylalanine (AzPhe) into a monoclonal antibody for conversion to an ADC via metal-free click chemistry. Also, in this case, an ADC with a DAR of 2, or a DAR of 4 if two AzPhe amino acids are introduced first, is prepared. It has also been shown by Nairn et al., Bioconj. Chem. 2012, 23, 2087-2097, which is incorporated by reference, that methionine analogs such as azidohomoalanine (Aha) can be introduced into proteins by auxotrophic bacteria and further converted into protein conjugates by (copper-catalyzed) click chemistry. Finally, pyrrolysyl-tRNA synthetase / tRNA CUA The genetic coding of aliphatic azides into recombinant proteins using the pair was demonstrated by Nguyen et al., J. Am. Chem. Soc. 2009, 131, 8720-8721, which is incorporated by reference, and labeling was achieved by click chemistry. This latter method should also be applicable to generating DAR2 ADCs, similar to the method reported by Oller-Salvia et al., Angew. Chem. Int. Ed. 2018, 57, 2831-2834.

[0027]

[0027] It has also been shown by Bruins et al., Bioconjugate Chem. 2017, 28, 1189-1193, which is incorporated by reference, that antibodies can be site-specifically conjugated to cytotoxic payloads by tyrosinase-mediated oxidation of appropriately positioned tyrosines via intermediate 1,2-quinones that can subsequently undergo cycloaddition with strained alkynes or alkenes.

[0028]

[0028] Chemical approaches have also been developed for the site-specific modification of antibodies without prior genetic modification, as highlighted, for example, by Yamada and Ito, ChemBioChem. 2019, 20, 2729-2737.

[0029]

[0029] Chemical conjugation with affinity peptides (CCAP) for site-specific modification has been developed by Kishimoto et al., Bioconj. Chem. 2019, using peptides that bind with high affinity to human IgG-Fc, thereby enabling the selective modification of a single lysine in the Fc fragment with a biotin moiety or a cytotoxic payload. Similarly, Matsuda et al., ACS Omega 2019, 4, 20564-20570, demonstrated that a similar approach (AJICAP™ technology) can be applied to the site-specific introduction of a thiol group into a single lysine of an antibody heavy chain. CCAP or AJICAP™ technology can also be employed to introduce azide groups or other functional groups.

[0030]

[0030] While the majority of ADCs on the market and in clinical settings have a drug load of approximately 2-8, as noted above, a lower DAR may be preferable for most PBD dimers, related IGN-type payloads, and some highly cytotoxic payloads, such as enediyne-based payloads, such as amanitin. It has been shown that the maximum tolerated dose in humans for highly potent payloads can be well below 1 mg / kg, typically even below 300 μg / kg or even below 100 μg / kg. As a result, in vivo receptor saturation is not achieved after administration (typically intravenous), leading to suboptimal tumor uptake and enhanced clearance. In such cases, a DAR1 format with the same payload may be preferable, as the MTD is likely two-fold higher compared to a similar DAR2 version. Ruddle et al., ChemMedChem 2019, 14, 1185-1195, describes a DAR1 conjugate as a C H1 and C L We recently demonstrated that DAR1-type Fab fragments can be prepared from antibody Fab fragments (prepared by papain digestion of whole antibodies or recombinant expression) by selective reduction of the interchain disulfide chains followed by re-crosslinking of the fragments by treatment 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. In the follow-up publications White et al., MAbs 2019, 11, 500-515, and also WO 2019034764, both of which are incorporated by reference, it was shown that DAR1 conjugates can also be prepared from full IgG antibodies after pre-design of the antibody: either antibodies with only one intrachain disulfide bridge in the hinge region are used (Flexmab technology, as reported in Dimasi et al., J. Mol. Biol. 2009, 393, 672-692, which is incorporated by reference), or antibodies with an additional free cysteine ​​are used, which can be obtained by mutation of a natural amino acid (e.g., HC-S239C) or by insertion into the sequence (e.g., HC-i239C, as reported by Dimasi et al., Mol. Pharmaceut. 2017, 14, 1501-1516). Both engineered antibodies were shown to enable the generation of DAR1 ADCs by reacting the resulting cysteine-engineered ADC with a bis-maleimide-derived PBD dimer. The Flexmab-derived DAR1 ADC was shown to be highly resistant to payload loss in serum and exhibited potent antitumor activity in a HER2-positive gastric cancer xenograft model. Furthermore, this ADC was well tolerated in rats at twice the dose compared to a site-specific DAR2 ADC prepared using a single maleimide-containing PBD dimer. However, the minimal effective dose (MED) of the DAR1 ADC increased by the same factor of 2 compared to the DAR2 ADC, so no improvement in the therapeutic window was observed.

[0031] To date, no DAR1 technology has been reported that improves the therapeutic index compared to DAR2 ADCs. Furthermore, no technology has been reported for generating DAR1 ADCs from intact antibodies without requiring monoclonal antibody redesign. Both improving the therapeutic index and / or non-genetic approaches toward DAR1 ADCs would be significant contributions to the development of superior ADCs with faster time to clinic. Summary of the Invention

[0032] A technology is presented for converting full-length antibodies into stable, site-specific ADCs with a single drug load (DAR1) without requiring prior antibody redesign. This technology is applicable to any IgG isotype and allows the attachment of payloads ranging from small molecule cytotoxic agents to protein scaffolds (cytokines, scFvs), oligonucleotides, and more, to antibodies via a cycloaddition conjugation reaction. The procedure, according to a preferred embodiment, involves prior trimming of glycans with endoglycosidases, with the concomitant abrogation of Fc gamma receptor binding, thereby eliminating effector function.

[0033] The antibody-payload conjugate according to the present invention has the structure (1): [ka] (In the formula, a, b, and c are each independently 0 or 1; L 1 , L 2 and L 3 is a linker; D is the payload; BM is the branching part; Z is a connecting group that can be obtained by cycloaddition reaction. This is due to the following.

[0034]

[0034] The present invention further provides a method for preparing an antibody-payload conjugate according to the present invention, an intermediate compound in the preparation method, and a medical use of an antibody-payload conjugate according to the present invention. [Brief explanation of the drawings]

[0035] [Figure 1]

[0023] Figure 1 shows a representative (but not comprehensive) set of functional groups (F) in biomolecules, either naturally occurring or introduced by design, that, upon reaction with a reactive group, yield a connecting group Z. The functional group F can be artificially introduced (designed) into a biomolecule at any selected position. The pyridazine connecting group (bottom row) is the product of rearrangement of the tetraazabicyclo[2.2.2]octane connecting group, formed by reaction of a tetrazine with an alkyne, with loss of N. As used herein, X can be a halogen, and X can be H, alkyl, or pyridyl. The connecting groups Z of structures (10e)-(10h) are preferred connecting groups for use in the present invention. [Figure 2] Figure 1 shows the structures of some derivatives of the UDP sugar of galactosamine, which can be modified, for example, with an azidoacetyl group (11b) or an azidodifluoroacetyl group (11c) at position 2, or with an azido group (11d) at position 6 of N-acetylgalactosamine. Monosaccharides (i.e., with the UDP removed) are the preferred moieties Su for use in the present invention. [Figure 3] Figure 1 shows a general method for non-genetic conversion of monoclonal antibodies into glycan-remodeling antibodies containing two azide groups (one at either native glycosylation site). Upon reaction with a bivalent cyclooctyne construct, a single payload (R) is attached to the bis-azide antibody. Such clipping can also be achieved by copper-catalyzed click reaction using a bivalent construct bearing two terminal acetylene groups (not shown). [Figure 4]Figure 1 shows cyclooctynes ​​suitable for metal-free click chemistry. This list is not comprehensive; for example, alkynes can be further activated by fluorination, substitution of the aromatic ring, or introduction of heteroatoms into the aromatic ring. [Figure 5]

[0023] Figures 1A and 1B show examples of R groups present in the bivalent constructs of Figures 3 and 4, which are defined as payloads in antibody-drug conjugates. The R group can be attached to the bivalent construct via a cleavable moiety, such as the peptide cleavable linker depicted in the top structure. Acid-cleavable linkers or disulfide-based linkers, or linkers that cleave by yet another mechanism, can also be used (not shown). The R group can also be attached via a non-cleavable linker (bottom structure). The R group itself can be, for example, but not limited to, a cytotoxic molecule. [Figure 6]

[0023] Figure 1 shows a bivalent cyclooctyne construct suitable for generating a DAR1 ADC by clipping onto a bis-azido antibody in which two cyclooctyne moieties are attached to two sites of a payload with a dimeric structure, such as a PBD dimer or a duocarmycin dimer. The linker may be cleavable or non-cleavable, as illustrated for the PBD dimer. The dimeric cytotoxic payload need not necessarily be symmetrical in nature as in the illustrated example; for example, a combination of a duocarmycin monomer and a PBD monomer is also possible. [Figure 7] FIG. 1 shows an indirect approach to attaching payloads in the DAR1 format by using a trivalent cyclooctyne construct that reacts with a bisazide-mAb, leaving one cyclooctyne free for subsequent click chemistry (exemplified with an azide-modified payload; other options could be click chemistry with nitrones, nitrile oxides, diazo compounds, tetrazines, etc.). [Figure 8]Figure 1 shows various options for trivalent constructs for reaction with bis-glycan-modified mAbs. Trivalent constructs can be homotrivalent or heterotrivalent (2 + 1 format). Homotrivalent constructs (X = Y) can consist of 3 x cyclooctyne, 3 x acetylene, 3 x maleimide, or 3 x other thiol-reactive groups. Heterotrivalent constructs (X ≠ Y) can consist of, for example, two cyclooctyne groups and one maleimide group, or one trans-cyclooctene group. Heterotrivalent constructs with any combination of X and Y can exist, as long as X and Y are not mutually reactive (e.g., BCN + tetrazine). [Figure 9] FIG. 1 shows various bivalent BCN reagents (105, 107, 118, 125, 129, 134), trivalent BCN reagents (143, 145, 150), monovalent BCN reagents for sortagging (157, 161, 163, 168) or monovalent tetrazine reagents for sortagging (154). [Figure 10] FIG. 1 shows various bivalent or trivalent cross-linkers (XL07-XL13). [Figure 11] FIG. 1 shows various antibody variants as starting materials for subsequent conversion into antibody conjugates. [Figure 12] FIG. 1 shows various bis-BCN-modified cytotoxic drugs based on MMAE or MMAF for crosslinking with bis-azide-modified antibodies to generate DAR1 ADCs. [Figure 13] FIG. 1 shows various additional bis-BCN-modified cytotoxic drugs based on MMAE (303), PBD dimer (304), calicheamicin (305), or PNU159,682 (306) for crosslinking with bis-azide-modified antibodies to generate DAR1 ADCs. [Figure 14] FIG. 1 shows various MMAE- or MMAF-based cyclooctynes ​​(BCN, DIBO, DBCO, with various inter-cyclooctyne linker variations) or azide-containing bivalent cytotoxic drugs for crosslinking with bis-azide- or bis-alkyne-modified antibodies to generate DAR1 ADCs. [Figure 15] FIG. 1 shows the structures of two monovalent linear linker-drugs based on BCN-MMAE (312) or azido-MMAF (313). [Figure 16] Figure 1 shows SDS-PAGE analysis: Lane 1 - rituximab; Lane 2 - rit-v1a; Lane 3 - rit-v1a-145; Lane 4 - rit-v1a-(201)2; Lane 5 - rit-v1a-145-204; Lane 6 - rit-v1a-145-PF01; Lane 7 - rit-v1a-145-PF02. Gels were stained with Coomassie to visualize total protein. Samples were analyzed by 6% SDS-PAGE under non-reducing conditions (left) and 12% SDS-PAGE under reducing conditions (right). [Figure 17] Figure 1 shows RP-HPLC traces of B12-v1a (top trace) and B12-v1a-145 (bottom trace). Samples were digested with IdeS prior to RP-HPLC analysis. [Figure 18] Figure 1 shows SDS-PAGE analysis: lane 1—trast-v1a; lane 2—trast-v1a-XL11; lanes 3 and 4—trast-v1a-XL11-PF01; lane 5—rit-v1a; lane 6—rit-v1a-XL11; lanes 7 and 8—rit-v1a-XL11-PF01. Gels were stained with Coomassie to visualize total protein. Samples were analyzed by 6% SDS-PAGE under non-reducing conditions (left) and 12% SDS-PAGE under reducing conditions (right). [Figure 19] FIG. 1 shows RP-HPLC data for deglycosylated trastuzumab after treatment with bis-BCN-MMAE LD03(=303). [Figure 20] Figure 1 shows SDS-PAGE analysis on a 6% gel under non-reducing conditions: Lane 1 - rituximab; Lane 2 - rit-v1a-(201)2; Lane 3 - rit-v1a-145-PF08; Lane 4 - B12-v1a-145-PF01; Lane 5 - B12-v1a-145-PF08. The gel was stained with Coomassie to visualize total protein. Lanes 1 and 2 are included as references for the unconjugated mAb and the 2:2 molecule format. [Figure 21] Figure 1 shows SDS-PAGE analysis on a 6% gel under non-reducing conditions: Lane 1 - rit-v1a-(201)2; Lane 2 - rit-v1a-145-PF01; Lane 3 - rit-v1a; Lane 4 - rit-v1a-PF22; Lane 5 - trast-v1a-PF22. The gel was stained with Coomassie to visualize total protein. Lanes 1 and 2 are included as references for the unconjugated mAb and 2:2 molecule format. [Figure 22] Figure 1 shows SDS-PAGE analysis on a 6% gel under non-reducing conditions: Lane 1 - trast-v1a; Lane 2 - trast-v1a-PF23. The gel was stained with Coomassie to visualize total protein. Lane 1 is included as a reference for unconjugated mAb. [Figure 23] Figure 1 shows SDS-PAGE analysis on a 6% gel under non-reducing conditions: lane 1 - rit-v1a; lane 2 - rit-v1a-(201)2; lane 3 - rit-v1a-145-PF01; lane 4 - rit-v1a-PF22; lane 5 - rit-v1a-PF23. The gel was stained with Coomassie to visualize total protein. Lanes 1-4 are included as references for unconjugated mAb, 2:1, and 2:2 molecule formats. [Figure 24] Figure 1 shows SDS-PAGE analysis on a 6% gel under non-reducing conditions: lane 1 - rit-v1a-145; lane 2 - rit-v1a-145-PF09; lane 3 - trast-v1a-145; lane 4 - trast-v1a-145-PF09; lane 5 - rit-v1a; lane 6 - rit-v1a-(PF07)2; lane 7 - trast-v1a; lane 8 - trast-v1a-(PF07)2. The gel was stained with Coomassie to visualize total protein. [Figure 25]Non-reducing SDS-page analysis: lane 1 - Trast-v1a-(PF.)1-2; lane 2 - Trast-v1a-(209)1-2; lane 3 - Trast-v1a-(PF11)1-2; lane 4 - Trast-v1a; lane 5 - Trast-v1a-145-PF12; lane 6 - Trast-v1a-145. The gel was stained with Coomassie to visualize total protein. [Figure 26] SDS-PAGE analysis on a 6% gel under non-reducing conditions: lane 1 - rit-v1a-145; lane 2 - rit-v1a-145-PF17; lane 3 - trast-v1a-145; lane 4 - trast-v1a-145-PF17. The gel was stained with Coomassie to visualize total protein. [Figure 27] Figure 1 shows SDS-PAGE analysis on a 6% gel under non-reducing conditions: lane 1 - trast-v1a; lane 2 - trast-v1a-PF29; lane 3 - rit-v1a; lane 4 - rit-v1a-PF29. The gel was stained with Coomassie to visualize total protein. [Figure 28] Figure 1 shows the effect of hOKT3 200-based bispecific antibodies on RajiB tumor cell killing using human PBMCs. Bispecific antibodies and calculated EC50 values ​​are shown in the legend. B12-v1a-145-PF01 was included as a negative control. [Figure 29] Figure 1 shows the effect of anti-4-1BB PF31-based bispecific antibodies on RajiB tumor cell killing using human PBMCs. Bispecific antibodies and calculated EC50 values ​​are shown in the legend. B12-v1a-145-PF31 was included as a negative control. [Figure 30] Figure 1 shows cytokine levels in the supernatant of RajiB-PBMC co-cultures after incubation with a bispecific antibody based on hOKT3 200. Mouse OKT3 mIgG2a antibody (Invitrogen 16-0037-81) was included as a positive control. [Figure 31]Figure 1 shows cytokine levels in the supernatant of RajiB-PBMC co-cultures after incubation with anti-4-1BB PF31-based bispecific antibody. Mouse OKT3 mIgG2a antibody (Invitrogen 16-0037-81) was included as a positive control. DETAILED DESCRIPTION OF THE INVENTION

[0036] definition

[0066] The verb "to comprise" and its conjugations, when used in this specification and claims, are used in an open-ended sense to mean that items following this word are included, but items not specifically mentioned are not excluded. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that one and only one element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0037]

[0067] The compounds disclosed herein may contain one or more asymmetric centers, and various diastereomers and / or enantiomers of the compounds may exist. Any description of a compound herein is intended to include all diastereomers and mixtures thereof, unless otherwise specified. Furthermore, any description of a compound herein is intended to include both individual enantiomers and any mixtures of enantiomers, such as racemates, unless otherwise specified. When the structure of a compound is depicted as a specific enantiomer, it should be understood that the invention of this application is not limited to that specific enantiomer.

[0038]

[0068] Compounds can occur in various tautomeric forms. The compounds according to the present invention are meant to include all tautomeric forms unless otherwise specified. When the structure of a compound is depicted as a specific tautomer, it should be understood that the invention of this application is not limited to that specific tautomer.

[0039]

[0069] The compounds disclosed herein and in the claims may further exist as exo and endo diastereoisomers. Unless otherwise specified, any description of a compound in the specification and claims is meant to include both the individual exo diastereoisomers of the compound and the individual endo diastereoisomers and mixtures thereof. When the structure of a compound is depicted as a particular endo or exo diastereomer, it should be understood that the invention of this application is not limited to that particular endo or exo diastereomer.

[0040]

[0070] Furthermore, the compounds disclosed herein may exist as cis and trans isomers. Unless otherwise specified, any description of a compound in this specification and claims is intended to include both the individual cis and trans isomers of the compound and mixtures thereof. For example, if the structure of a compound is depicted as a cis isomer, it should be understood that the corresponding trans isomer or mixtures of cis and trans isomers are not excluded from the present invention. If the structure of a compound is depicted as a specific cis or trans isomer, it should be understood that the present invention is not limited to that specific cis or trans isomer.

[0041]

[0071] The compounds according to the present invention may exist in the form of salts, which are also encompassed by the present invention. The salts are typically pharmaceutically acceptable salts containing pharmaceutically acceptable anions. The term "salt thereof" refers to a compound formed when an acidic proton, typically an acid proton, is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts not intended for administration to patients. For example, in the salt of a compound, the compound can be protonated with an inorganic or organic acid to form a cation with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0042]

[0072] The term "pharmaceutically acceptable" salt refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt with a counterion that has acceptable mammalian safety for a given dosage regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counterions known in the art, including, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, includes salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0043]

[0073] The term "protein" is used herein in its ordinary scientific sense. A polypeptide containing about 10 or more amino acids is considered a protein herein. Proteins can contain not only natural amino acids, but also unnatural amino acids.

[0044]

[0074] The term "monosaccharide" is used herein in its usual scientific sense to refer to an oxygen-containing heterocycle resulting from intramolecular hemiacetal formation upon cyclization of a chain of 5 to 9 (hydroxylated) carbon atoms, most commonly containing 5 carbon atoms (pentoses), 6 carbon atoms (hexoses), 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).

[0045]

[0075] The term "antibody" is used herein in its usual 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 is used broadly herein and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" is also used herein to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" includes whole immunoglobulins, but also antigen-binding fragments of antibodies. Furthermore, the term includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art. Typical examples of antibodies include abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, efalizumab, alemtuzumab, adalimumab, tositumomab-I131, cetuximab, ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, certolizumab pegol, golimumab, canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab, ipilimumab, and brentuximab, among others.

[0046]

[0076] An "antibody fragment" is defined herein as a portion of an intact antibody comprising its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments, diabodies, minibodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment(s), fragment(s) produced by a Fab expression library, or epitope-binding fragments of any of the above that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).

[0047]

[0077] An "antigen" is defined herein as the entity to which an antibody specifically binds.

[0048]

[0078] The terms "specific binding" and "specifically bind" are defined herein as a highly selective manner in which an antibody binds to the corresponding epitope of a target antigen but does not bind to many other antigens. Typically, an antibody or antibody derivative binds to at least about 1 x 10 -7 M, preferably 10 -8 M~10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M, and binds to a given antigen with an affinity that is at least two-fold higher than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein).

[0049]

[0079] The term "substantial" or "substantially" is defined herein as the majority of a population, mixture, or sample, i.e., greater than 50%, preferably greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the population.

[0050]

[0080] A "linker" is defined herein as a moiety that connects two or more elements of a compound. For example, in an antibody conjugate, the antibody and the payload are covalently connected to each other via a linker. The linker may include one or more linkers and spacer moieties that connect various moieties within the linker.

[0051]

[0081] A "polar linker" is defined herein as a linker containing a structural element with the specific purpose of increasing the polarity of the linker, thereby improving its aqueous solubility. Polar linkers may include, for example, one or more units selected from ethylene glycol, a carboxylic acid moiety, a sulfonate moiety, a sulfone moiety, an acylated sulfamide moiety, a phosphate moiety, a phosphinate moiety, an amino group, or an ammonium group, or a combination thereof.

[0052]

[0082] A "spacer" or spacer moiety is defined herein as a moiety that spaces (i.e., provides distance between) and covalently connects two (or more) portions of a linker. A linker can be, for example, part of a linker construct, a linker conjugate, or a bioconjugate, as defined below.

[0053]

[0083] A "self-immolative group" is defined herein as a portion of the linker in an antibody-drug conjugate that functions to conditionally release a free drug at the site targeted by the ligand unit. The activatable self-immolative moiety comprises an activatable group (AG) and a self-immolative spacer unit. Activation of the activatable group, for example by enzymatic conversion of an amide group to an amino group or by reduction of a disulfide to a free thiol group, initiates a self-immolative reaction sequence that results in the release of the free drug by one or more of a variety of mechanisms, which may involve (transient) 1,6-elimination of a p-aminobenzyl group to a p-quinone methide, optionally with the release of carbon dioxide and / or followed by a second cyclization-release mechanism. The self-immolative building block may be part of a chemical spacer (via a functional group) connecting the antibody and payload. Alternatively, the self-immolative group is not an inherent part of the chemical spacer, but is branched from the chemical spacer connecting the antibody and payload.

[0054]

[0084] An "activatable group" is defined herein as a functional group attached to an aromatic group that can undergo a biochemical processing step, such as proteolytic hydrolysis of an amide bond or reduction of a disulfide bond, which initiates a self-immolative process of the aromatic group. An activatable group may also be referred to as an "activating group."

[0055]

[0085] A "bioconjugate" is defined herein as a compound in which a biomolecule is covalently attached to a payload via a linker. A bioconjugate comprises one or more biomolecules and / or one or more payloads. Antibody conjugates, such as antibody-payload conjugates and antibody-drug conjugates, are bioconjugates in which the biomolecule is an antibody.

[0056]

[0086] A "biomolecule" is defined herein as any molecule that can be isolated from nature or that is composed of small building blocks that are constituents of naturally occurring macromolecular structures, particularly nucleic acids, proteins, glycans, and lipids. Examples of biomolecules include enzymes, (non-catalytic) proteins, polypeptides, peptides, amino acids, oligonucleotides, monosaccharides, oligosaccharides, polysaccharides, glycans, lipids, and hormones.

[0057]

[0087] The term "payload" refers to a moiety that is covalently attached to a targeting moiety, such as an antibody, but also to a molecule that is released from the conjugate upon cleavage of the linker. Thus, payload refers to a monovalent moiety with one open end that is covalently attached to a targeting moiety via a linker, referred to in the context of the present invention as D, and also to a molecule that is released therefrom.

[0058]

[0088] The term "2:1 molecular format" refers to a protein conjugate consisting of a bivalent monoclonal antibody (IgG type) conjugated to a monofunctional payload.

[0059] Antibody-payload conjugates according to the present invention

[0089] The present invention relates to a compound having the structure (1): [ka] (In the formula, a, b, and c are each independently 0 or 1; L 1 , L 2 and L 3 is a linker; D is the payload; BM is the branching part; Z is a connecting group that can be obtained by cycloaddition reaction. The present invention relates to an antibody-payload conjugate having the formula:

[0060]

[0090] In the antibody-payload conjugate (1), the payload D comprises a connecting group Z, an optional linker L 1 , L 2 and L 3 and a branching moiety BM, which is connected to the antibody AB. In (1), a, b, and c are each independently selected from 0 and 1. A preferred antibody-payload conjugate according to the present invention has a=b=1, i.e., L 1 and L 2 and more preferably both L 1 and L 2 are the same. Particularly preferred are the occurrences of Z, a / b and L 1 / L 2 are symmetric antibody-payload conjugates in which each of

[0061]

[0091] In a preferred embodiment, the antibody is conjugated to the payload D via a glycan, in which case the antibody-payload conjugate according to the invention has the structure (5): [ka] (In the formula, e is an integer ranging from 0 to 10; Su is a monosaccharide; G is a monosaccharide moiety; GlcNAc is an N-acetylglucosamine moiety; Fuc is a fucose moiety; d is 0 or 1) It has.

[0062] Antibody AB

[0092] In (1), AB is an antibody. Preferably, AB 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 of 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 may also be an Fc fragment.

[0063]

[0093] The GlcNAc moiety in (5) is preferably present at a natural N-glycosylation site in the Fc fragment of antibody AB. Preferably, said GlcNAc moiety is attached to an asparagine amino acid in the region 290-305 of AB. In a further preferred embodiment, the antibody is an IgG type antibody, and depending on the particular IgG type antibody, said GlcNAc moiety is present on amino acid asparagine 297 (Asn297 or N297) of AB.

[0064] Connecting group Z

[0094] In antibody-payload conjugate (1), Z is a connecting group. As described in more detail above, the term "connecting group" refers to a structural element that connects one portion of a compound to another portion of the same compound. In (1), Z, when present, is L 1 and / or L 2 The antibody is connected to the branching moiety BM via a spacer, possibly via a spacer. 1 and / or L 2 Whether is present or absent depends on the values ​​of a and b. In a preferred embodiment, both occurrences of Z are the same.

[0065]

[0095] As will be appreciated by those skilled in the art, the nature of the connecting group will depend on the type of cycloaddition reaction by which the connection between the moieties of the compound is obtained, for example, Z can be obtained by a [4+2] cycloaddition or a 1,3-dipolar cycloaddition.

[0066]

[0096] Cycloaddition reactions for attaching a reactive group Q to a reactive group F are known in the art. As a result, a wide variety of connecting groups Z may be present in conjugates according to the invention. In one embodiment, the connecting group Z is preferably selected from the options described above, as depicted in Figure 1.

[0067]

[0097] For example, when F comprises or is an alkynyl group, the complementary group Q comprises an azide group and the corresponding connecting group Z is as shown in FIG.

[0068]

[0098] For example, when F comprises or is an azido group, the complementary group Q comprises an alkynyl group and the corresponding connecting group Z is as shown in FIG.

[0069]

[0099] For example, when F comprises or is a cyclopropenyl, trans-cyclooctene, or cycloalkyne group, the complementary group Q comprises a tetrazinyl group, and the corresponding connecting group Z is as shown in Figure 1. In certain cases, Z is simply an intermediate structure that expels N2, thereby producing a dihydropyridazine (from reaction with an alkene) or a pyridazine (from reaction with an alkyne).

[0070]

[0100] For example, when F comprises or is a tetrazinyl group, the complementary group Q comprises a cyclopropenyl group, a trans-cyclooctene group, or a cycloalkyne group, and the corresponding connecting group Z is as shown in Figure 1. In certain cases, Z is simply an intermediate structure that expels N2, thereby generating a dihydropyridazine (from reaction with an alkene) or a pyridazine (from reaction with an alkyne).

[0071]

[0101] Additional suitable combinations of F and Q, and the nature of the resulting connecting group Z, are known to those skilled in the art and are described, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd Edition 2013 (ISBN: 978-0-12-382239-0), which is incorporated by reference, in particular Chapter 3, pages 229 to 258. A list of complementary reactive groups suitable for bioconjugation methods is disclosed in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd Edition 2013 (ISBN: 978-0-12-382239-0), Chapter 3, pages 230 to 232, Table 3.1, the contents of which are expressly incorporated herein by reference.

[0072]

[0102] In a preferred embodiment, the connecting group Z is according to any one of the structures (Za), (Ze) to (Zh), (Zj) and (Zk) defined below. Preferably, Z is according to the structure (Za), (Ze) or (Zj): [ka] This is due to the following.

[0073] In this specification, X 9 is H, C 1~12 alkyl and pyridyl; 1~12 Alkyl is preferably C 1~4 It is preferably alkyl, and most preferably methyl.

[0074] In structures (Zg) and (Zh), [ka] The bond may represent either a single or double bond and may be connected to the linker L via either side of the bond.

[0075] The wavy line indicates the connection to the linker L. The connectivity depends on the precise nature of Q and F. Any of the linking groups (Za) to (Zh) can be connected to L, but the leftmost of these groups drawn is (L 1 ) a / (L 2 ) b It is preferable that the oscillating element is connected to

[0076]

[0103] The connecting group (Zh) typically rearranges to (Zg) with the liberation of N2.

[0077]

[0104] In preferred embodiments, each Z independently contains a moiety selected from the group consisting of a triazole, cyclohexene, cyclohexadiene, isoxazoline, isoxazolidine, pyrazoline, piperazine, thioether, amide, or imide group. It is particularly preferred that a triazole moiety is present in Z.

[0078]

[0105] In a particularly preferred embodiment, the connecting group Z comprises a triazole moiety and has the structure (Zj): [ka] This is due to the following.

[0079] As used herein, R 15 , X 10 , u, u' and v are as defined for (Q36), all preferred embodiments of which apply equally to (Zj). The wavy lines indicate adjacent parts (Su and (L 1 ) a or (L 2 ) b ) and the connectivity depends on the precise nature of Q and F. Either part of the connecting group by (Zj) can be connected to (L 1 ) a / (L 2 ) b Although it is preferred that the wavy bond above depicted represents the connectivity to Su, the connecting groups according to structures (Zf) and (Zk) are preferred embodiments of the connecting group according to (Zj).

[0080]

[0106] In a particularly preferred embodiment, the connecting group Z comprises a triazole moiety and has the structure (Zk): [ka] This is due to the following.

[0081]

[0107] As used herein, R 15 , R 18 , R 19 , and l are as defined for (Q37), all preferred embodiments of which apply equally to (Zj). The wavy lines denote adjacent moieties (Su and (L 1 ) a or (L 2 ) b ) and the connectivity depends on the precise nature of Q and F. Either part of the connecting group by (Zj) can be connected to (L 1 ) a , but it is preferred that the left wavy line join depicted represents connectivity with Su.

[0082]

[0108] In a preferred embodiment, Q comprises or is an alkyne moiety and F is an azide moiety, such that the connecting group Z comprises a triazole moiety. Preferred connecting groups comprising a triazole moiety are connecting groups according to structure (Ze) or (Zj), and connecting groups according to structure (Zj) are preferably according to structure (Zk) or (Zf). In a preferred embodiment, the connecting group is according to structure (Zj), more preferably according to structure (Zk) or (Zf).

[0083] Branching part BM

[0109] A "branched moiety" in the context of the present invention refers to a moiety that is embedded in a linker that connects three moieties. In other words, the branched moiety contains at least three bonds to other moieties: one bond to a reactive group F, a connecting group Z, or a payload D, one bond to a reactive group Q or a connecting group Z, and one bond to a reactive group Q or a connecting group Z.

[0084]

[0110] Any moiety containing at least three bonds to other moieties is suitable as a branched moiety in the context of the present invention. Suitable branched moieties include carbon atoms (BM-1), nitrogen atoms (BM-3), phosphorus atoms (phosphines (BM-5) and phosphine oxides (BM-6)), aromatic rings such as phenyl rings (e.g., BM-7) or pyridyl rings (e.g., BM-9), (hetero)cyclic rings (e.g., BM-11 and BM-12), and polycyclic moieties (e.g., BM-13, BM-14, and BM-15). Preferred branched moieties are selected from carbon atoms and phenyl rings, with BM being most preferably a carbon atom. Structures (BM-1) to (BM-15) are depicted below, where the three branches, i.e., the bonds to other moieties as defined above, are shown. * It is shown by ( * (bonds labeled with ).

[0085] [ka]

[0086]

[0111] In (BM-1), * one of the branches labeled with can be a single or double bond, [ka] In (BM-11) to (BM-15), the following applies: n, p, q, and q are each independently an integer ranging from 0 to 5, preferably 0 or 1, most preferably 1; W 1 , W 2 and W 3 Each of C(R 21 ) w and N independently selected; W 4 , W 5 and W 6 Each of C(R 21 ) w+1 , N(R 22 ) w , independently selected from O and S; each [ka] represents a single or double bond; w is 0 or 1 or 2, preferably 0 or 1; Each R 21 are hydrogen, OH, C1-C 24 Alkyl groups, C1-C 24 Alkoxy group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, independently selected from the group consisting of C1-C 24 Alkyl groups, C1-C 24 Alkoxy group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted and include O, S, and NR 3 and R is optionally interrupted by one or more heteroatoms selected from 3 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; Each R 22 is hydrogen, C1 to C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, independently selected from the group consisting of C1-C 24 Alkyl groups, C1-C 24 Alkoxy group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted and include O, S, and NR 3and R is optionally interrupted by one or more heteroatoms selected from 3 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.

[0087]

[0112] Those skilled in the art will recognize the value of w and [ka] are interdependent. Thus, whenever an occurrence of W is attached to an endocyclic double bond, w=1 for that occurrence of W, and whenever an occurrence of W is attached to two endocyclic single bonds, w=0 for that occurrence of W. For BM-12, at least one of o and p is not 0.

[0088]

[0113] Representative examples of branched moieties according to structures (BM-11) and (BM-12) include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, aziridine, azetidine, diazetidine, oxetane, thietane, pyrrolidine, dihydropyrrolyl, tetrahydrofuranyl, dihydrofuranyl, thiolanyl, imidazolinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, piperidinyl, oxanyl, thianyl, piperazinyl, morpholino, thiomorpholino, dioxanyl, trioxanyl, dithianyl, trithianyl, azepanyl, oxepanyl, and thiepanyl. Preferred cyclic moieties for use as branching moieties include cyclopropenyl, cyclohexyl, oxanyl (tetrahydropyran), and dioxanyl. The substitution pattern of the three branches determines whether the branching moiety is of structure (BM-11) or structure (BM-12).

[0089]

[0114] Representative examples of branched moieties having structures (BM-13) to (BM-15) include decalin, tetralin, dialin, naphthalene, indene, indane, isoindene, indole, isoindole, indoline, and isoindoline.

[0090]

[0115] In a preferred embodiment, BM is a carbon atom. If the carbon atom is of structure (BM-1) and has a total of four bonds with separate moieties, the carbon atom is chiral. The stereochemistry of the carbon atom is not important to the present invention and may be S or R. The same applies to phosphine (BM-6). Most preferably, the carbon atom is of structure (BM-1). In the carbon atom of structure (BM-1), * One of the branches indicated by may be a double bond, in which case the carbon atom may be part of an alkene or imine. When BM is a carbon atom, it may be part of a larger functional group such as an acetal, ketal, hemiketal, orthoester, orthocarbonate, amino acid, etc. This also applies when BM is a nitrogen or phosphorus atom, in which case the nitrogen or phosphorus atom may be part of an amide, imide, imine, phosphine oxide (as in BM-6), or phosphotriester.

[0091]

[0116] In a preferred embodiment, BM is a phenyl ring. Most preferably, the phenyl ring is according to structure (BM-7). The substitution pattern of the phenyl ring can be of any positional chemistry, such as a 1,2,3-substituted phenyl ring, a 1,2,4-substituted phenyl ring, or a 1,3,5-substituted phenyl ring. To allow optimal flexibility and conformational freedom, it is preferred that the phenyl ring is according to structure (BM-7), and most preferably the phenyl ring is 1,3,5-substituted. The same applies to the pyridine ring of (BM-9).

[0092]

[0117] In a preferred embodiment, the branching moiety BM is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cyclic or a polycyclic moiety.

[0093] Linker

[0118] L 1 , L 2 and L 3 Each of the linking units may be absent or present, but preferably all three linking units are present. 1 , L 2 and L 3 Each of the atoms, when present, is independently a chain of at least two, preferably 5 to 100, atoms selected from C, N, O, S, and P. As used herein, a chain of atoms refers to the shortest chain of atoms emerging from the end of a linking unit. The atoms in the chain may also be referred to as backbone atoms. As one skilled in the art will recognize, atoms with three or more valences, such as C, N, and P, may be appropriately functionalized to satisfy the valences of these atoms. In other words, the backbone atoms are optionally functionalized. In a preferred embodiment, L 1 , L 2 and L 3 Each, when present, is independently a chain of at least 5-50, preferably 6-25 atoms selected from C, N, O, S and P. The backbone atoms are preferably selected from C, N and O.

[0094]

[0119] Linker L 1 and L 2 connects the BM to the reactive moiety Q or the connecting group Z. 1 and L 2 are both present, i.e., a=b=1, and more preferably, L 1 and L 2 In a particularly preferred embodiment, (L 1 ) a -Z is (L 2 ) b -Z, and (L 1 ) a -Q is (L 2 ) b Same as -Q.

[0095]

[0120] L 1 , L 2 and L 3 is a straight chain or branched C1-C200 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 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group and C9-C 200 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted and include O, S, and NR 3 and R is optionally interrupted by one or more heteroatoms selected from the group 3 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 and cycloalkyl groups, wherein the alkyl, alkenyl, alkynyl, and cycloalkyl groups are optionally substituted. When the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are interrupted by one or more heteroatoms as defined above, it is preferred that said groups are interrupted by one or more O atoms and / or one or more S-S groups.

[0096]

[0121] More preferably, L 1 and L 2 If present, linear or branched C1-C 100 Alkylene group, C2-C 100 Alkenylene group, C2-C 100 Alkynylene group, C3-C100 Cycloalkylene group, C5-C 100 Cycloalkenylene group, C8-C 100 Cycloalkynylene group, C7-C 100 Alkylarylene group, C7-C 100 Aryl alkylene group, C8-C 100 Arylalkenylene group and C9-C 100 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted, and wherein O, S, and NR 3 and R is optionally interrupted by one or more heteroatoms selected from the group 3 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 and independently selected from the group consisting of cycloalkyl groups, wherein alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0097]

[0122] Even more preferably, L 1 and L 2 If present, 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 Cycloalkynylene group, C7-C 50 Alkylarylene group, C7-C 50 Aryl alkylene group, C8-C 50 Arylalkenylene group and C9-C 50and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted, and wherein O, S, and NR 3 and R is optionally interrupted by one or more heteroatoms selected from the group 3 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 and independently selected from the group consisting of cycloalkyl groups, wherein alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0098]

[0123] Even more preferably, L 1 and L 2 If present, 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 Cycloalkynylene group, C7-C 20 Alkylarylene group, C7-C 20 Aryl alkylene group, C8-C 20 Arylalkenylene group and C9-C 20 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted, and wherein O, S, and NR 3 and R is optionally interrupted by one or more heteroatoms selected from the group 3 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 and independently selected from the group consisting of cycloalkyl groups, wherein alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0099]

[0124] In these preferred embodiments, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are unsubstituted and are not substituted with O, S, or NR 3 , optionally interrupted by one or more heteroatoms, preferably selected from the group of O, and R 3 It is further preferred that are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0100]

[0125] Most preferably, L 1 and L 2 If present, linear or branched C1-C 20 alkylene groups, wherein the alkylene groups are optionally substituted; and 3 and R is optionally interrupted by one or more heteroatoms selected from the group 3 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 In this embodiment, the alkylene group is unsubstituted and is selected from the group consisting of O, S, and NR 3 , optionally interrupted by one or more heteroatoms, preferably selected from the group O and / or SS, and R 3 It is further preferred that are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0101]

[0126] Preferred linkers L 1 and L 2 -(CH2) n1 -, -(CH2CH2) n1 -, -(CH2CH2O) n1 -, -(OCH2CH2) n1 -, -(CH2CH2O) n1 CH2CH2-, -CH2CH2(OCH2CH2) n1 -, -(CH2CH2CH2O) n1 -, -(OCH2CH2CH2) n1 -, -(CH2CH2CH2O) n1 CH2CH2CH2- and -CH2CH2CH2(OCH2CH2CH2) n1 - (wherein n1 is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, and even more preferably in the range of 1 to 15). More preferably, n1 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.

[0102]

[0127] In one embodiment, L 3 is absent and c=0. In an alternative more preferred embodiment, L 3 exists and c=1. L 3 If there is L 3 L 1 and L 2 may be the same as or different from, and are preferably different from

[0103]

[0128] In a preferred embodiment, L 3 L 4 , L 5 , L 6 and L 7 Thus, in one embodiment, L 3 But-(L 4 ) n -(L 5 ) o-(L 6 ) p -(L 7 ) q -(In the formula, L 4 , L 5 , L 6 and L 7 are linkers which together form the linker L, further defined below; and n, o, p, and q are independently 0 or 1. In a preferred embodiment, at least the linker L 4 and L 5 is present (i.e., n=1; o=1; p=0 or 1; q=0 or 1), and more preferably a linker L 4 , L 5 and L 6 exists, and L 7 is present or absent (i.e., n=1; o=1; p=1; q=0 or 1). In one embodiment, the linker L 4 , L 5 , L 6 and L 7 (i.e., n=1; o=1; p=1; q=1). In one embodiment, the linker L 4 , L 5 and L 6 exists, and L 7 is absent (i.e., n=1; o=1; p=1; q=0). In one embodiment, n+o+p+q=1, 2, 3 or 4, preferably 2, 3 or 4, more preferably 3 or 4. In a preferred embodiment, L 5 and L 6 are present together, i.e., o+p=2. Most preferably, n+o+p+q=4.

[0104]

[0129] Linker L 3 is the connecting group Z formed when the payload D is connected to the linker construct, which can be either before or after reaction of the linker construct (particularly the reactive moiety Q) with the functionalized antibody (particularly the reactive moiety F). 3 The linker L 3 The connecting group in the 4 , L 5 , L 6 and L 7or a linker L 3 For example, L 3 Ha-Z 3 -(L 4 ) n -(L 5 ) o -(L 6 ) p -(L 7 ) q -or-(L 4 ) n -Z 3 -(L 5 ) o -(L 6 ) p -(L 7 ) q As used herein, Z can take any form, and is preferably as further defined below for the connecting group obtained by reaction of Q with F.

[0105] Linker L 4

[0130] Linker L 4 is absent (n=0) or present (n=1). Preferably, the linker L 4 where n=1. L 4 is, for example, a 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 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200Optionally, the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, and the heteroatoms are preferably O, S(O), y and NR 15 wherein y is 0, 1 or 2, preferably y=2; and R 15 are hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups.

[0106]

[0131] L 4 may contain (poly)ethylene glycol diamines (e.g., 1,8-diamino-3,6-dioxaoctane or equivalents containing longer ethylene glycol chains), polyethylene glycol or polyethylene oxide chains, polypropylene glycol or polypropylene oxide chains, and 1,z-diaminoalkanes, where z is the number of carbon atoms in the alkane (z may be an integer in the range of, for example, 1 to 10).

[0107]

[0132] In a preferred embodiment, the linker L 4 contains an ethylene glycol group, a carboxylic acid moiety, a sulfonate moiety, a sulfone moiety, a phosphate moiety, a phosphinate moiety, an amino group, an ammonium group, or a sulfamide group.

[0108]

[0133] In a preferred embodiment, the linker L 4 is a sulfamide group, preferably structure (23): [ka] Contains a sulfamide group according to

[0109]

[0134] The wavy line indicates the remainder of the compound, typically BM and L 5 , L 6 , L 7 or D, preferably BM and L 5 Preferably, (O) a The C(O) moiety is connected to the BM, and the NR 13 The part is L 5 , L 6 , L 7 or D, preferably L 5 is connected to.

[0110]

[0135] In structure (23), a1=0 or 1, preferably a1=1, and R 13 is hydrogen, C1 to C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted and include O, S, and NR 14 and R is optionally interrupted by one or more heteroatoms selected from 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.

[0111]

[0136] Or, R 13 is a D connected to N, possibly via a spacer moiety. In one embodiment, this connection is via a spacer moiety Sp 2 and preferably D is further defined below, -(B) e1 -(A)f1 -(B) g1 via -C(O)- or -(B) e1 -(A) f1 -(B) g1 -C(O)-(L 5 ) o -(L 6 ) p -(L 7 ) q In another embodiment, R 13 is also connected to the first instance of payload D such that a cyclic structure is formed. For example, N is part of a piperazine moiety that is connected to D through a carbon or nitrogen atom, preferably through the second nitrogen atom of the piperazine ring. Preferably, the cyclic structure, e.g., the piperazine ring, is -(B) e1 -(A) f1 -(B) g1 via -C(O)- or -(B) e1 -(A) f1 -(B) g1 -C(O)-(L 5 ) o -(L 6 ) p -(L 7 ) q - is connected to D.

[0112]

[0137] In a preferred embodiment, R 13 is hydrogen or C1-C 20 is an alkyl group, more preferably R 13 is hydrogen or C1-C 16 alkyl group, and even more preferably R 13 is hydrogen or C1-C 10 alkyl groups, wherein the alkyl groups are optionally substituted, and O, S, and NR 14 , and optionally interrupted by one or more heteroatoms selected from O, 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. In a preferred embodiment, R 13 is hydrogen. In another preferred embodiment, R 13 C1~C20 Alkyl groups, more preferably C1 to C 16 Alkyl groups, even more preferably C1-C 10 is an alkyl group, optionally interrupted by one or more O atoms, and optionally substituted with an —OH group, preferably a terminal —OH group. In this embodiment, R 13 It is further preferred that R is a (poly)ethylene glycol chain containing a terminal -OH group. 13 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl, more preferably from the group consisting of hydrogen, methyl, ethyl, n-propyl and i-propyl, even more preferably from the group consisting of hydrogen, methyl and ethyl. Even more preferably, R 13 is hydrogen or methyl, and most preferably R 13 is hydrogen.

[0113]

[0138] In a preferred embodiment, L 4 Structure (24): [ka] This is due to the following.

[0114]

[0139] In this specification, a and R 13 is as defined above, and Sp 1 and Sp 2 are independently a spacer moiety, and b1 and c1 are independently 0 or 1. Preferably, b1=0 or 1 and c1=1, more preferably b1=0 and c1=1. In one embodiment, the spacer Sp 1 and Sp 2 Is straight chain 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 200Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group and C9-C 200 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted, and wherein O, S, and NR 16 and R is optionally interrupted by one or more heteroatoms selected from the group 16 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 and cycloalkyl groups, wherein the alkyl, alkenyl, alkynyl, and cycloalkyl groups are optionally substituted. When the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are interrupted by one or more heteroatoms as defined above, it is preferred that said groups are interrupted by one or more O atoms and / or by one or more S-S groups.

[0115]

[0140] More preferably, the spacer moiety Sp 1 and Sp 2 If present, 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-C 100 Cycloalkynylene group, C7-C 100 Alkylarylene group, C7-C 100Aryl alkylene group, C8-C 100 Arylalkenylene group and C9-C 100 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted, and wherein O, S, and NR 16 and R is optionally interrupted by one or more heteroatoms selected from the group 16 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 and independently selected from the group consisting of cycloalkyl groups, wherein alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0116]

[0141] Even more preferably, the spacer moiety Sp 1 and Sp 2 If present, 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 Cycloalkynylene group, C7-C 50 Alkylarylene group, C7-C 50 Aryl alkylene group, C8-C 50 Arylalkenylene group and C9-C 50 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted, and wherein O, S, and NR 16and R is optionally interrupted by one or more heteroatoms selected from the group 16 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 and independently selected from the group consisting of cycloalkyl groups, wherein alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0117]

[0142] Even more preferably, the spacer moiety Sp 1 and Sp 2 If present, 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 Cycloalkynylene group, C7-C 20 Alkylarylene group, C7-C 20 Aryl alkylene group, C8-C 20 Arylalkenylene group and C9-C 20 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are optionally substituted, and wherein O, S, and NR 16 and R is optionally interrupted by one or more heteroatoms selected from the group 16 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 and independently selected from the group consisting of cycloalkyl groups, wherein alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0118]

[0143] In these preferred embodiments, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are unsubstituted and are not substituted with O, S, or NR 16 , optionally interrupted by one or more heteroatoms, preferably selected from the group of O, and R 16 It is further preferred that are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0119]

[0144] Most preferably, the spacer moiety Sp 1 and Sp 2 If present, linear or branched C1-C 20 alkylene groups, wherein the alkylene groups are optionally substituted; and 16 and R is optionally interrupted by one or more heteroatoms selected from the group 16 is hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 In this embodiment, the alkylene group is unsubstituted and is selected from the group consisting of O, S, and NR 16 , optionally interrupted by one or more heteroatoms, preferably selected from the group O and / or SS, and R 3 It is further preferred that are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0120]

[0145] Therefore, the preferred spacer moiety Sp 1 and Sp 2 Ha-(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 - (wherein r is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, and even more preferably in the range of 1 to 15). More preferably, r 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.

[0121]

[0146] Alternatively, the preferred linker L 4 is -(W) k1 -(A) d1 -(B) e1 -(A) f1 -(C(O)) g1 -(In the formula, d1=0 or 1, preferably d1=1; e1=an integer in the range of 0 to 10, preferably e1=0, 1, 2, 3, 4, 5 or 6, preferably an integer in the range of 1 to 10, most preferably e1=1, 2, 3 or 4; f1=0 or 1, preferably f1=0; d1+e1+f1 is at least 1, preferably in the range of 1 to 5; preferably d1+f1 is at least 1, preferably d1+f1=1; g1=0 or 1, preferably g1=1; k1=0 or 1, preferably k1=1; A is a sulfamide group according to structure (23); B is a —CH—CH—O— or —O—CH—CH— moiety, or (B) e1 -(CH2-CH2-O)e3 a -CH2-CH2- moiety, where e3 is defined in the same way as e1; W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -C(O)(CH2) m C(O)-, -C(O)(CH2) m C(O)NH- or -(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, preferably W is -OC(O)NH-, -C(O)(CH2) m C(O)NH— or —C(O)NH—, where m is an integer ranging from 0 to 10, preferably m=0, 1, 2, 3, 4, 5, or 6, and most preferably m=2 or 3; Preferably, L 4 (A) d1 -(B) e1 to the BM via (C(O)) g1 , preferably via C(O) (L 5 ) o connected to It can be represented by:

[0122]

[0147] In the context of this embodiment, the wavy line in structure (23) represents (W) k1 , (B) e1 and (C(O)) g1 A is according to structure (23), a1=1, and R 13 =H or C1~C 20 Preferably, R is an alkyl group, more preferably R 13 =H or methyl, most preferably R 13 =H.

[0123]

[0148] Preferred linkers L 4 is as follows: (a) k1=0; d1=1; g1=1; f1=0; B=-CH2-CH2-O-; e1=1, 2, 3 or 4, preferably e1=2.

[0124] (b) k1 = 1; W = -C(O)(CH2) mC(O)NH-; m=2; d1=0; (B) e1 =-(CH2-CH2-O) e3 -CH2-CH2-; f1=0; g1=1; e3=1, 2, 3 or 4, preferably e1=1.

[0125] (c) k1 = 1; W = -OC(O)NH-; d1 = 0; B = -CH2-CH2-O-; g1 = 1; f1 = 0; e1 = 1, 2, 3 or 4, preferably e1 = 2.

[0126] (d) k1 = 1; W = -C(O)(CH2) m C(O)NH-; m=2; d1=0; (B) e1 =-(CH2-CH2-O) e3 -CH2-CH2-; f1=0; g1=1; e3=1, 2, 3 or 4, preferably e3=4.

[0127] (e) k1 = 1; W = -OC(O)NH-; d1 = 0; (B) e1 =-(CH2-CH2-O) e3 -CH2-CH2-; g1=1; f1=0; e3=1, 2, 3 or 4, preferably e3=4.

[0128] (f)k1=1;W=-(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, m=3; d1=0; (B) e1 =-(CH2-CH2-O) e3 -CH2-CH2-; g1=1; f1=0; e3=1, 2, 3 or 4, preferably e3=4.

[0129] (g) k1=0; d1=0; g1=1; f1=0; B=-CH2-CH2-O-; e1=1, 2, 3 or 4, preferably e1=2.

[0130] (h) k1 = 1; W = -C(O)NH-; d1 = 0; g1 = 1; f1 = 0; B = -CH2-CH2-O-; e1 = 1, 2, 3 or 4, preferably e1 = 2.

[0131]

[0149] In one embodiment, the linker L 4 However, BM and (L 5 ) oand preferably linked to the branched nitrogen atom via a linker. An example of a branched nitrogen atom is the nitrogen atom NR 13 and R 13 is connected to the second occurrence of D via a spacer moiety. Alternatively, the branched nitrogen atom may be a group having the structure -(W) k1 -(A) d1 -(B) e1 -(A) f1 -(C(O)) g1 -By L 4 In one embodiment, L 4 Ga-(W) k1 -(A) d1 -(B) e1 -(A) f1 -(C(O)) g1 -N * [-(A) d1 -(B) e1 -(A) f1 -(C(O)) g1 -]2, where A, B, W, d1, e1, f1, g1, and k1 are as defined above and are selected independently for each occurrence; * is -(A) d1 -(B) e1 -(A) f1 -(C(O)) g1 - is a branched nitrogen atom to which two instances of - are connected. As used herein, both (C(O)) g1 Part is -(L 5 ) o -(L 6 ) p -(L 7 ) q -D(in the formula, L 5 , L 6 , L 7 , o, p, q and D are as defined above and are each independently selected). In the most preferred embodiment, there are no such branch atoms and the linker L 4 does not contain a connection to a further moiety D.

[0132] Linker L 5

[0150] Linker L 5 is absent (o=0) or present (o=1). Preferably, the linker L 5 is present and o=1. A linker L 5 is a peptide spacer known in the art, preferably containing 2 to 5 amino acids, more preferably a dipeptide or tripeptide spacer, most preferably a dipeptide spacer. Although any peptide spacer can be used, preferably the linker L 5 is selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, more preferably Val-Cit, Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably Val-Cit, Val-Ala, Ala-Ala-Asn. 5 In one embodiment, L 5 =Val-Ala.

[0133]

[0151] In a preferred embodiment, L 5 The general structure (27): [ka] is expressed by

[0134]

[0152] As used herein, R 17 =CH3 or CH2CH2CH2NHC(O)NH2. The wavy line indicates (L 4 ) n and (L 6 ) p and preferably by structure (27) 5 through NH (L 4 ) n and through C(O) (L 6 ) p is connected to.

[0135] Linker L 6

[0153] Linker L 6 is absent (p=0) or present (p=1). Preferably, the linker L 6 is present and p=1. 6 is a self-cleavable spacer, also called a self-immolative spacer. Preferably, L 6 is a para-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (25).

[0136] [ka]

[0137]

[0154] In this specification, the wavy line represents (L 5 ) n and (L 7 ) p Typically, the PABC derivative is connected to (L 5 ) n to, and through O (L 7 ) p is connected to.

[0138]

[0155] R 3 is H, R 4 or C(O)R 4 and R 4 is optionally substituted and is O, S, and NR 5 (In the formula, R 5 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 24 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 (Hetero)aryl groups, C3-C 10 Alkyl (hetero)aryl groups and C3-C 10 (hetero)arylalkyl groups. Preferably, R 4 C3~C10 (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H. The polyalkylene glycol is most preferably polyethylene glycol, preferably —(CHCHO) s H (wherein s is an integer ranging from 1 to 10, preferably from 1 to 5, and most preferably s=1, 2, 3 or 4). More preferably, R 3 is H or C(O)R 4 and R 4 is 4-methyl-piperazine or morpholine. Most preferably, R 3 is H.

[0139] Linker L 7

[0156] Linker L 7 is absent (q=0) or present (q=1). Preferably, the linker L 7 is present and q=1. 7 is an aminoalkanoic acid spacer, i.e., -N-(C h -alkylene)-C(O)- (wherein h is an integer ranging from 1 to 20, preferably from 1 to 10, and most preferably from 1 to 6). 6 and to D via a carbonyl moiety. Preferred linkers L 7 is selected from 6-aminohexanoic acid (Ahx, h=6), β-alanine (h=2) and glycine (Gly, h=1), even more preferably 6-aminohexanoic acid or glycine. 7 = 6-aminohexanoic acid. In one embodiment, L 7 = glycine. Alternatively, the linker L 7 The structure is -N-(CH2-CH2-O) e6 -(CH2) e7An ethylene glycol spacer is -(C(O)- (wherein e6 is an integer ranging from 1 to 10, and e7 is an integer ranging from 1 to 3).

[0140] Payload D

[0157] In a preferred embodiment of the linker-conjugate according to the invention, the payload is selected from the group consisting of an active substance, a reporter molecule, a polymer, a solid surface, a hydrogel, a nanoparticle, a microparticle and a biomolecule. Particularly preferred payloads are active substances and reporter molecules, in particular active substances.

[0141]

[0158] The term "active substance" as used herein relates to pharmacological and / or biological substances, i.e., substances that are biologically and / or pharmaceutically active, such as drugs, prodrugs, diagnostic agents, proteins, peptides, polypeptides, peptide tags, amino acids, glycans, lipids, vitamins, steroids, nucleotides, nucleosides, polynucleotides, RNA or DNA. Examples of peptide tags include human lactoferrin or cell membrane-penetrating peptides such as polyarginine. An example of a glycan is oligomannose. An example of an amino acid is lysine.

[0142]

[0159] When the payload is an active substance, the active substance is preferably selected from the group consisting of drugs and prodrugs. More preferably, the active substance is selected from the group consisting of pharmaceutically active compounds, particularly low- to medium-molecular-weight compounds (e.g., about 200 to about 2500 Da, preferably about 300 to about 1750 Da). In a further preferred embodiment, the active substance is selected from the group consisting of cytotoxins, antivirals, antibacterials, peptides, and oligonucleotides. Examples of cytotoxins include colchicine, vinca alkaloids, anthracyclines, camptothecin, doxorubicin, daunorubicin, taxanes, calicheamicin, tublysin, irinotecan, inhibitory peptides, amanitin, debouganin, duocarmycin, maytansine, auristatins, enediynes, pyrrolobenzodiazepines (PBDs), indolinobenzodiazepine dimers (IGNs), and PNU159,682.

[0143]

[0160] The term "reporter molecule" as used herein refers to a molecule whose presence is easily detected, such as a diagnostic agent, dye, fluorophore, radioisotope label, contrast agent, magnetic resonance imaging agent or mass label.

[0144]

[0161] A wide variety of fluorophores, also called fluorescent probes, are known to those skilled in the art. Some fluorophores are described in more detail, for example, in G.T. Hermanson, "Bioconjugate Techniques," Elsevier, 3rd Edition 2013, Chapter 10: "Fluorescent probes," pp. 395-463, which is incorporated by reference. Examples of fluorophores include all types of Alexa Fluor (e.g., Alexa Fluor 555), cyanine dyes (e.g., Cy3 or Cy5) and cyanine dye derivatives, coumarin derivatives, fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, boron dipyrromethene derivatives, pyrene derivatives, naphthalimide derivatives, phycobiliprotein derivatives (e.g., allophycocyanin), chromomycin, lanthanide chelates, and quantum dot nanocrystals.

[0145]

[0162] Examples of radioisotope labels include DTPA (diethylenetriaminepentaacetic anhydride), DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid), DTTA (N 1 -(p-Isothiocyanatobenzyl)-diethylenetriamine-N 1 ,N 2 ,N 3 ,N 3 -tetraacetic acid), deferoxamine or DFA (N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide) or HYNIC (hydrazinonicotinamide) 99m Tc, 111 In, 114m In, 115 In, 18 F, 14 C. 64 Cu, 131 I, 125 I, 123 I, 212 Bi, 88 Y, 90 Y, 67 Cu, 186 Rh, 188 Rh, 66 Ga, 67 Ga and 10 Isotopic labeling techniques are known to those skilled in the art and are described in more detail, for example, in GT Hermanson, "Bioconjugate Techniques," Elsevier, 3rd Edition 2013, Chapter 12: "Isotopic labeling techniques," pp. 507-534, which is incorporated by reference.

[0146]

[0163] Polymers suitable for use as payload D in the compounds according to the invention are known to those skilled in the art, and some examples are described in more detail, for example, in G.T. Hermanson, "Bioconjugate Techniques," Elsevier, 3rd Edition 2013, Chapter 18: "PEGylation and synthetic polymer modification," pp. 787-838, which is incorporated by reference. When payload D is a polymer, it is preferably independently selected from the group consisting of poly(ethylene glycol) (PEG), polyethylene oxide (PEO), polypropylene glycol (PPG), polypropylene oxide (PPO), 1,x-diaminoalkane polymers (where x is the number of carbon atoms in the alkane, preferably x is an integer ranging from 2 to 200, preferably from 2 to 10), (poly)ethylene glycol diamines (e.g., 1,8-diamino-3,6-dioxaoctane and equivalents containing longer ethylene glycol chains), polysaccharides (e.g., dextran), poly(amino acids) (e.g., poly(L-lysine)), and poly(vinyl alcohol).

[0147]

[0164] Solid surfaces suitable for use as payload D are known to those skilled in the art. Solid surfaces include, for example, functional surfaces (e.g., surfaces of nanomaterials, carbon nanotubes, fullerenes, or viral capsids), metal surfaces (e.g., titanium, gold, silver, copper, nickel, tin, rhodium, or zinc surfaces), metal alloy surfaces (alloys derived from, for example, aluminum, bismuth, chromium, cobalt, copper, gallium, gold, indium, iron, lead, magnesium, mercury, nickel, potassium, plutonium, rhodium, scandium, silver, sodium, titanium, tin, uranium, zinc, and / or zirconium), polymer surfaces (polymers are, for example, polystyrene, polyvinyl chloride, polyethylene, polypropylene, poly(dimethylsiloxane), or polymethyl methacrylate, polyacrylamide), glass surfaces, silicone surfaces, chromatographic support surfaces (chromatographic supports are, for example, silica supports, agarose supports, cellulose supports, or alumina supports), etc. When payload D is a solid surface, it is preferred that D is independently selected from the group consisting of functional surfaces or polymer surfaces.

[0148]

[0165] Hydrogels are known to those skilled in the art. Hydrogels are water-swollen networks formed by cross-linking between polymer components. See, for example, A S. Offman, Adv. Drug Delivery Rev. 2012, 64, 18, which is incorporated by reference. When the payload is a hydrogel, it is preferred that the hydrogel be composed of poly(ethylene) glycol (PEG) as the polymer base.

[0149]

[0166] Suitable microparticles and nanoparticles for use as payload D are known to those skilled in the art. Various suitable microparticles and nanoparticles are described, for example, in G.T. Hermanson, "Bioconjugate Techniques," Elsevier, 3rd Edition 2013, Chapter 14: "Microparticles and nanoparticles," pp. 549-587, which is incorporated by reference. The microparticles or nanoparticles can be of any shape, such as spheres, rods, tubes, cubes, triangles, and cones. Preferably, the microparticles or nanoparticles are spherical in shape. The chemical composition of the microparticles and nanoparticles can vary. When payload D is a microparticle or nanoparticle, the microparticle or nanoparticle can be, for example, a polymer microparticle or nanoparticle, a silica microparticle or nanoparticle, or a gold microparticle or nanoparticle. When the particles are polymeric microparticles or nanoparticles, the polymer is preferably polystyrene or a copolymer of styrene (e.g., a copolymer of styrene and divinylbenzene, butadiene, acrylate, and / or vinyltoluene), polymethyl methacrylate (PMMA), polyvinyltoluene, poly(hydroxyethyl methacrylate (pHEMA) or poly(ethylene glycol dimethacrylate / 2-hydroxyethyl methacrylate) [poly(EDGMA / HEMA)]. Optionally, the surface of the microparticle or nanoparticle is modified, for example with a surfactant, such as by grafting a secondary polymer or covalent attachment of another polymer or spacer moiety.

[0150]

[0167] Payload D may be a biomolecule. Biomolecules, and preferred embodiments thereof, are described in more detail below. When payload D is a biomolecule, it is preferred that the biomolecule is selected from the group consisting of proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids, and monosaccharides.

[0151]

[0168] The DAR1 antibody-payload conjugate according to the present invention is particularly suitable for use with highly potent cytotoxins such as PBD dimers, indolinobenzodiazepine dimers (IGNs), enediynes, PNU159,682, duocarmycin dimers, amanitin, and auristatins, preferably PBD dimers, indolinobenzodiazepine dimers (IGNs), enediynes, or PNU159,682. In particularly preferred embodiments, the payload is selected from the group consisting of PBD dimers, indolinobenzodiazepine dimers (IGNs), enediynes, PNU159,682, duocarmycin dimers, amanitin, and auristatins, preferably PBD dimers, indolinobenzodiazepine dimers (IGNs), enediynes, or PNU159,682. In a further preferred embodiment, the payload is neither a symmetric payload nor a dimeric payload.

[0152]

[0169] In a preferred embodiment, the antibody-payload conjugate according to the invention is according to structure (5). The conjugate according to this embodiment is (G), further defined below. e and Su.

[0153]

[0170] Each of the two GlcNAc moieties in (4) is preferably present at a natural N-glycosylation site in the Fc fragment of antibody AB. Preferably, said GlcNAc moieties are attached to an asparagine amino acid in the region 290-305 of AB. In a further preferred embodiment, the antibody is an IgG type antibody, and, depending on the particular IgG type antibody, said GlcNAc moiety is present on amino acid asparagine 297 (Asn297 or N297) of the antibody.

[0154]

[0171] G is a monosaccharide moiety, and e is an integer ranging from 0 to 10. G is preferably selected from the group consisting of glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc), sialic acid, and xylose (Xyl). More preferably, G is selected from the group consisting of glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc).

[0155]

[0172] In a preferred embodiment, e is 0 and G is absent. If the antibody glycans have been trimmed, G is typically absent. Trimming refers to treatment with an endoglycosidase such that only the core GlcNAc moiety of the glycan remains.

[0156]

[0173] In another preferred embodiment, e is an integer ranging from 1 to 10. In this embodiment, it is even more preferred that G is selected from the group consisting of glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc) or sialic acid and xylose (Xyl), more preferably from the group consisting of glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc).

[0157]

[0174] If e is 3 to 10, (G) e can be linear or branched. Branched oligosaccharides (G) e Preferred examples are (a), (b), (c), (d), (e), (f), (h) and (h) shown below.

[0158] [ka]

[0159]

[0175] When G is present, it is preferred that G terminates with GlcNAc. In other words, the monosaccharide residue directly connected to Su is GlcNAc. The presence of a GlcNAc moiety facilitates the synthesis of functionalized antibodies, since the monosaccharide derivative Su can be easily introduced onto the terminal GlcNAc residue by glycosyltransfer. (G) having structures (a) to (h) e In the above preferred embodiment for, the moiety Su can be attached to any of the terminal GlcNAc residues, i.e., not those with a wavy bond, which are attached to a core GlcNAc residue on the antibody.

[0160]

[0176] It is particularly preferred that G is absent, i.e., e = 0. The advantage of antibody-payload conjugates (1) in which e = 0 is that binding to the Fc gamma receptors CD16, CD32 and CD64 is significantly reduced or completely abrogated when such conjugates are used clinically.

[0161]

[0177] Su is a monosaccharide derivative, also referred to as a sugar derivative. Preferably, the sugar derivative can be incorporated into the functionalized antibody by glycosyl transfer. See Figure 2 for some preferred examples of nucleotide-sugar derivatives that can be introduced. More preferably, Su is Gal, Glc, GalNAc, or GlcNAc, more preferably Gal or GalNAc, and most preferably GalNAc. The term derivative refers to (G) e and F.

[0162] Preparation method

[0178] The present invention also provides a method for preparing an antibody-payload conjugate having a hypothetical payload-antibody ratio of 1, comprising the steps of: (a) reacting a compound having structure (2) containing at least two reactive groups Q with an antibody having structure (3) functionalized with two reactive groups F to form: [ka] (In the formula, Ab is antibody; a, b, and c are each independently 0 or 1; L 1 , L 2 and L 3 is a linker; V is a reactive group Q' or a payload D; BM is the branching part; Q and F are reactive groups capable of undergoing a cycloaddition reaction that links them to form the connecting group Z. Functionalized antibody according to structure (1'): [ka] (wherein Z is a connecting group obtained by reaction of Q with F; The functionalized antibody according to structure (1') is an antibody-payload conjugate when V is a payload D; Or the functionalized antibody according to structure (1') is further reacted according to step (b) to obtain an antibody-payload conjugate where V is a payload D when V is a reactive group Q'. and obtaining; (b) if V=Q′, reacting the reactive group Q′ with a payload containing a reactive group F′ to obtain an antibody-payload conjugate wherein V is payload D; The present invention relates to a method comprising:

[0163]

[0179] In a preferred embodiment, the antibody having structure (3) has structure (3b): [ka] It has.

[0164]

[0180] In a preferred embodiment, the functionalized antibody according to structure (1) has the structure (5b): [ka] It has.

[0165]

[0181] The method according to the invention can take two main forms: one in which step (b) is not performed and one in which step (b) is performed.

[0166]

[0182] In one embodiment, step (b) is not performed and V present on the compound having structure (2) is payload D. In that case, step (a) directly provides the final conjugate (structure (1)). The method according to this preferred embodiment can be represented by Scheme 1:

[0167]

[0183] Scheme 1 [ka]

[0168]

[0184] As used herein, L B represents a trivalent linker according to structure (9), further defined above.

[0169] [ka]

[0170]

[0185] Thus, in a preferred embodiment, a functionalized antibody according to structure (1) is obtained in step (a), D is the payload, and step (b) is not performed.

[0171]

[0186] In one embodiment, step (b) is performed where V present on the compound having structure (2) is a reactive group Q'. In that case, step (a) provides an intermediate functionalized antibody having structure (1) where V = Q' (hereinafter depicted as (1b)). This intermediate functionalized antibody contains an additional reactive group Q' that reacts with an appropriately functionalized payload bearing a reactive group F to provide a final conjugate having structure (1) where V = D. The method according to this preferred embodiment can be represented by Scheme 2.

[0172]

[0187] Scheme 2 [ka]

[0173]

[0188] In this specification, Q 1 and F 1 is a reactive moiety, just like Q and F, and definitions and preferred embodiments of Q and F are 1 and F 1 The presence of Q' in the linker compound (2) should not interfere with the reaction, which is 1 and F 1 This can be achieved by Q' being inert in the reaction between 1 and Q' are the same reactive moiety, and Ab(F 1 )2 and the reaction is two combinations Q 1 It was found that only the third reactive moiety / Q' occurs, leaving the third reactive moiety unreacted. Further reduction of the third reaction occurring with the linker compound is achieved by carrying out the reaction at dilute concentrations.

[0174]

[0189] Thus, in a preferred embodiment, a functionalized antibody according to structure (1') is obtained in step (a), V is a reactive group Q', ie structure (1b), and step (b) is carried out.

[0175]

[0190] The "payload antibody ratio," also known as the drug-antibody ratio (DAR), refers to the ratio of payload molecules to antibody molecules in a conjugate. The present invention provides an efficient route to conjugates with a DAR of 1 (i.e., one payload molecule is conjugated to one antibody molecule). Because not all functionalized antibodies can react with the linker compound of structure (2), and as a result, the actual payload antibody ratio may deviate somewhat from (i.e., be somewhat lower than) the assumed payload antibody ratio, the payload antibody ratio of the product may be slightly lower than the assumed payload antibody ratio. The method according to the present invention provides product mixtures with payload antibody ratios close to the assumed ratio of 1.

[0176]

[0191] The present invention provides a greatly improved method for preparing antibody conjugates with a payload-to-antibody ratio of 1 compared to conventional methods. Conventional methods struggle with introducing only a single attachment point onto antibodies. Antibodies contain many amino acids, so random conjugation, such as maleimide-cysteine ​​conjugation, typically provides a wide range of conjugates carrying up to eight or even more payloads. Other conjugation methods suffer from the fact that antibodies are symmetrical, thus providing at least two potentially usable attachment points. Thus, genetic engineering can be relied upon to engineer recombinant antibodies containing only a single attachment point.

[0177]

[0192] An alternative prior art approach involves the use of symmetrically functionalized payloads, in which a symmetric payload (dimer) is symmetrically functionalized with two identical reactive moieties via a linker, such that these two reactive moieties react with the two attachment points provided on the antibody.

[0178]

[0193] The method according to the present invention elegantly converts two attachment points of an antibody into a single attachment point by clipping a bifunctional linker compound to the two attachment points on the antibody. As demonstrated in the examples, conjugates with a payload-to-antibody ratio of 1 can be easily obtained. Furthermore, the branching moiety allows any payload to be conjugated to the antibody, and as a result, the method is not limited to symmetrical payloads.

[0179]

[0194] If V=D, then the reaction in step (a) is a conjugation reaction. Otherwise, if V=Q', then the reaction in step (b) is a conjugation reaction. The method according to the invention is compatible with any conjugation technique, and any such technique, if implemented, can be used for both step (a) and step (b).

[0180]

[0195] In a preferred embodiment, the reaction in step (a) is a [4+2] cycloaddition or a 1,3-dipolar cycloaddition.

[0181]

[0196] Antibodies according to structure (3) can be prepared by any means known in the art. For example, reduction of the antibody's interchain disulfide bond followed by reaction with a defined number of reactive moieties F containing a maleimide construct (or other thiol-reactive construct) results in the bearing of groups F, which can be matched by stoichiometry. A more controlled, site-specific method of antibody conjugation can be achieved, for example, by genetic engineering of an antibody containing two unpaired cysteines (one per heavy chain or one per light chain) by subjecting the antibody to an F-containing maleimide construct to provide the antibody with exactly two reactive moieties F. The genetic code, by applying the AMBER stop codon, allows for the direct expression of antibodies containing a predefined number of reactive moieties F at specific sites. Various enzymatic approaches for attaching a defined number of reactive moieties F to antibodies have also been reported, based, for example, on transglutaminase (TGase), sortase, formyl-glycine generating enzyme (FGE), etc. Thus, in one embodiment, functionalized antibodies are prepared by reduction of interchain disulfide bonds, followed by reaction with an F-containing thiol-reactive construct, introduction of an unpaired cysteine ​​residue, followed by reaction with an F-containing thiol-reactive construct, enzymatic introduction of a reactive moiety F, and introduction of the reactive moiety by genetic modification. The use of genetic modification is least preferred in the context of the present application, and enzymatic introduction of a reactive moiety F is most preferred.

[0182]

[0197] In a preferred embodiment, GlycoConnect technology (see, e.g., WO 2014 / 065661 and van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, incorporated by reference) utilizes naturally occurring glycans on the heavy chain of a monoclonal antibody to introduce a number of click probes, particularly azides. Thus, in a preferred embodiment, functionalized antibodies are prepared by (i) optionally trimming the native glycans with a suitable endoglycosidase, thereby liberating the core GlcNAc typically present on Asn-297, followed by (ii) transferring an unnatural azide-bearing sugar substrate from the corresponding UDP-sugar under the action of a suitable glycosyltransferase, e.g., transferring GalNAz with the galactosyltransferase mutant Gal-T(Y289L) or 6-azidoGalNAc with GalNAc-transferase (GalNAc-T). Alternatively, GalNAc-T can be applied to attach GalNAc derivatives bearing an aromatic moiety or a thiol functional group on the Ac group onto the core GlcNAc. Functionalized antibodies according to structure (5) can be obtained by this technique, where trimming step (i) can be employed to obtain a functionalized antibody with e=0, or trimming step (i) can be omitted to obtain a functionalized antibody with e=1-10. Preferably, step (i) is performed and e=0.

[0183] Reactive moieties Q and F

[0198] In the context of the present invention, the term "reactive moiety" can refer to a chemical moiety that contains a functional group, but can also refer to the functional group itself. For example, a cyclooctynyl group is a functional group, i.e., a reactive group that contains a C-C triple bond. However, functional groups such as an azide functional group, a thiol functional group, or an alkynyl functional group can also be referred to as reactive groups herein.

[0184]

[0199] To be reactive in the method according to the present invention, a reactive moiety Q should be capable of reacting with a reactive moiety F present on the functionalized antibody. In other words, the reactive moiety Q is reactive to a reactive moiety F present on the functionalized antibody. As used herein, a reactive moiety is defined as "reactive" with another reactive moiety if said first reactive moiety selectively reacts with said second reactive moiety, optionally in the presence of other functional groups. Complementary reactive moieties are known to those skilled in the art and are described in more detail below and illustrated in FIG. 1. Thus, a conjugation reaction is a chemical reaction between Q and F to form a conjugate comprising a covalent connection between the antibody and the payload. The definition of a reactive moiety Q provided herein is used to refer to a reactive moiety F, Q 1 , F 1 and Q' equally.

[0185]

[0200] In preferred embodiments, the reactive moiety is selected from the group consisting of optionally substituted alkenyl, alkynyl, tetrazinyl, azide, nitrile oxide, nitrone, nitrile imine, diazo, ketone, (O-alkyl)hydroxylamino, hydrazine, arenamide, triazine, and phosphonamidite groups. In particularly preferred embodiments, the reactive moiety Q is an azide or alkynyl group, and most preferably, the reactive moiety Q is an alkynyl group. When Q is an alkynyl group, it is preferred that Q be selected from a terminal alkyne group, a (hetero)cycloalkynyl group, and a bicyclo[6.1.0]non-4-yn-9-yl group.

[0186]

[0201] In another preferred embodiment, Q comprises an alkenyl group containing a cycloalkenyl group or is an alkenyl group containing a cycloalkenyl group, preferably Q is an alkenyl group. The alkenyl group may be linear or branched and optionally substituted. The alkenyl group may be a terminal or internal alkenyl group. The alkenyl group may contain two or more C-C double bonds, preferably one or two C-C double bonds. When the alkenyl group is a dienyl group, it is more preferred that the two C-C double bonds are separated by one C-C single bond (i.e., the dienyl group is preferably a conjugated dienyl group). Preferably, the alkenyl group is a C2-C 24 Alkenyl groups, more preferably C2-C 12 It is an alkenyl group, even more preferably a C2-C6 alkenyl group. It is further preferred that the alkenyl group is a terminal alkenyl group. More preferably, the alkenyl group is according to structure (Q8) shown below, where l is an integer ranging from 0 to 10, preferably from 0 to 6, and p is an integer ranging from 0 to 10, preferably from 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. More preferably, p is 0, 1, 2, 3, or 4, more preferably p is 0, 1, or 2, and most preferably p is 0 or 1. It is particularly preferred that p is 0 and l is 0 or 1, or p is 1 and l is 0 or 1.

[0187]

[0202] Particularly preferred alkenyl groups are cycloalkenyl groups, including heterocycloalkenyl groups, which are optionally substituted. Preferably, the cycloalkenyl groups are C3 to C6 24 Cycloalkenyl group, more preferably C3-C 12

[0033] In a preferred embodiment, the cycloalkenyl group is a trans-cycloalkenyl group, more preferably a trans-cyclooctenyl group (also referred to as a TCO group), most preferably a trans-cyclooctenyl group according to the structures (Q9) or (Q10) shown below. In another preferred embodiment, the cycloalkenyl group is a cyclopropenyl group, which is optionally substituted. In another preferred embodiment, the cycloalkenyl group is a norbornenyl group, an oxanorbornenyl group, a norbornadienyl group, or an oxanorbornadienyl group, which is optionally substituted. In a further preferred embodiment, the cycloalkenyl group is according to the structures (Q11), (Q12), (Q13), or (Q14) shown below, where X 4 is CH2 or O, and R 27 is hydrogen, straight chain or branched C1-C 12 Alkyl group or C4-C 12 (hetero)aryl groups; R 14 is selected from the group consisting of hydrogen and fluorinated hydrocarbons. 27 are independently hydrogen or a C1 to C6 alkyl group, and more preferably R 27 are independently hydrogen or a C1-C4 alkyl group. Even more preferably, R 27 are independently hydrogen or methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Even more preferably, R 27 are independently hydrogen or methyl. In a further preferred embodiment, R 14 is selected from the group of hydrogen and -CF, -C2F5, -C3F7 and -C4F9, more preferably hydrogen and -CF. In a further preferred embodiment, the cycloalkenyl group is according to structure (Q11), and one R 27 is hydrogen, and the other R 27 is a methyl group. In another more preferred embodiment, the cycloalkenyl group is according to structure (Q12), and both R27 is hydrogen. In these embodiments, it is even more preferred that l is 0 or 1. In another even more preferred embodiment, the cycloalkenyl group is a norbornenyl (X) according to structure (Q13): 4 is CH2) or oxanorbornenyl (X 4 is O) group, or norbornadienyl (X 4 is CH2) or oxanorbornadienyl (X 4 is O), and R 27 is hydrogen and R 14 is hydrogen or -CF3, preferably -CF3.

[0188]

[0203] In another preferred embodiment, Q comprises an alkynyl group containing a cycloalkynyl group or is an alkynyl group containing a cycloalkynyl group, preferably Q comprises an alkynyl group. The alkynyl group may be linear or branched and optionally substituted. The alkynyl group may be a terminal or internal alkynyl group. Preferably, the alkynyl group is a C2-C 24 Alkynyl groups, more preferably C2-C 12 It is preferably an alkynyl group, even more preferably a C2-C6 alkynyl group. It is even more preferably that the alkynyl group is a terminal alkynyl group. More preferably, the alkynyl group is according to the structure (Q15) shown below, where 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 0, 1, or 2, and most preferably 1 is 0 or 1.

[0189]

[0204] Particularly preferred alkynyl groups are cycloalkynyl groups, including heterocycloalkynyl groups, where the cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, i.e., a heterocyclooctynyl group or a cyclooctynyl group, where the (hetero)cyclooctynyl group is optionally substituted. In a further preferred embodiment, the (hetero)cyclooctynyl group is according to structure (Q36), which is further defined below. Preferred examples of (hetero)cyclooctynyl groups include structures (Q16), also known as DIBO groups, (Q17), also known as DIBAC groups, or (Q18), also known as BARAC groups, (Q19), also known as COMBO groups, and (Q20), also known as BCN groups, all shown below, where X 5 is O or NR 27 and R 27 Preferred embodiments of (Q16) are as defined above. The aromatic ring in (Q16) is optionally O-sulfonylated at one or more positions, preferably at two positions, most preferably as in (Q40) (sulfonylated dibenzocyclooctyne (s-DIBO)), while the rings in (Q17) and (Q18) can be halogenated at one or more positions. A particularly preferred cycloalkynyl group is the optionally substituted bicyclo[6.1.0]non-4-yn-9-yl group (BCN group). Preferably, the bicyclo[6.1.0]non-4-yn-9-yl group is according to the structure (Q20) shown below.

[0190]

[0205] In another preferred embodiment, Q comprises or is a conjugated (hetero)diene group, preferably Q is a conjugated (hetero)diene group capable of reacting in a Diels-Alder reaction. Preferred (hetero)diene groups include optionally substituted tetrazinyl groups, optionally substituted 1,2-quinone groups, and optionally substituted triazine groups. More preferably, the tetrazinyl group is according to the structure (Q21) shown below, and R 27 is hydrogen, straight chain or branched C1-C 12 Alkyl group or C4-C12 (hetero)aryl groups. Preferably, R 27 is hydrogen, a C1-C6 alkyl group or a C4-C 10 (hetero)aryl groups, more preferably R 27 is hydrogen, a C1-C4 alkyl group, or a C4-C6 (hetero)aryl group. Even more preferably, R 27 is hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl or pyridyl. Even more preferably, R 27 is hydrogen, methyl, or pyridyl. More preferably, the 1,2-quinone group is according to structure (Q22) or (Q23). The triazine group can be any positional isomer. More preferably, the triazine group is a 1,2,3-triazine group or a 1,2,4-triazine group, which can be attached via any available position, as shown in structure (Q24). 1,2,3-triazine is the most preferred triazine group.

[0191]

[0206] In another preferred embodiment, Q comprises or is an azide group, preferably Q is an azide group. Preferably, the azide group is according to structure (Q25) shown below.

[0192]

[0207] In another preferred embodiment, Q comprises or is a nitrile oxide group, preferably Q is a nitrile oxide group. Preferably, the nitrile oxide group is according to structure (Q27) shown below.

[0193]

[0208] In another preferred embodiment, Q comprises or is a nitrone group, preferably Q is a nitrone group. Preferably, the nitrone group is according to the structure (Q28) shown below, and R 29 Is straight chain or branched C1-C 12 Alkyl groups and C6-C 12 aryl groups. Preferably, R 29 is a C1 to C6 alkyl group, and more preferably R 29is a C1 to C4 alkyl group. Even more preferably, R 29 is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Even more preferably, R 29 is methyl.

[0194]

[0209] In another preferred embodiment, Q comprises or is a nitrile imine group, preferably Q is a nitrile imine group. Preferably, the nitrile imine group is according to structure (Q29) or (Q30) shown below, and R 30 Is straight chain or branched C1-C 12 Alkyl groups and C6-C 12 aryl groups. Preferably, R 30 is a C1 to C6 alkyl group, and more preferably R 30 is a C1 to C4 alkyl group. Even more preferably, R 30 is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Even more preferably, R 30 is methyl.

[0195]

[0210] In another preferred embodiment, Q comprises or is a diazo group, preferably Q is a diazo group. Preferably, the diazo group is according to the structure (Q31) shown below, and R 33 is selected from the group consisting of hydrogen or carbonyl derivatives. More preferably, R 33 is hydrogen.

[0196]

[0211] In another preferred embodiment, Q comprises or is a ketone group, preferably Q is a ketone group. Preferably, the ketone group is according to structure (Q32) shown below, and R 34 Is straight chain or branched C1-C 12 Alkyl groups and C6-C 12 aryl groups. Preferably, R 34 is a C1 to C6 alkyl group, and more preferably R 34is a C1-C4 alkyl group. Even more preferably, R 34 is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Even more preferably, R 34 is methyl.

[0197]

[0212] In another preferred embodiment, Q comprises or is an (O-alkyl)hydroxylamino group, preferably Q is an (O-alkyl)hydroxylamino group. Preferably, the (O-alkyl)hydroxylamino group is according to structure (Q33) shown below.

[0198]

[0213] In another preferred embodiment, Q comprises or is a hydrazine group, preferably Q is a hydrazine group. Preferably, the hydrazine group is according to structure (Q34) shown below.

[0199]

[0214] In another preferred embodiment, Q comprises or is an allenamide group, preferably Q is an allenamide group. Preferably, the allenamide group is according to structure (Q35).

[0200]

[0215] In another preferred embodiment, Q comprises or is a phosphonamidite group, preferably Q is a phosphonamidite group. Preferably, the phosphonamidite group is according to structure (Q36).

[0201] [ka]

[0202]

[0216] Herein, the aromatic ring in (Q16) is optionally O-sulfonylated at one or more positions, whereas the rings of (Q17) and (Q18) may be halogenated at one or more positions.

[0203]

[0217] When Q is a (hetero)cycloalkynyl group, Q is (Q42) to (Q60): [ka] It is preferably selected from the group consisting of:

[0204]

[0218] The connection to the rest of the molecule, depicted herein as a wavy bond, can be any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q50), (Q53), (Q54) and (Q55) can bear a connection or contain a hydrogen atom or can optionally be functionalized. B (-) is an anion, preferably (-) OTf, Cl (-) , Br (-) or I (-) and most preferably B (-) but (-) OTf. In the conjugation reaction, B (-) will exchange with any anion present in the reaction mixture, so B (-) does not need to be a pharmaceutically acceptable anion. When (Q59) is used for Q, the negatively charged counterion is preferably pharmaceutically acceptable for isolation of the antibody conjugates according to the invention, so that the antibody conjugates can be readily used as pharmaceuticals.

[0205]

[0219] Some representative examples of reactions between F and Q and their corresponding products (connecting group Z) are depicted in FIG.

[0206]

[0220] The conjugation is achieved by cycloaddition. In a preferred embodiment, the conjugation is achieved by [4+2] cycloaddition or 1,3-dipolar cycloaddition, and the nucleophilic reaction is a Michael addition or a nucleophilic substitution. Thus, in a preferred embodiment of the conjugation method according to the present invention, the conjugation is achieved via [4+2] cycloaddition or 1,3-dipolar cycloaddition, preferably 1,3-dipolar cycloaddition.

[0207]

[0221] A typical [4 + 2] cycloaddition is the Diels-Alder reaction, where Q is a diene or a dienophile. As will be recognized by those skilled in the art, the term "diene" in the context of the Diels-Alder reaction refers to 1,3-(hetero)dienes, including conjugated dienes (RC=CR-CR=CR), imines (e.g., RC=CR-N=CR or RC=CR-CR=NR, RC=NN=CR), and carbonyls (e.g., RC=CR-CR=O or O=CR-CR=O). Hetero-Diels-Alder reactions with N- and O-containing dienes are known in the art. Any diene known in the art to be suitable for [4 + 2] cycloaddition can be used as the reactive group Q. Preferred dienes include the tetrazines, 1,2-quinones, and triazines described above. Any dienophile known in the art to be suitable for [4+2] cycloaddition can be used as the reactive group Q, although the dienophile is preferably an alkene or alkyne group as described above, most preferably an alkyne group. For conjugation via [4+2] cycloaddition, it is preferred that Q is a dienophile (and F is a diene), and more preferably Q is or includes an alkynyl group.

[0208]

[0222] For 1,3-dipolar cycloaddition, Q is a 1,3-dipole or dipolarophile. Any 1,3-dipole known in the art to be suitable for 1,3-dipolar cycloaddition can be used as the reactive group Q. Preferred 1,3-dipoles include azide groups, nitrone groups, nitrile oxide groups, nitrile imine groups, and diazo groups. Any dipolarophile known in the art to be suitable for 1,3-dipolar cycloaddition can be used as the reactive group Q, but the dipolarophile is preferably an alkene or alkyne group, most preferably an alkyne group. For conjugation via 1,3-dipolar cycloaddition, it is preferred that Q is a dipolarophile (and F is a 1,3-dipole), and more preferably Q is or includes an alkynyl group.

[0209]

[0223] Thus, in a preferred embodiment, Q is selected from dipolarophiles and dienophiles. Preferably, Q is an alkene or alkyne group. In a particularly preferred embodiment, Q comprises an alkyne group, preferably selected from the above-mentioned alkynyl groups, the above-mentioned cycloalkenyl groups, the above-mentioned (hetero)cycloalkynyl groups, and the bicyclo[6.1.0]non-4-yn-9-yl group. More preferably, Q comprises a terminal alkyne or cyclooctyne moiety, preferably bicyclononyne (BCN), azadibenzocyclooctyne (DIBAC / DBCO), or dibenzocyclooctyne (DIBO), more preferably BCN or DIBAC / DBCO, most preferably BCN. In an alternative preferred embodiment, Q is selected from formulae (Q5), (Q6), (Q7), (Q8), (Q20), and (Q9), more preferably selected from formulae (Q6), (Q7), (Q8), (Q20), and (Q9). Most preferably, Q is a bicyclo[6.1.0]non-4-yn-9-yl] group, preferably of formula (Q20): These groups are known to be highly effective in conjugation with azide-functionalized antibodies.

[0210]

[0224] In a particularly preferred embodiment, the reactive group Q comprises an alkynyl group and has the structure (Q36): [ka] This is due to the following.

[0211] In this specification, R 15 is hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , C1~C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl, and (hetero)arylalkyl groups are optionally substituted, and two substituents R15 may be linked to form a fused cycloalkyl or fused (hetero)arene substituent, R 16 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; X 10 is C(R 17 )2, O, S or NR 17 and R 17 is R 15 and; u is 0, 1, 2, 3, 4 or 5; u' is 0, 1, 2, 3, 4 or 5; u+u'=5; v=9 or 10.

[0212]

[0225] Preferred embodiments of reactive groups according to structure (Q36) are reactive groups according to structures (Q37), (Q6), (Q7), (Q8), (Q9) and (Q20).

[0213]

[0226] In a particularly preferred embodiment, the reactive group Q comprises an alkynyl group and has the structure (Q37): [ka] This is due to the following.

[0214] In this specification, R 15 is hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24(hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl, and (hetero)arylalkyl groups are optionally substituted, and two substituents R 15 may be linked to form a fused cycloalkyl or fused (hetero)arene substituent, R 16 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; R 18 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; R 19 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted 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; l is an integer ranging from 0 to 10.

[0215]

[0227] In a preferred embodiment of the reactive group according to structure (Q37), R 15 But hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 16 is hydrogen or C1-C6 alkyl, more preferably R 15are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably all R 15 is H. In a preferred embodiment of the reactive group according to structure (Q37), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of the reactive group according to structure (Q37), R 19 is H. In preferred embodiments of reactive groups according to structure (Q37), l is 0 or 1, more preferably l is 1. Particularly preferred embodiments of reactive groups according to structure (Q37) are reactive groups according to structure (Q20).

[0216] compound

[0228] In a further aspect, the present invention provides a compound of structure (2): [ka] (In the formula, a, b, and c are each independently 0 or 1; L 1 , L 2 and L 3 is a linker; D is the payload; BM is the branching part; Q contains a (hetero)cyclooctyne moiety The present invention relates to the compound

[0217]

[0229] Part a, b, c, L 1 , L 2 , L 3 , D, BM, and Q are as further defined above and apply equally to this aspect, including the preferred embodiments as defined above. In preferred embodiments, D is a cytotoxin as further defined above. Preferred compounds of structure (2) are symmetrical, i.e., the a / b, L, and Q occurring 1 / L 2 and Q are each the same. Preferably, a=b=1, and more preferably also c=1.

[0218]

[0230] In the context of this embodiment, Q comprises a (hetero)cyclooctyne moiety, which may be optionally substituted, a heterocyclooctynyl group or a cyclooctynyl group, preferably a cyclooctynyl group. In a further preferred embodiment, the (hetero)cyclooctynyl group is according to structure (Q36). Preferred examples of (hetero)cyclooctynyl groups include structures (Q16), also known as DIBO groups, (Q17), also known as DIBAC groups, or (Q18), also known as BARAC groups, (Q19), also known as COMBO groups, and (Q20), also known as BCN groups; X 5 is O or NR 27 and R 27 Preferred embodiments of (Q16) are as defined above. The aromatic ring of (Q16) is optionally O-sulfonylated at one or more positions, preferably at two positions, most preferably with (Q37), and the rings of (Q17) and (Q18) may be halogenated at one or more positions. A particularly preferred cyclooctynyl group is the optionally substituted bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group). Preferably, the bicyclo[6.1.0]non-4-yn-9-yl] group is according to the structure (Q20) shown below. In one embodiment, Q is bicyclononyne (BCN), azadibenzocyclooctyne (DIBAC / DBCO), dibenzocyclooctyne (DIBO) or sulfonylated dibenzocyclooctyne (s-DIBO), more preferably BCN or DIBAC / DBCO, most preferably BCN.

[0219]

[0231] Compounds according to this aspect are ideally suited as intermediates in the preparation of antibody-payload conjugates according to the invention.

[0220] Purpose

[0232] The conjugates according to the present invention are particularly suitable for the treatment of cancer. Thus, the present invention further relates to the use of the conjugates according to the present invention in medicine. In a further aspect, the present invention also relates to a method for treating a subject in need thereof, the method comprising administering a conjugate according to the present invention to the subject. The method according to this aspect can also be expressed as the conjugate according to the present invention for use in treatment. The method according to this aspect can also be expressed as the use of the conjugate according to the present invention for the manufacture of a medicine. Herein, administration is typically carried out using a therapeutically effective amount of the conjugate according to the present invention.

[0221]

[0233] The present invention further relates to a method for treating a specific disease in a subject in need thereof, the method comprising administering a conjugate according to the present invention as defined above. The specific disease may be selected from cancer, viral infection, bacterial infection, neurological disease, autoimmune disease, eye disease, hypercholesterolemia, and amyloidosis, more preferably cancer and viral infection, and most preferably the disease is cancer. The subject in need thereof is typically a cancer patient. The use of conjugates according to the present invention in such treatments is well known, particularly in the field of cancer treatment, and conjugates according to the present invention are particularly suitable in this regard. In the method according to this aspect, the conjugate is typically administered in a therapeutically effective amount. This aspect of the present invention can also be expressed as a conjugate according to the present invention for use in treating a specific disease in a subject in need thereof, preferably for treating cancer. In other words, this aspect relates to the use of a conjugate according to the present invention for preparing a medicament or pharmaceutical composition for use in treating a specific disease in a subject in need thereof, preferably for treating cancer.

[0222]

[0234] It is preferred that the conjugates according to the invention are Fc silent, i.e., when used clinically, do not significantly bind to the Fc gamma receptors CD16, CD32 and CD64. This is when G is absent, i.e., e=0.

[0223]

[0235] Administration in the context of the present invention refers to systemic administration.Therefore, in one embodiment, the method defined herein is for systemic administration of conjugates.In light of the specificity of conjugates, conjugates can be administered systemically, but these antibodies can still exert their activity in or near the target tissue (e.g., tumor).Systemic administration has a great advantage compared to local administration, because the drug can reach tumor metastases that are not detectable by imaging technology and can be applied to blood tumors.

[0224]

[0236] The present invention further relates to a pharmaceutical composition comprising an antibody-payload conjugate according to the invention and a pharmaceutically acceptable carrier. [Example]

[0225] The invention is illustrated by the following examples. General Procedure Chemicals were purchased from commonly used suppliers (Sigma-Aldrich, Acros, Alfa Aesar, Fluorochem, Apollo Scientific Ltd, and TCI) and used without further purification. Solvents (including dehydrated solvents) for chemical transformations, workup, and chromatography were purchased from Aldrich (Dorset, UK) at HPLC grade and used without further distillation. Silica gel 60 F254 analytical thin-layer chromatography (TLC) plates were from Merck (Darmstadt, Germany) and visualized under UV light using potassium permanganate stain or anisaldehyde stain. Chromatographic purification was performed using Acros silica gel (0.06–0.200, 60A) or prepacked columns (Silicycle) coupled with a Buchi Sepacore C660 fraction collector (Frawil, Switzerland). Reverse-phase HPLC purification was performed using an Agilent 1200 system equipped with a Waters Xbridge C18 column (5 μm OBD, 30 × 100 mm, PN 186002982). Deuterated solvents used for NMR spectroscopy were obtained from Cambridge Isotope Laboratories. H-Val-Ala-PABC-MMAF.TFA was obtained from Levena Biopharm, bis-mal-Lys-PEG4-TFP ester (177) was obtained from Quanta Biodesign, O-(2-aminoethyl)-O'-(2-azidoethyl)diethylene glycol (XL07) and compounds 344 and 179 were obtained from Broadpharm, 2,3-bis(bromomethyl)-6-quinoxalinecarboxylic acid (178) was obtained from ChemScene, and 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanoic acid (348) was obtained from Carbosynth.

[0226] General Procedure for Mass Spectrometric Analysis of Monoclonal Antibodies and ADCs Prior to mass spectrometry analysis, IgG was treated with IdeS (Fabricator™) for analysis of the Fc / 2 fragment. A solution of 20 μg of (modified) IgG was incubated with 0.5 μL of IdeS (50 U / μL) in phosphate-buffered saline (PBS) pH 6.6 in a total volume of 10 μL for 1 h at 37°C. Samples were diluted to 40 μL and subsequently subjected to electrospray ionization time-of-flight (ESI-TOF) analysis on a JEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.

[0227] General procedure for analytical RP-HPLC Prior to RP-HPLC analysis, the IgG was treated with IdeS, which allows for analysis of the Fc / 2 fragment. A solution of (modified) IgG (100 μL, 1 mg / mL in PBS pH 7.4) was incubated with 1.5 μL of IdeS / Fabricator™ (50 U / μL) in phosphate-buffered saline (PBS) pH 6.6 for 1 hour at 37°C. The reaction was quenched by adding 100 μL of 49% acetonitrile, 49% water, and 2% formic acid. RP-HPLC analysis was performed on an Agilent 1100 series (Hewlett Packard). Samples (10 μL) were injected at 0.5 mL / min onto a ZORBAX Poroshell 300SB-C8 column (1 × 75 mm, 5 μm, Agilent) at a column temperature of 70°C. A linear gradient was applied from 30 to 54% acetonitrile and water in 0.1% TFA for 25 min.

[0228] General procedure for analytical HPLC-SEC HPLC-SEC analysis was performed on an Agilent 1100 series (Hewlett Packard). Samples (4 μL, 1 mg / mL) were injected onto an Xbridge BEH200A (3.5 μM, 7.8 × 300 mm, PN186007640 Waters) column at 0.86 mL / min. Isocratic elution was performed for 16 min using 0.1 M sodium phosphate buffer pH 6.9 (NaH2PO4 / Na2HPO4).

[0229] Example 1. Synthesis of Compound 102 [ka] To a cooled (0 °C) solution of 4-nitrophenyl chloroformate (30.5 g, 151 mmol) in DCM (500 mL) was added pyridine (24.2 mL, 23.7 g, 299 mmol). A solution of BCN-OH (101, 18.0 g, 120 mmol) in DCM (200 mL) was added dropwise to the reaction mixture. After the addition was complete, a saturated aqueous solution of NH4Cl (500 mL) and water (200 mL) were added. After separation, the aqueous phase was extracted with DCM (2 × 500 mL). The combined organic phases were dried (Na2SO4) and concentrated. The crude material was purified by silica gel chromatography to give the desired product 102 as an off-white solid (18.7 g, 59 mmol, 39%). 1 H NMR (400 MHz, CDCl3) δ (ppm) 8.32-8.23 (m, 2H), 7.45-7.34 (m, 2H), 4.40 (d, J = 8.3 Hz, 2H), 2.40-2.18 (m, 6H), 1.69- 1.54 (m, 2H), 1.51 (quintet, J = 9.0 Hz, 1H), 1.12-1.00 (m, 2H)

[0230] [ka]

[0231] Example 2. Synthesis of Compound 104 Azide-PEG 11To a cooled (-5 °C) solution of amine (103) (182 mg, 0.319 mmol) in THF (3 mL) was added 10% aqueous NaHCO (1.5 mL) and 9-fluorenylmethoxycarbonyl chloride (99 mg, 0.34 mmol) dissolved in THF (2 mL). After 2 h, EtOAc (20 mL) was added, and the mixture was washed with brine (2 × 6 mL), dried over MgSO, and concentrated. Purification by silica gel column chromatography (0 → 11% MeOH in DCM) afforded 104 as a clear oil in 98% yield (251 mg, 0.316 mmol). LCMS (ESI+) C 39 H 60 N4O 13 + (M+Na + ) calculated value 815.42, actual value 815.53.

[0232] Example 3. Synthesis of Compound 105 A solution of 104 (48 mg, 0.060 mmol) in THF (3 mL) and water (0.2 mL) was prepared and cooled to 0 °C. Trimethylphosphine (1 M in toluene, 0.24 mL, 0.24 mmol) was added, and the mixture was left stirring for 23 h. Water was removed via extraction with DCM (6 mL). To this solution was added (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate (102) (25 mg, 0.079 mmol) and triethylamine (10 μL, 0.070 mmol). After 27 h, the mixture was concentrated, and the residue was dissolved in DMF (3 mL), followed by the addition of piperidine (400 μL). After 1 h, the mixture was concentrated and the residue was purified by silica gel column chromatography (0→21% MeOH in DCM) to give 105 as a colorless oil (8.3 mg, 0.0092 mmol). LCMS (ESI+) C 46 H 76 N2O 15 + (M+NH4 + ) calculated value 914.52, actual value 914.73.

[0233] [ka]

[0234] Example 4. Synthesis of Compound 107 A solution of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate (102) (4.1 mg, 0.013 mmol) in 500 μL of dry DCM was slowly added to a solution of amino-PEG-23-amine (106) (12.3 mg, 0.0114 mmol) in 500 μL of dry DCM. After 20 h, the mixture was concentrated, and the residue was purified by silica gel column chromatography (0→25% MeOH in DCM) to give the desired compound 107 in 73% yield (12 mg, 0.0080 mmol). LCMS (ESI+) C 70 H 124 N2O 27 + (M+ NH4 + ) Calculated value 1443.73 Measured value 1444.08.

[0235] [ka]

[0236] Example 5. Synthesis of Compound 108 To a solution of BCN-OH (101, 21.0 g, 0.14 mol) in MeCN (450 mL) was added disuccinimidyl carbonate (53.8 g, 0.21 mol) and triethylamine (58.5 mL, 0.42 mol). After stirring the mixture for 140 min, it was concentrated in vacuo, and the residue was coevaporated once with MeCN (400 mL). The residue was dissolved in EtOAc (600 mL) and washed with HO (3 × 200 mL). The organic layer was dried over NaSO and concentrated in vacuo. The residue was purified by silica gel column chromatography (0 → 4% EtOAc in DCM) to give 108 (11.2 g, 38.4 mmol, 27% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ (ppm) 4.45 (d, 2H, J = 8.4 Hz), 2.85 (s, 4H), 2.38- 2.18 (m, 6H), 1.65- 1.44 (m, 3H), 1.12-1.00 (m, 2H).

[0237] Example 6. Synthesis of Compound 110 To a solution of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (108) (500 mg, 1.71 mmol) in DCM (15 mL) was added triethylamine (718 μL, 5.14 mmol) and mono-Fmoc ethylenediamine hydrochloride (109) (657 mg, 2.06 mmol). The mixture was stirred for 45 min, diluted with EtOAc (150 mL), and washed with 50% saturated aqueous NH4Cl (50 mL). The aqueous layer was extracted with EtOAc (50 mL), and the combined organic layers were washed with HO (10 mL). The combined organic extracts were concentrated in vacuo, and half of the residue was purified by silica gel column chromatography (0→3% MeOH in DCM) to give the desired compound 110 in 42% yield (332 mg, 0.72 mmol). 1 H NMR (400 MHz, CDCl3) δ (ppm) 7.77 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.4 Hz, 2H), 7.44-7.37 (m, 2H), 7.36-7.28 (m, 2H), 5.12 (br s, 1H), 4.97 (br s, 1H), 44.41 (d, J = 6.8 Hz, 2H), 4.21 (t, J = 6.7 Hz, 1H), 4.13 (d, J = 8.0 Hz, 2H), 3.33 (br s, 4H), 2.36-2.09 (m, 6H), 1.67-1.45 (m, 2H), 1.33 (quintet, J = 8.6 Hz, 1H), 1.01- 0.85 (m, 2H). LCMS (ESI+) C 28 H 31 N2O4 + (M+ H + ) calculated value 459.23, actual value 459.52.

[0238] Example 7. Synthesis of Compound 111 Compound 110 (327 mg, 0.713 mmol) was dissolved in DMF (6 mL) and piperidine (0.5 mL) was added. After 2 h, the mixture was concentrated, and the residue was purified by silica gel column chromatography (0 to 32% 0.7 N NH3MeOH in DCM) to give the desired compound 111 as a yellow oil (128 mg, 0.542 mmol, 76%). 1 H-NMR (400 MHz, CDCl3) δ (ppm, rotamers) 5.2 (bs, 1H), 4.15 (d, J = 8.0 Hz, 2H), 3.48-3.40 (m, 2 / 3H), 3.33-3.27 (m, 2 / 3H), 3.27-3.19 (m, 1 1 / 3H), 2.85-2.80 (m, 1 1 / 3H), 2.36-2.17 (m, 6H), 1.67-1.50 (m, 2H), 1.36 (quintet, J = 8.5 Hz, 1H), 1.01-0.89 (m, 2H)

[0239] [ka]

[0240] Example 8. Synthesis of Compound 114 To a solution of diethanolamine 112 (208 mg, 1.98 mmol) in water (20 mL) was added MeCN (20 mL), NaHCO (250 mg, 2.97 mmol), and a solution of Fmoc-OSu 113 (601 mg, 1.78 mmol) in MeCN (20 mL). The mixture was stirred for 2 h, and DCM (50 mL) was added. After separation, the organic phase was washed with water (20 mL), dried (NaSO), and concentrated. The desired product 114 was obtained as a colorless thick oil (573 mg, 1.75 mmol, 98%). 1H NMR (400 MHz, CDCl3) δ (ppm) 7.79-7.74 (m, 2H), 7.60-7.54 (m, 2H), 7.44-7.37 (m, 2H), 7.36-7.30 (m, 2H), 4.58 (d, J = 5.4 Hz, 2H), 4.23 (t, J = 5.3 Hz, 1H), 3.82-3.72 (m, 2H), 3.48-3.33 (m, 4H), 3.25-3.11 (m, 2H).

[0241] Example 9. Synthesis of Compound 116 To a solution of 114 (567 mg, 1.73 mmol) in DCM (50 mL) was added 4-nitrophenyl chloroformate (115) (768 mg, 3.81 mmol) and EtN (1.2 mL, 875 mg). The mixture was stirred for 18 h and concentrated. The residue was purified by silica gel chromatography (0% → 10% MeOH in DCM, then 20% → 70% EtOAc in heptane) to give 32 mg (49 μmol, 2.8%) of the desired product 116. 1 H NMR (400 MHz, CDCl3) δ (ppm) 8.31-8.20 (m, 4H), 7.80-7.74 (m, 2H), 7.59-7.54 (m, 2H), 7.44-7.37 (m, 2H), 7.37-7.29 (m, 6H), 4.61 (d, J = 5.4 Hz, 2H), 4.39 (t, J = 5.1 Hz, 2H), 4.25 (t, J = 5.5 Hz, 1H), 4.02 (t, J = 5.0 Hz, 2H), 3.67 (t, J = 4.8 Hz, 2H), 3.45 (t, J = 5.2Hz, 2H).

[0242] [ka]

[0243] Example 10. Synthesis of Compound 117 To a solution of 116 (34 mg, 0.050 mmol) in DCM (2 mL) was added 111 (49 mg, 0.21 mmol) and triethylamine (20 μL, 0.14 mmol). The mixture was left stirring at room temperature overnight. After 23 h, the mixture was concentrated. Purification by silica gel column chromatography (0→40% MeOH in DCM) afforded 117 as a white solid in 61% yield (27 mg, 0.031 mmol). LCMS (ESI+) C 47 H 57 N5O 10 + (M+H + ) calculated value 851.41, actual value 852.49.

[0244] Example 11. Synthesis of Compound 118 Compound 118 was obtained during the preparation of 117 (3.8 mg, 0.0060 mmol). LCMS (ESI+) C 32 H 47 N5O8 + (M+H + ) calculated value 629.34, actual value 630.54.

[0245] [ka]

[0246] Example 12. Synthesis of Compound 121 A solution of diethylenetriamine (119) (73 μL, 0.67 mmol) and triethylamine (283 μL, 2.03 mmol) in THF (6 mL) was cooled to −5 °C and placed under a nitrogen atmosphere. 2-(Boc-oximino)-2-phenylacetonitrile (120) (334 mg, 1.35 mmol) was dissolved in THF (4 mL) and slowly added to the cooled solution. After 2.5 h, the ice bath was removed, and the mixture was stirred at room temperature for an additional 2.5 h and concentrated in vacuo. The residue was redissolved in DCM (15 mL), washed with 5% aqueous NaOH (2 × 5 mL), brine (2 × 5 mL), and dried over MgSO. Purification by silica gel column chromatography (0 → 14% MeOH in DCM) afforded 121 as a colorless oil in 91% yield (185 mg, 0.610 mmol). 1 H-NMR (400 MHz, CDCl3) δ (ppm) 5.08 (s, 2H), 3.30-3.12 (m, 4H), 2.74 (t, J = 5.9 Hz, 4H), 1.45 (s, 18H).

[0247] Example 13. Synthesis of Compound 123 To a cooled solution (-10 °C) of 121 (33.5 mg, 0.110 mmol) in THF (2 mL) was added 10% aqueous NaHCO (500 μL) and 9-fluorenylmethoxycarbonyl chloride (122) (34 mg, 0.13 mmol) dissolved in THF (1 mL). After 1 h, the mixture was concentrated, and the residue was redissolved in EtOAc (10 mL), washed with brine (2 × 5 mL), dried over NaSO, and concentrated. Purification by silica gel column chromatography (0 → 50% MeOH in DCM) afforded 123 in 86% yield (50 mg, 0.090 mmol). 1H-NMR (400 MHz, CDCl3) δ (ppm) 7.77 (d, J = 7.4 Hz, 2H), 7.57 (d, J = 7.4 Hz, 2H), 7.43-7.38 (m, 2H), 7.36-7.31 (m, 2H), 5.57 (d, J = 5.2 Hz, 2H), 4.23 (t, J = 5.1 Hz, 1H), 3.40-2.83 (m, 8H), 1.41 (s, 18H).

[0248] [ka]

[0249] Example 14. Synthesis of Compound 124 To a solution of 123 (50 mg, 0.095 mmol) in DCM (3 mL) was added 4 M HCl in dioxane (200 μL). The mixture was stirred for 19 h and concentrated to give a white solid (35 mg). Without further purification, the deprotected intermediate and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate (102) (70 mg, 0.22 mmol) were dissolved in DMF (3 mL) and triethylamine (34 μL, 0.24 mmol) was added. After 2 h, the mixture was concentrated and the residue was purified by silica gel column chromatography (0→25% MeOH in DCM) to give 124 in 48% yield (31 mg, 0.045 mmol). LCMS (ESI+) C 41 H 47 N3O6 + (M+H + ) calculated value 677.35, actual value 678.57.

[0250] Example 15. Synthesis of Compound 125 To a solution of 124 (10 mg, 0.014 mmol) in DMF (500 μL) was added piperidine (20 μL). After 3.5 h, the mixture was concentrated. Purification by silica gel column chromatography (0→20% MeOH in DCM) afforded 125 in 58% yield (3.7 mg, 0.0080 mmol). LCMS (ESI+) C26 H 37 N3O4 + (M+H + ) calculated value 455.28, actual value 456.41.

[0251] [ka]

[0252] Example 16. Synthesis of compounds 127 and 128 To a solution of diethylene glycol 126 (446 μL, 0.50 g, 4.71 mmol) in DCM (20 mL) was added 4-nitrophenol chloroformate 115 (1.4 g, 7.07 mmol) and EtN (3.3 mL, 2.4 g, 23.6 mmol). The mixture was stirred, filtered, and concentrated in vacuo (at 55 °C). The residue was purified by silica gel chromatography (15% → 75% EtOAc in heptane), and two products were isolated. Product 127 was obtained as a white solid (511 mg, 1.17 mmol, 25%). 1 H NMR (400 MHz, CDCl) 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). The product 128 was obtained as a colorless oil (321 mg, 1.18 mmol, 25%). 1 H NMR (400 MHz, CDCl3) δ (ppm) 8.32-8.24 (m, 2H), 7.43-7.36 (m, 2H), 4.50-4.44 (m, 2H), 3.86-3.80 (m, 2H), 3.81-3.74 (m, 2H), 3.69-3.64 (m, 2H).

[0253] [ka]

[0254] Example 17. Synthesis of Compound 131 To a solution of 118 (2.3 mg, 3.7 μmol) in DMF (295 μL) was added a solution of 127 (3.2 mg, 7.4 μmol) in DMF (65 μL) and EtN (1.6 μL, 1.1 mg, 11.1 μmol). The mixture was allowed to stand for 17 h, and a solution of HOBt (0.5 mg, 3.7 μmol) in DMF (14 μL) was added. After 4 h, a solution of EtN (5.2 μL, 3.8 mg, 37 μmol) and vc-PABC-MMAE.TFA (130, 13.8 mg, 11 μmol) in DMF (276 μL) was added. After 3 days, the mixture was purified by RP HPLC (C18, 30% → 90% MeCN (1% AcOH) in water (1% AcOH)). The desired product 131 was obtained as a colorless film (1.5 mg, 0.78 μmol, 21%). LCMS (ESI+) C 96 H 148 N 15 O 25 + (M+H + ) Calculated value 1911.08 Actual value 1912.08.

[0255] [ka]

[0256] Example 18. Synthesis of Compound 132 To a solution of 121 (168 mg, 0.554 mmol) in DCM (2 mL) was added a solution of 128 (240 mg, 0.89 mmol) in DCM (1 mL), DCM (1 mL), and EtN (169 mg, 233 μL). The mixture was stirred for 17 h, concentrated, and purified by silica gel chromatography (a gradient of EtOAc in heptane). The desired product 132 was obtained as a slightly yellow oil (85 mg, 0.20 mmol, 35%). 1 H NMR (400 MHz, CDCl3) δ (ppm) 5.24-5.02 (m, 2H), 4.36-4.20 (m, 3H), 3.84-3.67 (m, 4H), 3.65-3.58 (m, 2H), 3.47-3.34 (m, 4H), 3.34-3.18 (m, 4H), 1.44 (bs, 18H).

[0257] Example 19. Synthesis of Compound 134 To a solution of 132 (81 mg, 0.19 mmol) in DCM (3 mL) was added 4 N HCl in dioxane (700 μL). The mixture was stirred for 19 h, concentrated, and the residue was dissolved in DMF (0.5 mL). EtN (132 μL, 96 mg, 0.95 mmol), DMF (0.5 mL), and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate (102) (132 mg, 0.42 mmol) were added, and the resulting mixture was stirred for 2 h. The mixture was concentrated, and the residue was purified by silica gel chromatography (0% → 3% MeOH in DCM). The desired product 134 was obtained as a colorless film (64 mg, 0.11 mmol, 57%). 1 H NMR (400 MHz, CDCl3) δ (ppm) 4.31-4.23 (m, 2H), 4.22-4.08 (m, 4H), 3.80-3.68 (m, 4H), 3.66-3.58 (m, 2H), 3.50-3.28 (m, 8H), 2.80-2.65 (m, 1H), 2.40-2.10 (m, 12H), 1.68-1.48 (m, 4H), 1.35 (quintet, J = 8.1 Hz, 1H), 1.02-0.87 (m, 2H). LCMS (ESI+) C 31 H 46 N3O8 + (M+H + ) calculated value 588.33, actual value 588.43.

[0258] [ka]

[0259] Example 20. Synthesis of Compound 137 To a solution of 134 (63 mg, 0.11 mmol) in DCM (1 mL) was added bis(4-nitrophenyl)carbonate (35) (32.6 mg, 0.107 mmol) and EtN (32.5 mg, 45 μL, 0.32 mmol). After 2 h, 77 μL was removed from the main reaction mixture, and a solution of vc-PABC-MMAE.TFA (130, 10 mg, 8.1 μmol) in DMF (200 μL) and EtN (3.4 μL, 2.5 mg, 24 μmol) was added. After 18 h, 2,2′-(ethylenedioxy)bis(ethylamine) (4.9 μL, 5.0 mg, 34 μmol) was added, and the mixture was allowed to stand for 45 min. The mixture was purified by RP HPLC (C18, 30% → 90% MeCN (1% AcOH) in water (1% AcOH)). The desired product 137 was obtained as a colorless film (8.7 mg, 5.0 μmol, 61%). LCMS (ESI+) C 90 H 138 N 13 O 21 + (M+H + ) Calculated value 1737.01 Actual value 1738.01.

[0260] [ka]

[0261] Example 21. Synthesis of Compound 139 To a solution of 134 (63 mg, 0.11 mmol) in DCM (1 mL) was added bis(4-nitrophenyl)carbonate (35) (32.6 mg, 0.107 mmol) and EtN (32.5 mg, 45 μL, 0.32 mmol). After 20 h, 77 μL was removed from the main reaction mixture, and a solution of vc-PABC-MMAF.TFA (138, 9.6 mg, 8.2 μmol) in DMF (240 μL) and EtN (3.4 μL, 2.5 mg, 24 μmol) was added. After 3 h, 2,2′-(ethylenedioxy)bis(ethylamine) (20 μL, 20 mg, 0.14 mmol) was added, and the mixture was allowed to stand for 20 min. The mixture was purified by RP HPLC (C18, 30% → 90% MeCN (1% AcOH) in water (1% AcOH)). The desired product 139 was obtained as a colorless film (5.3 mg, 3.2 μmol, 39%). LCMS (ESI+) C 87 H 130 N 11 O 21 + (M+H + ) Calculated value 1664.94 Measured value 1665.99.

[0262] [ka]

[0263] Example 22. Synthesis of Compound 141 To a solution of (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (108) (16.35 g, 56.13 mmol) in DCM (400 mL) was added 2-(2-aminoethoxy)ethanol (140) (6.76 mL, 67.35 mmol) and triethylamine (23.47 mL, 168.39 mmol). The resulting pale yellow solution was stirred at room temperature for 90 min. The mixture was concentrated in vacuo, and the residue was coevaporated once with acetonitrile (400 mL). The resulting oil was dissolved in EtOAc (400 mL) and washed with HO (3 × 200 mL). The organic layer was concentrated in vacuo. The residue was purified by silica gel column chromatography (50%→88% EtOAc in heptane) to give 141 (11.2 g, 39.81 mmol, 71% yield) as a pale yellow oil. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 5.01 (br s, 1H), 4.17 (d, 2H, J = 12.0 Hz), 3.79-3.68 (m, 2H), 3.64-3.50 (m, 4H), 3.47-3.30 (m, 2H), 2.36-2.14 (m, 6H), 1.93 (br s, 1H), 1.68- 1.49 (m, 2H), 1.37 (quintet, 1H, J = 8.0 Hz), 1.01-0.89 (m, 2H).

[0264] Example 23. Synthesis of Compound 142 To a solution of 141 (663 mg, 2.36 mmol) in DCM (15 mL) was added triethylamine (986 μL, 7.07 mmol) and 4-nitrophenyl chloroformate (115) (712 mg, 3.53 mmol). The mixture was stirred for 4 h and concentrated in vacuo. Purification by silica gel column chromatography (0→20% EtOAc in heptane) afforded 142 (400 mg, 0.9 mmol, 38% yield) as a pale yellow oil. 1H-NMR (400 MHz, CDCl3) δ (ppm) 8.29 (d, J = 9.4 Hz, 2H), 7.40 (d, J = 9.3 Hz, 2H), 5.05 (br s, 1H), 4.48-4.41 (m, 2H), 4.16 (d, J = 8.0 Hz, 2H), 3.81-3.75 (m, 2H), 3.61 (t, J = 5.0 Hz, 2H), 3.42 (q, J = 5.4 Hz, 2H), 2.35-2.16 (m, 6H), 1.66-1.50 (m, 2H), 1.35 (quintet, J = 8.6 Hz, 1H), 1.02-0.88 (m, 2H). LCMS (ESI+) C 22 H 26 N2NaO8 + (M+Na + ) calculated value 469.16, actual value 469.36.

[0265] [ka]

[0266] Example 24. Synthesis of Compound 143 A solution of 142 (2.7 mg, 6.0 μmol) in DMF (48 μL) and EtN (2.1 μL, 1.5 mg, 15 μmol) were added to a solution of 125 (2.3 mg, 5.0 μmol) in DMF (0.32 mL). The mixture was allowed to stand for 4 days, diluted with DMF (100 μL), and purified by RP HPLC (C18, 30% → 100% MeCN (1% AcOH) in water (1% AcOH)). The product 143 was obtained as a colorless film (2.8 mg, 3.7 μmol, 74%). LCMS (ESI+) C 42 H 59 N4O9 + (M+H + ) calculated value 763.43, actual value 763.53.

[0267] [ka]

[0268] Example 25. Synthesis of Compound 145 To a solution of 128 (200 mg, 0.45 mmol) in DCM (1 mL) was added triethylamine (41.6 μL, 0.30 mmol) and tris(2-aminoethyl)amine 144 (14.9 μL, 0.10 mmol). After stirring for 150 min, the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (25% to 100% EtOAc in DCM, then 0% to 10% MeOH in DCM) to give 145 in 43% yield (45.4 mg, 42.5 μmol) as a yellow oil. 1 H NMR (400 MHz, CDCl3): δ (ppm) 5.68-5.18 (m, 6H), 4.32-4.18 (m, 6H), 4.18-4.11 (d, J = 7.9 Hz, 6H), 3.74-3.61 (m, 6H), 3.61-3.51 (m, 6H), 3.43- 3.29 (m, 6H), 3.29-3.15 (m, 6H), 2.65-2.47 (m, 6H), 2.37-2.16 (m, 18H), 1.69-1.49 (m, 6H), 1.35 (quintet, J = 8.9 Hz, 3H), 1.03-0.87 (m, 6H).

[0269] [ka]

[0270] Example 26. Synthesis of Compound 148 To a solution of BCN-OH (101) (3.0 g, 20 mmol) in DCM (300 mL) was added CSI (146) (1.74 mL, 2.83 g, 20 mmol). The mixture was stirred for 15 min, and then EtN (5.6 mL, 4.0 g, 40 mmol) was added. The mixture was stirred for 5 min, and 2-(2-aminoethoxy)ethanol (147) (2.2 mL, 2.3 g, 22 mmol) was added. The resulting mixture was stirred for 15 min, and then saturated aqueous NH4Cl (300 mL) was added. The layers were separated, and the aqueous phase was extracted with DCM (200 mL). The combined organic layers were dried (Na2SO4) and concentrated. The residue was purified by silica gel chromatography (0% to 10% MeOH in DCM). The fractions containing the desired product were concentrated. The residue was dissolved in EtOAc (100 mL) and concentrated. The desired product 148 was obtained as a slightly yellow oil (4.24 g, 11.8 mmol, 59%). 1 H NMR (400 MHz, CDCl3) δ (ppm) 5.99-5.79 (bs, 1H), 4.29 (d, J = 8.3 Hz, 2H), 3.78-3.74 (m, 2H), 3.66-3.56 (m, 4H), 3.37-3.30 (m, 2H), 2.36-2.16 (m, 6H), 1.63-1.49 (m, 2H), 1.40 (quintet, J = 8.7 Hz, 1H), 1.05-0.94 (m, 2H).

[0271] Example 27. Synthesis of Compound 149 To a solution of 148 (3.62 g, 10.0 mmol) in DCM (200 mL) was added 4-nitrophenyl chloroformate (15) (2.02 g, 10.0 mmol) and EtN (4.2 mL, 3.04 g, 30.0 mmol). The mixture was stirred for 1.5 h and concentrated. The residue was purified by silica gel chromatography (20% → 70% EtOAc (1% AcOH) in heptane (1% AcOH)). The product 149 was obtained as a white foam (4.07 g, 7.74 mmol, 74%). 1H NMR (400 MHz, CDCl3) δ (ppm) 8.32-8.26 (m, 2H), 7.45-7.40 (m, 2H), 5.62-5.52 (m, 1H), 4.48-4.42 (m, 2H), 4.28 (d, J = 8.2 Hz, 2H), 3.81-3.76 (m, 2H), 3.70-3.65 (m, 2H), 3.38-3.30 (m, 2H), 2.35-2.16 (m, 6H), 1.62-1.46 (m, 2H), 1.38 (quintet, J = 8.7 Hz, 1H), 1.04-0.93 (m, 2H).

[0272] [ka]

[0273] Example 28. Synthesis of Compound 150 To a solution of 149 (200 mg, 0.38 mmol) in DCM (1 mL) was added triethylamine (35.4 μL, 0.24 mmol) and tris(2-aminoethyl)amine (144) (12.6 μL, 84.6 μmol). The mixture was stirred for 120 min and concentrated in vacuo. The residue was purified by silica gel column chromatography (25% to 100% EtOAc in DCM, then 0% to 10% MeOH in DCM) to give 150 in 36% yield (40.0 mg, 30.6 μmol) as a white foam. 1 H NMR (400 MHz, CDCl3): δ (ppm) 6.34-5.72 (m, 6H), 4.34-4.18 (m, 12H), 3.76-3.58 (m, 12H), 3.43-3.30 (m, 6H), 3.30-3.18 (m, 6H), 2.64-2.49 (m, 6H), 2.38-2.14 (m, 18H), 1.65-1.47 (m, 6H), 1.39 (quintet, J = 9.1 Hz, 3H), 1.06-0.90 (m, 6H).

[0274] [ka]

[0275] Example 29. Synthesis of Compound 153 To a mixture of Fmoc-Gly-Gly-Gly-OH (151) (31.2 mg, 75.8 μmol) in anhydrous DMF (1 mL) was added N,N-diisopropylethylamine (40 μL, 29 mg, 0.23 mmol) and HATU (30.3 mg, 79.6 μmol). After 10 min, tetrazine-PEG3-ethylamine (152) (30.3 mg, 75.8 μmol) was added and the mixture was vortexed. After 2 h, the mixture was purified by RP HPLC (C18, 30% → 90% MeCN (1% AcOH) in water (1% AcOH)). The desired product was obtained as a pink film (24.1 mg, 31.8 μmol, 42%). LCMS (ESI+) C 38 H 45 N8O9 + (M+H + ) calculated value 757.33, actual value 757.46.

[0276] Example 30. Synthesis of Compound 154 To a solution of 153 (24.1 mg, 31.8 μmol) in DMF (500 μL) was added diethylamine (20 μL, 14 mg, 191 μmol). The mixture was allowed to stand for 2 h and purified by RP HPLC (C18, 5% → 90% MeCN (1% AcOH) in water (1% AcOH)). The desired product 154 was obtained as a pink film (17.5 mg, 32.7 μmol, quantitative). LCMS (ESI+) C 23 H 35 N8O7 + (M+H + ) calculated value 535.26, actual value 535.37.

[0277] [ka]

[0278] Example 31. Synthesis of Compound 156 A solution of N-[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane (155) (68 mg, 0.21 mmol) in 2 mL of dry DMF was transferred to a solution of Fmoc-Gly-Gly-Gly-OH (151) (86 mg, 0.21 mmol) in 2 mL of dry DMF. DIPEA (100 μL, 0.630 mmol) and HATU (79 mg, 0.21 mmol) were added. After 1.5 h, the mixture was concentrated, and the residue was purified by silica gel column chromatography (0→11% MeOH in DCM) to give the desired compound 156 in 34% yield (52 mg, 0.072 mmol). LCMS (ESI+) C 35 H 47 N5O9 + (M+ H+) calculated 717.34, found 718.39.

[0279] Example 32. Synthesis of Compound 157 Compound 156 (21 mg, 0.029 mmol) was dissolved in DMF (2.4 mL) and piperidine (600 μL) was added. After 20 min, the mixture was concentrated and the residue was purified by preparative HPLC to give the desired compound 157 as a white solid (9.3 mg, 0.018 mmol, 64%). LCMS (ESI+) C 23 H 37 N5O7 + (M+ H + ) calculated value 495.27, actual value 496.56.

[0280] [ka]

[0281] Example 33. Synthesis of Compound 159 Amino-PEG 11To a solution of 158 (143 mg, 0.260 mmol) in DCM (5 mL) was slowly added (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate (102) (41 mg, 0.13 mmol) dissolved in DCM (5 mL). After 1.5 h, the mixture was reduced and the residue was purified by silica gel column chromatography (0→20% 0.7 N NH3MeOH in DCM) to give the desired compound 159 as a clear oil (62 mg, 0.086 mmol, 66%). LCMS (ESI+) C 35 H 46 N2O 13 + (M+ H + ) calculated value 720.44, actual value 721.56.

[0282] Example 34. Synthesis of Compound 160 A solution of 159 (62 mg, 0.086 mmol) in anhydrous DMF (2 mL) was transferred to a solution of Fmoc-Gly-Gly-Gly-OH (151) (36 mg, 0.086 mmol) in anhydrous DMF (2 mL). DIPEA (43 μL, 0.25 mmol) and HATU (33 mg, 0.086 mmol) were added. After 18 h, the mixture was concentrated, and the residue was purified by silica gel column chromatography (0→20% MeOH in DCM) to give the desired compound 160 in 62% yield (60 mg, 0.054 mmol). LCMS (ESI+) C 56 H 83 N5O 18 + (M+H + ) calculated value 1113.57, actual value 1114.93.

[0283] Example 35. Synthesis of Compound 161 Compound 160 (36 mg, 0.032 mmol) was dissolved in DMF (2 mL) and piperidine (200 μL) was added. After 2 h, the mixture was concentrated and the residue was purified by silica gel column chromatography (0→40% 0.7 N NH3MeOH in DCM) to give the desired compound 161 as a yellow oil (16.7 mg, 0.0187 mmol, 58%). LCMS (ESI+) C 41 H 73 N5O 16 + (M+H + ) calculated value 891.51, actual value 892.82.

[0284] [ka]

[0285] Example 36. Synthesis of Compound 162 To a solution of amino-PEG-amine 106 (60 mg, 0.056 mmol) in DCM (3 mL) was slowly added (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate 102 (12 mg, 0.037 mmol) dissolved in DCM (5 mL). After 4 h, the mixture was concentrated and redissolved in DMF (2 mL), followed by the addition of Fmoc-Gly-Gly-Gly-OH 51 (23 mg, 0.056 mmol), HATU (21 mg, 0.056 mmol), and DIPEA (27 μL, 0.16 mmol). After 20 h, the mixture was concentrated and the residue was purified by silica gel column chromatography (0→27% MeOH in DCM) to give the desired compound 162 in 93% (57 mg, 0.043 mmol). LCMS (ESI+) C 80 H 131 N5O 30 + (M+NH4 + ) calculated value 1641.89 Measured value 1659.92.

[0286] Example 37. Synthesis of Compound 163 Compound 162 (57 mg, 0.034 mmol) was dissolved in DMF (1 mL) and piperidine (120 μL) was added. After 2 h, the mixture was concentrated, redissolved in water, and the Fmoc-piperidine by-product was removed by extraction with diethyl ether (3 × 10 mL). After lyophilization, 163 was obtained as a yellow oil (46.1 mg, 0.032 mmol, 95%). LCMS (ESI+) C 65 H 121 N5O 28 + (M+H + ) calculated value 1419.82, actual value 1420.91.

[0287] [ka]

[0288] Example 38. Synthesis of Compound 165 To a solution of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate (102) (204 mg, 0.650 mmol) was added amino-PEG-12-alcohol (164) (496 mg, 0.908 mmol) and triethylamine (350 μL, 2.27 mmol). After 19 h, the mixture was concentrated, and the residue was purified by silica gel column chromatography (2→20% MeOH in DCM) to give 165 as a clear yellow oil (410 mg, 0.560 mmol, 87%). LCMS (ESI+) C 35 H 63 NO 14 + (M+ Na + ) calculated value 721.42, actual value 744.43.

[0289] Example 39. Synthesis of Compound 166 To a solution of 165 (410 mg, 0.560 mmol) in DCM (6 mL) was added 4-nitrophenyl chloroformate (171, 0.848 mmol) and triethylamine (260 μL, 1.89 mmol). After 18 h, the mixture was concentrated and the residue was purified by silica gel column chromatography (0→7% MeOH in DCM) to give the desired compound 166 as a clear oil (350 mg, 0.394 mmol, 70%). LCMS (ESI+) C 42 H 66 N2O 18 + (M+ Na + ) calculated value 886.43, actual value 909.61.

[0290] [ka]

[0291] Example 40. Synthesis of Compound 168 To a solution of 166 (15 mg, 0.017 mmol) in DMF (2 mL) was added peptide LPETGG (167) (9.7 mg, 0.017 mmol) and triethylamine (7 μL, 0.05 mmol). After 46 h, the mixture was concentrated and the residue was purified by preparative HPLC to give the desired compound 168 in 63% (14 mg, 0.010 mmol). LCMS (ESI+) C 60 H 101 N7O 25 + (M+H + ) calculated value 1319.68, actual value 1320.92.

[0292] [ka]

[0293] Synthesis of Example 42.182 To a solution of 180 (methyltetrazine-NHS ester, 19 mg, 0.058 mmol) in DCM (0.8 mL) was added 181 (33.6 mg, 0.061 mmol) and EtN (24 μL, 0.17 mmol). After stirring at room temperature for 2.5 h, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→15% MeOH in DCM) to give the desired compound 182 in 93% yield (41 mg, 0.054 mmol). LCMS (ESI+) C 35 H 60 N5O 13 + (M+H + ) calculated value 758.88, actual value 758.64.

[0294] Synthesis of Example 43.183 To a solution of 182 (41 mg, 0.054 mmol) in DCM (3 mL) was added 4-nitrophenyl chloroformate (16 mg, 0.081 mmol) and EtN (23 μL, 0.16 mmol). After stirring at room temperature for 21 h, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (gradient: A. 0% to 20% EtOAc in DCM (until p-nitrophenol escapes), followed by gradient B. 0% to 13% MeOH in DCM) to give the desired compound 183 in 76% yield (37.9 mg, 0.041 mmol). LCMS (ESI+) C 42 H 63 NO 17 + (M+H + ) calculated value 923.98 Measured value 923.61.

[0295] Example 44. Synthesis of XL10 To a solution of 184 (5.6 mg, 0.023 mmol), prepared by MacDonald et al., Nat. Chem. Biol. 2015, 11, 326-334, incorporated by reference, in anhydrous DMF (0.1 mL) was added 183 (14.3 mg, 0.015 mmol) dissolved in anhydrous DMF (0.3 mL) and EtN (7 μL, 0.046 mmol). After stirring at room temperature for 2 h, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→15% MeOH in DCM) to give the desired compound XL10 in 50% yield (7.5 mg, 0.0076 mmol). LCMS (ESI+) C 47 H 73 N8O 15 + (M+H + ) Calculated value 990.13 Measured value 990.66.

[0296] [ka] Synthesis of Example 45.186 To a solution of octa-ethylene glycol 185 in DCM (10 mL) was added triethylamine (1.0 mL, 7.24 mmol, 2.5 equiv.), followed by the dropwise addition of a solution of 4-nitrophenyl chloroformate (0.58 g, 2.90 mmol, 1 equiv.) in DCM (5 mL) over 28 min. After stirring the mixture for 90 min, the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (75% → 0% EtOAc in DCM, followed by 0% → 7% MeOH in DCM). The product 186 was obtained as a colorless oil (584.6 mg, 1.09 mmol) in 38% yield. LCMS (ESI+) C 23 H 38 NO 13 + (M+H + ) calculated value 536.23, actual value 536.93. 1H-NMR (400 MHz, CDCl3): δ (ppm) 8.28 (d, J = 12.0 Hz, 2H), 7.40 (d, J = 12.0 Hz, 2H), 4.47 - 4.42 (m, 2H), 3.84 - 3.79 (m, 2H), 3.75 - 3.63 (m, 26H), 3.63 - 3.59 (m, 2H), 2.70 - 2.55 (bs, 1H).

[0297] Example 46. Synthesis of 188 To a solution of 187 (BocNH-PEG2)2NH (202 mg, 0.42 mmol) in DCM (1 mL) was added a portion (0.5 mL, 0.54 mmol, 1.3 equiv) of the prepared stock solution of 186 (584 mg in DCM (1 mL)), followed by triethylamine (176 μL, 1.26 mmol, 3 equiv) and HOBt (57 mg, 0.42 mmol, 1 equiv). After stirring for 8 days, the mixture was concentrated in vacuo. The residue was dissolved in a mixture of acetonitrile (4.2 mL) and 0.1 N NaOH (aq) (4.2 mL, 1 equiv) with an additional amount of solid NaOH (91.5 mg). After stirring for an additional 21.5 h, the mixture was extracted with DCM (3 × 40 mL). The combined organic layers were concentrated in vacuo, and the residue was purified by silica gel column chromatography (0% → 15% MeOH in DCM). The product 188 was obtained in 87% yield as a pale yellow oil (320.4 mg, 0.37 mmol). LCMS (ESI+) C 39 H 78 N3O 18 + (M+H + ) calculated value 876.53, actual value 876.54. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 5.15 - 5.02 (bs, 2H), 4.25 - 4.19 (m, 2H), 3.76 - 3.46 (m, 50H), 3.35 - 3.26 (m, 4H), 2.79 - 2.69 (br. s, 1H), 1.44 (s, 18H).

[0298] Synthesis of Example 47.189 188 (320 mg, 0.37 mmol) was dissolved in DCM (1 mL). 4M HCl in dioxane (456 μL, 1.83 mmol, 5 equiv) was then added. The mixture was stirred for 3.5 h, after which additional 4M HCl in dioxane (450 μL, 1.80 mmol, 4.9 equiv) was added. The mixture was stirred for another 3.5 h, after which additional 4M HCl in dioxane (450 μL, 1.80 mmol, 4.9 equiv) was added. The mixture was stirred for 16.5 h, after which the mixture was concentrated in vacuo. The product 189 was obtained as a white sticky solid in quantitative yield, which was used directly in the next step. 1 H-NMR (400 MHz, DMSO-d6): δ (ppm) 8.07 - 7.81 (bs, 6H), 4.15 - 4.06 (m, 2H), 3.75 - 3.66 (m, 2H), 3.65 - 3.48 (m, 48H), 3.03 - 2.92 (m, 4H).

[0299] [ka]

[0300] Example 48. Synthesis of 190 To a solution of BCN-OH (101, 164 mg, 1.10 mmol, 3 equiv.) in DCM (3 mL) was added CSI (76 μL, 0.88 mmol, 2.4 equiv.). After stirring for 15 min, triethylamine (255 μL, 5.50 mmol, 5 equiv.) was added. A solution of 189 was prepared by adding DCM (3 mL) and triethylamine (508 μL, 11.0 mmol, 10 equiv.). This stock solution was added to the original reaction mixture 6 min later. The mixture was stirred for 21.5 h, after which the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (0% → 10% MeOH in DCM). Product 190 was obtained in 39% yield as a pale yellow oil (165.0 mg, 139 μmol). LCMS (ESI+) C 43 H 72 N5O 18 S2 +(M+H + ) calculated value 1186.54, actual value 1186.65. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 6.09 - 5.87 (m, 2H), 4.31 - 4.19 (m, 6H), 3.76 - 3.50 (m, 50H), 3.40 - 3.29 (m, 4H), 2.38 - 2.16 (m, 12H), 1.66 - 1.47 (m, 4H), 1.40 (quintet, J = 8.0 Hz, 2H), 1.04 - 0.94 (m, 4H).

[0301] Example 49. Synthesis of 191 To a solution of 190 (101 mg, 0.085 mmol) in DCM (2.0 mL) was added bis(4-nitrophenyl)carbonate (39 mg, 0.127 mmol) and EtN (36 μL, 0.25 mmol). After stirring at room temperature for 42 h, the crude mixture was concentrated in vacuo and purified by silica gel flash column chromatography (A. 0% to 25% EtOAc in DCM (until p-nitrophenol escapes), followed by gradient B. 0% to 12% MeOH in DCM) to give 191 as a clear oil (49 mg, 0.036 mmol, 42%). LCMS (ESI+) C 58 H 91 NO 26 S2 + (M+H + ) calculated value 1352.50 Measured value 1352.78.

[0302] Example 50. Synthesis of XL11 To a solution of 191 (7 mg, 0.0059 mmol) in anhydrous DMF (130 μL) was added EtN (2.2 μL, 0.015 mmol) and TCO-amine hydrochloride (Broadpharm) (1.8 mg, 0.0068 mmol). After stirring at room temperature for 19 h, the crude mixture was purified by silica gel flash column chromatography (0% → 15% MeOH in DCM) to give XL11 as a clear oil (1.5 mg, 0.001 mmol, 17%). LCMS (ESI+) C64 H 111 N8O 25 S2 + (M+NH4 + ) calculated value 1456.73, actual value 1456.81.

[0303] [ka]

[0304] Synthesis of Example 51.194 To a solution of available 187 (638 mg, 1.33 mmol) in DCM (8.0 mL) was added 128 (470 mg, 1.73 mmol), EtN (556.0 μL, 4.0 mmol), and 1-hydroxybenzotriazole (179.0 mg, 1.33 mmol). After stirring at ambient temperature for 41 h, the mixture was concentrated in vacuo and redissolved in MeCN (10 mL), followed by the addition of 0.1 M aqueous NaOH (10 mL) and solid NaOH pellets (100.0 mg). After 1.5 h, DCM (20 mL) was added, and the desired compound was extracted four times. The organic layer was concentrated in vacuo, and the residue was purified by silica gel flash column chromatography (0% → 12% MeOH in DCM) to give 194 as a clear yellow oil (733 mg, 1.19 mmol, 90%). 1 H NMR (400 MHz, CDCl3) δ (ppm) 4.29 - 4.23 (m, 2H), 3.77 - 3.68 (m, 4H), 3.65 - 3.56 (m, 14H), 3.56 - 3.49 (m, 8H), 3.37 - 3.24 (m, 4H), 1.45 (s, 18H). LCMS (ESI+) C 27 H 54 N3O 12 + (M+H + ) calculated value 612.73, actual value 612.55.

[0305] Example 52. Synthesis of 195 To a solution of 194 (31.8 mg, 0.052 mmol) in DCM (1.0 mL) was added 4.0 M HCl in dioxane (0.4 mL). After stirring at ambient temperature for 2.5 h, the reaction mixture was concentrated in vacuo, in between redissolving in DCM (2 mL) and concentrating. Compound 195 was obtained as a clear oil in quantitative yield. LCMS (ESI+) C 17 H 38 N3O8 + (M+H + ) Calculated value 412.50 Measured value 412.45

[0306] Synthesis of Example 53.196 To a cooled solution (0 °C) of 195 (21.4 mg, 0.052 mmol) in DCM (1.0 mL) was added EtN (36 μL, 0.26 mmol) and 2-bromoacetyl bromide (10.5 μL, 0.12 mmol). After stirring on ice for 10 min, the ice bath was removed and 0.1 M aqueous NaOH (0.8 mL) was added. After stirring at room temperature for 20 min, the aqueous layer was extracted with DCM (2 × 5 mL). The organic layers were combined and concentrated in vacuo. The crude brown oil was purified by silica gel flash column chromatography (0% → 18% MeOH in DCM) to give 196 as a clear oil (6.9 mg, 0.011 mmol, 20%). LCMS (ESI+) C 21 H 40 Br2N3O 10 + (M+H + ) calculated value 654.36, actual value 654.29.

[0307] Example 54. Synthesis of XL12 To a solution of 196 (6.9 mg, 0.011 mmol) in DCM (0.8 mL) was added bis(4-nitrophenyl)carbonate (3.8 mg, 0.012 mmol) and EtN (5 μL, 0.03 mmol). After stirring at room temperature for 18 h, 155 (BCN-PEG-NH, 3.3 mg, 0.01 mmol) dissolved in DCM (0.5 mL) was added. After stirring for an additional 2 h, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (gradient: A. 0% to 30% EtOAc in DCM (until p-nitrophenol escapes), followed by gradient B. 0% to 20% MeOH in DCM) to give XL12 as a clear oil (1.0 mg, 0.001 mmol, 9%). LCMS (ESI+) C 39 H 66 Br2N5O 15 + (M+H + ) calculated value 1004.77, actual value 1004.51.

[0308] [ka]

[0309] Synthesis of Example 55.197 To a solution of 102 (204 mg, 0.647 mmol) in DCM (20 mL) was added 181 (496 mg, 0.909 mmol) and EtN (350 μL, 2.27 mmol). After stirring at room temperature for 19 h, the solvent was reduced in vacuo and the residue was purified by silica gel flash column chromatography (2 → 20% MeOH in DCM) to give the desired compound 197 as a yellow oil in 87% yield (410 mg, 0.567 mmol). LCMS (ESI+) C 35 H 63 NO 14 Na + (M+Na + ) calculated value 744.86, actual value 744.43.

[0310] Example 56. Synthesis of 198 To a solution of 197 (410 mg, 0.567 mmol) and 4-nitrophenyl chloroformate (172 mg, 0.853 mmol) in DCM (6 mL) was added EtN (260 μL, 1.88 mmol). After stirring at room temperature for 18 h, the solvent was reduced in vacuo and the residue was purified by silica gel flash column chromatography (0→7% MeOH in DCM) to give the desired compound 198 as a clear oil in 70% yield (350 mg, 0.394 mmol). LCMS (ESI+) C 42 H 66 N2O 18 Na + (M+Na + ) Calculated value 909.96 Measured value 909.61.

[0311] Example 57. Synthesis of XL13 To a solution of 198 (44.2 mg, 0.05 mmol) in DCM (5 mL) was added 199 (bis-aminooxy-PEG2, 33.3 mg, 0.18 mmol) and EtN (11 μL, 0.07 mmol). After stirring at room temperature for 67 h, the mixture was concentrated in vacuo and purified by RP HPLC (column XBridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN in HO (both containing 1% acetic acid)). The product XL13 was obtained as a clear oil (8.1 mg, 0.0087 μmol, 17%). LCMS (ESI+) C 42 H 78 N3O 19 + (M+H + ) Calculated value 929.08 Measured value 928.79.

[0312] [ka]

[0313] Example 58. Synthesis of 319 To a solution of compound 121 (442 mg, 1.46 mmol) in DCM (1 mL) and DMF (200 μL) was added a solution of compound 128 in DCM (1 mL) and triethylamine (609 μL, 4.37 mmol). After stirring for 16 h, the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (50% to 100% EtOAc in heptane) to give 319 (316 mg). This was further purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% to 90% MeCN (1% AcOH) in water (1% AcOH)). The product 319 was obtained as a colorless oil (110 mg, 0.25 mmol) in 17% yield. LCMS (ESI+) C 19 H 37 N3NaO8 + (M+Na + ) calculated value 458.25, actual value 458.33. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 5.41 - 4.89 (m, 2H), 4.31 - 4.24 (m, 2H), 3.78 - 3.68 (m, 4H), 3.65 - 3.59 (m, 2H), 3.44 - 3.34 (m, 4H), 3.34 - 3.19 (m, 4H), 1.43 (s, 18H).

[0314] Example 59. Synthesis of 320 Compound 319 (107 mg, 0.25 mmol) was dissolved in DCM (1 mL). 4 M HCl in dioxane (300 μL, 1.2 mmol, 4.8 equiv.) was then added. After stirring the mixture for 15 h, the mixture was decanted from the precipitate, which was washed once with DCM (2 mL). Product 320 was obtained in quantitative yield as a white sticky solid (89.9 mg, 0.29 mmol), which was used directly in the next step.

[0315] Synthesis of Example 60.321 To a solution of 101 (75 mg, 0.50 mmol, 2 equiv.) in DCM (1 mL) was added CSI (41 μL, 0.48 mmol, 1.9 equiv.). After stirring for 6 min, triethylamine (139 μL, 1.0 mmol, 4 equiv.) was added. A stock solution of 320 was prepared by adding DMF (200 μL) and DCM (2 mL), followed by triethylamine (139 μL, 0.75 mmol, 3 equiv.). A portion of this stock solution of 320 (32 μL, 0.25 mmol) was added to the original reaction mixture containing CSI. After stirring for 16 h, the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (0% → 10% MeOH in DCM). The product 321 was obtained as a colorless oil (11 mg, 14.2 μmol) in 3% yield. LCMS (ESI+) C 31 H 48 N5O 12 S2 + ((M+H + ) calculated value 746.27, actual value 746.96. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 6.36 - 5.94 (m, 2H), 4.38 - 4.17 (m, 6H), 3.84 - 3.79 (m, 2H), 3.77 - 3.72 (m, 2H), 3.68 - 3.63 (m, 2H), 3.54 - 3.45 (m, 4H), 3.39 - 3.27 (m, 4H), 2.38 - 2.16 (m, 12H), 1.67 - 1.47 (m, 5H), 1.40 (quintet, J = 8.0 Hz, 2H), 1.05 - 0.93 (m, 4H).

[0316] Example 61. Synthesis of 301 (LD01) To a solution of 321 (10.6 mg, 14.2 μmol) in DCM (100 μL) was added bis(4-nitrophenyl)carbonate (4.3 mg, 14.2 μmol, 1.0 equiv.) and triethylamine (5.9 μL, 42.6 μmol, 3.0 equiv.). After stirring for 66 h, a portion of this mixture was treated with a stock solution of vc-PABC-MMAE.TFA in DMF (200 μL, 50 mg / mL) and an additional amount of triethylamine (5.9 μL, 42.6 μmol, 3.0 equiv.). After 24 h, it was partially concentrated in vacuo. The residue was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN (1% AcOH) in water (1% AcOH)). Compound 301 was obtained as a film (3.4 mg, 1.9 μmol) in 28% yield. LCMS (ESI+) C 90 H 140 N 15 O 25 S2 + ((M+H + ) calculated value 1894.96, actual value 1895.00.

[0317] [ka] Synthesis of Example 62.322 To a solution of 185 (octaethylene glycol) in DCM (10 mL) was added triethylamine (1.0 mL, 7.24 mmol; 2.5 equiv.), followed by the dropwise addition of a solution of 4-nitrophenyl chloroformate (0.58 g; 2.90 mmol; 1 equiv.) in DCM (5 mL) over 28 min. The mixture was stirred for 90 min, after which the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (75% to 0% EtOAc in DCM, followed by 0% to 7% MeOH in DCM). The product 322 was obtained in 38% yield as a colorless oil (584.6 mg; 1.09 mmol). LCMS (ESI+) C 23 H 38 NO 13 + (M+H + ) calculated value 536.23, actual value 536.93.1 H-NMR (400 MHz, CDCl3): δ (ppm) 8.28 (d, J = 12.0 Hz, 2H), 7.40 (d, J = 12.0 Hz, 2H), 4.47 - 4.42 (m, 2H), 3.84 - 3.79 (m, 2H), 3.75 - 3.63 (m, 26H), 3.63 - 3.59 (m, 2H), 2.70 - 2.55 (br. s, 1H).

[0318] Synthesis of Example 63.323 To a solution of compound 121 (127 mg, 0.42 mmol) in DCM (1 mL) was added a portion (0.5 mL; 0.54 mmol; 1.3 equiv) of a prepared stock solution of 322 (584 mg in 1 mL DCM), followed by triethylamine (176 μL, 1.26 mmol; 3 equiv) and HOBt (57 mg; 0.42 mmol; 1 equiv). After stirring for 4.5 days, the mixture was concentrated in vacuo. The residue was dissolved in a mixture of acetonitrile (4.2 mL) and 0.1 N NaOH (4.2 mL, 1 equiv). After stirring for 24 h, additional solid NaOH (104.5 mg) was added. After stirring for an additional 5 h, the mixture was extracted with DCM (2 × 10 mL). The combined organic layers were concentrated in vacuo, and the residue was purified by silica gel column chromatography (0% → 15% MeOH in DCM). The product 323 was obtained in 54% yield as a pale yellow oil (164.5 mg, 0.23 mmol). LCMS (ESI+) C 26 H 54 N3O 12 + (M-BOC + ) calculated value 600.36, actual value 600.49. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 5.27 - 5.05 (m, 2H), 4.26 - 4.21 (m, 2H), 3.76 - 3.59 (m, 30H), 3.43 - 3.33 (m, 4H), 3.33 - 3.22 (m, 4H), 1.43 (s, 18H).

[0319] Synthesis of Example 64.324 Compound 323 (164 mg, 0.23 mmol) was dissolved in DCM (1 mL). 4M HCl in dioxane (293 μL, 1.17 mmol, 5 equiv.) was then added. The mixture was stirred for 18 h, after which additional 4M HCl in dioxane (293 μL, 1.17 mmol, 5 equiv.) was added. The mixture was stirred for an additional 5 h, after which the mixture was concentrated in vacuo. Product 324 was obtained in quantitative yield as a white sticky solid (132 mg, 0.23 mmol). This was used directly in the next step.

[0320] Example 65. Synthesis of 325 To a solution of 101 (81 mg, 0.54 mmol, 2.3 equiv) in DCM (2 mL) was added CSI (43 μL, 0.49 mmol, 2.1 equiv). After stirring for 15 min, triethylamine (164 μL, 1.17 mmol, 5 equiv) was added. A solution of 324 was prepared by adding DCM (2 mL) and triethylamine (164 μL, 1.17 mmol, 5 equiv). This stock solution was added to the original reaction mixture after 6 min. The mixture was stirred for 23 h, after which the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (0% → 12% MeOH in DCM). The product 325 was obtained as a pale yellow oil (73.0 mg, 72.2 μmol) in 31% yield. LCMS (ESI+) C 43 H 72 N5O 18 S2 + (M+H + ) calculated value 1010.43, actual value 1010.50. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 6.21 - 5.85 (m, 2H), 4.38 - 4.17 (m, 6H), 3.80 - 3.57 (m, 30H), 3.57 - 3.44 (m, 4H), 3.44 - 3.30 (m, 4H), 2.38 - 2.16 (m, 12H), 1.64 - 1.48 (m, 4H), 1.40 (quintet, J = 8.0 Hz, 2H), 1.05 - 0.91 (m, 4H).

[0321] Example 66. Synthesis of 302 (LD02) To a solution of 325 (19.5 mg, 19.7 μmol) in DCM (100 μL) was added bis(4-nitrophenyl)carbonate (6.0 mg, 19.7 μmol, 1.0 equiv.) and triethylamine (8.2 μL, 59.1 μmol, 3.0 equiv.). After stirring for 66 h, a portion of this mixture was treated with a stock solution of vc-PABC-MMAE.TFA in DMF (200 μL, 50 mg / mL) and an additional amount of triethylamine (8.2 μL, 59.1 μmol, 3.0 equiv.). After 95 h, it was partially concentrated in vacuo. The residue was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN (1% AcOH) in water (1% AcOH)). Compound 302 was obtained as a film (3.7 mg, 1.71 μmol) in 9% yield. LCMS (ESI+) C 102 H 165 N 15 O 31 S2 2+ (M+2H + ) calculated value 1080.56, actual value 1080.74.

[0322] [ka]

[0323] Synthesis of Example 67.329 To a solution of 101 (18 mg, 0.12 mmol) in DCM (1 mL) was added chlorosulfonyl isocyanate (CSI). After 30 min, EtN (37 μL, 27 mg, 0.27 mmol) was added. To a solution of 195 (26 mg, 0.054 mmol) in DCM (1 mL) was added EtN (37 μL, 27 mg, 0.27 mmol). This mixture was added to the reaction mixture. After 45 min, the reaction mixture was concentrated, and the residue was purified by silica gel chromatography (DCM → 7% MeOH in DCM). Product 329 was obtained as a colorless film (27 mg, 0.029 mmol, 54%). LCMS (ESI+) C 39 H 64 N5O 16 S2 + (M+H + ) calculated value 922.38, actual value 922.50.

[0324] Example 68. Synthesis of 330 To a solution of 329 in DCM (1 mL) was added bis(4-nitrophenyl)carbonate (8.9 mg, 29.3 μmol) and EtN (12.2 μL, 8.9 mg, 87.9 μmol). One day later, 0.28 mL was used to prepare compound 303. Two days later, extra bis(4-nitrophenyl)carbonate (7.0 mg, 23 μmol) was added to the main reaction mixture. One day later, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography. Product 330 was obtained as a colorless film (17.5 mg, 0.016 mmol, 55% (76% corrected)). LCMS (ESI+) C 46 H 67 NO 20 S2 + (M+H + ) calculated value 1087.38, actual value 1087.47.

[0325] Example 69. Synthesis of 303 (LD03) To the reaction mixture of 330 (0.28 mL, theoretically containing 8.8 mg, 8.1 μmol), a solution of EtN (3.4 μL, 2.5 mg, 24.3 μmol) and vc-PABC-MMAE.TFA (10 mg, 8.1 μmol) in DMF (200 μL) was added. After 21 h, 2,2'-(ethylenedioxy)bis(ethylamine) (4.7 μL, 4.8 mg, 32 μmol) was added. After 45 min, the reaction mixture was concentrated under a stream of nitrogen gas. The residue was purified by RP-HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% to 90% MeCN (1% AcOH) in water (1% AcOH)). Product 303 was obtained as a colorless film (5.6 mg, 2.7 μmol). LCMS (ESI+) C 98 H 157 N 15 O 29 S2 2+ ((M+2H + ) / 2) calculated value 1036.53, actual value 1036.70.

[0326] [ka]

[0327] Synthesis of Example 70.332 To a solution of Alloc2-va-PABC-PBD 331 (10.0 mg, 0.009 mmol) in degassed DCM (400 μL, obtained by purging N through DCM for 5 min) was added pyrrolidine (1.9 μL, 0.027 mmol) and Pd(PPh3)4 (1.6 mg, 0.0014 mmol). After stirring at ambient temperature for 15 min, the reaction mixture was diluted with DCM (10 mL) and saturated aqueous NH4Cl (10 mL) was added. The crude mixture was extracted with DCM (3 × 10 mL). The organic layers were combined, dried over Na2SO4, filtered, and concentrated in vacuo. The yellow residue was redissolved in DMF (450 μL) and MeCN (450 μL) and purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN in HO (both containing 0.1% formic acid)). Pure fractions were neutralized on an SPE column (PL-HCO3MP, 500 mg / 6 mL), concentrated, and coevaporated with MeCN (2 × 5 mL) to give 332 as a white solid (4.8 mg, 0.005 mmol, 58%). LCMS (ESI+) C 49 H 60 N7O 11 + (M+H + ) Calculated value 923.04 Measured value 923.61.

[0328] Example 71. Synthesis of 304 (LD04) To a solution of 332 (4.8 mg, 0.005 mmol) in anhydrous, degassed DMF (60 μL, obtained by purging N through DMF for 5 min) was added 330 (10 mg, 0.009 mmol, dissolved in 48 μL of anhydrous, degassed DMF), EtN (3.6 μL, 0.026 mmol), and HOBt (stock in anhydrous, degassed DMF, 5.1 μL, 0.35 mg, 0.0026 mmol, 0.5 equiv). After stirring in the dark at ambient temperature for 41 h, the crude reaction mixture was diluted with DCM (300 μL) and purified by silica gel flash column chromatography (0% → 12% MeOH in DCM) to give 304 as a clear, yellow oil (4.0 mg, 0.0021 mmol, 41%). LCMS (ESI+) C 89 H 121N 12 O 28 S2 + (M+H + ) Calculated value 1871.11 Actual value 1871.09.

[0329] [ka]

[0330] Example 72. Synthesis of 305 (LD05) To a solution of 333 (2.9 mg, 0.0013 mmol) in anhydrous DMF (60 μL), prepared according to Example 5-5 of International Publication No. 2019110725, incorporated by reference, were added 330 (1.45 mg, 0.0013 mmol) and EtN (1.2 μL, 0.023 mmol). After stirring at room temperature for 48 h, the reaction mixture was diluted with DMF (500 μL) and purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% → 100% MeCN in HO (both containing 1% acetic acid)). The product 305 was obtained as a colorless film (0.6 mg, 0.207 μmol, 16%). LCMS (ESI+) C 124 H 182 IN 14 O 46 S5 + (M / 2+H + ) Calculated value 1447.03 Measured value 1447.19.

[0331] [ka]

[0332] Example 73. Synthesis of 306 (LD06) To a solution of 330 (7 mg, 0.006 mmol) in anhydrous DMF (150 μL) was added vc-PABC-DMEA-PNU (334) stock in anhydrous DMF (125 μL, 5.7 mg, 0.005 mmol) and EtN (2 μL, 0.015 mmol). After stirring at room temperature for 25 h, the reaction mixture was diluted with DCM (0.3 mL) and purified by silica gel flash column chromatography (0% → 20% MeOH in DCM) to give 306 as a red film (5 mg, 0.0024 mmol, 47%). LCMS (ESI+) C 96 H 133 N 13 O 36 S3 + (M / 2+H + ) calculated value 1055.64 Measured value 1055.50.

[0333] [ka]

[0334] Synthesis of Example 74.337 Compound 336 (DIBO, 95 mg, 0.43 mmol) was dissolved in DCM (1.0 mL) and chlorosulfonyl isocyanate (33.0 μL, 0.37 mmol) was added at room temperature. After 2 min, insoluble material formed. After stirring for an additional 15 min at room temperature, EtN (120.0 μL, 0.85 mmol) was added, and all insoluble material disappeared. A mixture of 195 (71 mg, 0.0171) and EtN (120.0 μL, 0.85 mmol) dissolved in DCM (1.0 mL) was added. After stirring for 16 h at room temperature, the crude mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0% → 15% MeOH in DCM), which was then coevaporated with EtOAc (2x) to completely remove MeOH. The product 337 was obtained as a waxy white solid (136.0 mg, 0.12 mmol, 75%). LCMS (ESI+) C 51 H 63 NO 16 S2 + (M+NH4 +) calculated value 1080.21, actual value 1080.59.

[0335] Synthesis of Example 75.338 To a solution of 337 (136.0 mg, 0.12 mmol) in DCM (2.0 mL) was added bis(4-nitrophenyl)carbonate (47.0 mg, 0.15 mmol) and EtN (54.0 μL, 0.38 mmol). After stirring at room temperature for 18 h, the crude mixture was concentrated in vacuo and purified by silica gel flash column chromatography (gradient: A. 0% to 35% EtOAc in DCM (until p-nitrophenol escapes), followed by gradient B. 0% to 13% MeOH in DCM) to give 338 as a pale yellow oil (89.0 mg, 0.07 mmol, 60%). LCMS (ESI+) C 48 H 66 N7O 20 S2 + (M+NH4 + ) calculated value 1245.31, actual value 1245.64.

[0336] Example 76. Synthesis of 307 (LD07) To a solution of 338 (6.95 mg, 0.005 mmol) in anhydrous DMF (93.0 μL) was added EtN (2.4 μL, 0.017 mmol) and a stock solution of vc-PABC-MMAE.TFA (Levena Bioscience) in anhydrous DMF (70 μL, 7.0 mg, 0.005 mmol). After stirring at room temperature for 18 h, DMF (450 μL) was added and the crude mixture was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% → 100% MeCN in HO (both containing 1% acetic acid)). Product 307 was obtained as a colorless film (4.5 mg, 0.002 mmol, 36%). LCMS (ESI+) C 110 H 152 N 15 O 29 S2 + (M / 2+H + ) calculated value 1106.30, actual value 1106.79.

[0337] [ka]

[0338] Synthesis of Example 77.341 Compound 101 (16.3 mg, 0.10 mmol) was dissolved in DCM (0.8 mL) and chlorosulfonyl isocyanate (8.6 μL, 0.099 mmol) was added at room temperature. After stirring for 15 min at room temperature, EtN (69.0 μL, 0.49 mmol) was added, followed by a mixture of 335 (40 mg, 0.099 mmol) and EtN (69.0 μL, 0.49 mmol) dissolved in DCM (1.0 mL). This mixture was stirred at room temperature for 1.5 h (mixture 1) to give crude 339. In a separate vial, 340 (DBCO-C2-OH, Broadpharm) (34.0 mg, 0.099 mmol) was dissolved in DCM (0.8 mL) at room temperature and chlorosulfonyl isocyanate (7.75 μL, 0.089 mmol) was added. After stirring at room temperature for 15 min, EtN (69.0 μL, 0.49 mmol) was added followed by crude 339. After stirring at room temperature for an additional 2 h, the reaction mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0% → 15% MeOH in DCM), which was then co-evaporated with EtOAC (2×) to completely remove MeOH. The product 341 was obtained as a clear yellow oil (20.0 mg, 0.017 mmol, 17%). LCMS (ESI+) C 50 H 70 N7O 18 S2 + (M+H + ) calculated value 1121.26 Measured value 1121.59.

[0339] Synthesis of Example 78.342 To a solution of 341 (20.0 mg, 0.17 mmol) in DCM (1.0 mL) was added bis(4-nitrophenyl)carbonate (5.6 mg, 0.019 mmol) and EtN (7.5 μL, 0.053 mmol). After stirring at room temperature for 40 h, the crude mixture was concentrated in vacuo and purified by silica gel flash column chromatography (gradient: A. 0% to 30% EtOAc in DCM (until p-nitrophenol escapes), followed by gradient B. 0% to 20% MeOH in DCM) to give 342 as a clear, pale yellow oil (6.9 mg, 0.005 mmol, 30%). LCMS (ESI+) C 57 H 73 N8O 22 S2 + (M+H + ) calculated value 1286.36, actual value 1286.57.

[0340] Example 79. Synthesis of 308 (LD08) To a solution of 342 (3.6 mg, 0.0028 mmol) in anhydrous DMF (35.0 μL) was added EtN (1.2 μL, 0.008 mmol) and a stock solution of vc-PABC-MMAE.TFA (Levena Bioscience) in anhydrous DMF (34 μL, 3.4 mg, 0.0028 mmol). After stirring at room temperature for 27 h, DCM (400 μL) was added and the crude mixture was purified by silica gel flash column chromatography (0% → 30% MeOH in DCM) to give 308 as a colorless film (3.7 mg, 0.0016 mmol, 58%). LCMS (ESI+) C 109 H 161 N 17 O 31 S2 + (M / 2+H + ) calculated value 1135.84, actual value 1135.73.

[0341] [ka]

[0342] Example 80. Synthesis of 310 (LD10) To an Eppendorf vial containing 344 (4.3 mg, 6.0 μmol, 1.7 equiv.), vc-PABC-MMAF.TFA salt in DMF (4.00 mg, 100 μL, 34.31 mmol, 3.43 μmol, 1.0 equiv.) was added, followed by triethylamine (1.43 μL, 10.3 μmol, 3.0 equiv.). The mixture was mixed, and the resulting colorless solution was allowed to stand at room temperature for approximately 3 hours. The reaction mixture was then directly purified via RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% → 90% MeCN (1% AcOH) in water (1% AcOH)). The desired product 310 was obtained as a colorless residue (4.5 mg, 2.7 μmol, 79% yield). LCMS (ESI+) C 80 H 134 N 15 O 22 + (M+H + ) calculated value 1656.98. Actual value 1657.03.

[0343] [ka]

[0344] Synthesis of Example 81.346 To an Eppendorf vial containing 102 (54.7 mg, 1.00 equiv., 173 μmol) and 345 (triglycine, 28.8 mg, 0.878 equiv., 152 μmol), dry DMF (250 μL) and triethylamine (52.7 mg, 72.5 μL, 3 equiv., 520 μmol) were added. The resulting yellow suspension was stirred at room temperature for 21 h, after which 50 μL of HO was added to the reaction mixture. The reaction mixture was stirred at room temperature for an additional day, and additional HO (200 μL) was added, and the reaction mixture was stirred at room temperature for an additional 3 days. Next, MeCN (approximately 0.5 mL) and additional EtN (approximately 10 drops) were added, and the resulting suspension was stirred at room temperature for 1 h before being concentrated in vacuo. The yellow residue was dissolved in DMF (600 μL), and the resulting yellow suspension was filtered through a membrane filter. The membrane filter was washed with an additional 200 μL of DMF, and the combined filtrates were directly purified via RP HPLC (column Xbridge prep C18 5 μm OBD, 30×100 mm, 30%→90% MeCN (1% AcOH) in water (1% AcOH)). The desired product 346 was obtained as a brown oil (41.5 mg, 114 μmol, 66% yield). LCMS (ESI+) C 17 H 24 N3O6 + (M+H + ) calculated value: 366.17. Measured value: 366.27.

[0345] Synthesis of Example 82.347 To a solution of 346 (21.6 mg, 0.056 mmol) in anhydrous DMF (0.3 mL) were added DIPEA (30 μL, 0.171 mmol) and HATU (21.6 mg, 0.056 mmol). After stirring at room temperature for 10 min, 320 (7.37 mg, 0.031 mmol) dissolved in DCM (310 μL) was added. After stirring at room temperature for 24 h, the mixture was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% → 100% MeCN in HO (both containing 1% AcOH)). The product 347 was obtained as an off-white oil (5.2 mg, 0.005 mmol, 20%). LCMS (ESI+) C 43 H64 N9O 14 + (M+H + ) Calculated value 931.02 Measured value 931.68.

[0346] Example 83. Synthesis of 311 (LD13) To a solution of 347 (5.2 mg, 0.0056 mmol) in anhydrous DMF (200 μL) was added bis(4-nitrophenyl)carbonate (1.9 mg, 0.006 mmol) and EtN (2.4 μL, 0.016 mmol). After stirring at room temperature for 27 h, a stock solution of vc-PABC-MMAE.TFA (Levena Bioscience) (66 μL, 6.6 mg, 0.0053 mmol) and EtN (2 μL, 0.014 mmol) were added. After stirring at room temperature for an additional 17 h, the crude mixture was diluted with DMF (250 μL) and purified by RP HPLC (Xbridge prep C18 5 μm OBD, 30 × 100 mm column, 5% → 90% MeCN in HO (both containing 1% AcOH)). The product 311 was obtained as a clear oil (0.6 mg, 0.28 μmol, 5%). LCMS (ESI+) C 102 H 156 N 19 O 27 + (M / 2+H + ) calculated value 1040.71, actual value 1040.85.

[0347] Example 84. Synthesis of Compound 312 (LD11) Compound 312 (LD11) was prepared by the procedure described by Verkade et al., Antibodies 2018, 7, doi:10.3390 / antib7010012, which is incorporated by reference.

[0348] [ka]

[0349] Example 85. Synthesis of 313 (LD12) To a vial containing 348 (2.7 mg, 1.1 equiv., 4.9 μmol), DMF (60 μL) and neat triethylamine (1.9 μL, 3 equiv., 13 μmol) were added. Next, a solution of HBTU in dry DMF (2.0 mg, 11 μL, 472 mmol, 1.2 equiv., 5.3 μmol) was added and the mixture was mixed. The reaction mixture was allowed to stand at room temperature for 30 min, followed by the addition of va-PABC-MMAF.TFA salt (5.2 mg, 0.13 mL, 34.31 mmol, 1 equiv., 4.4 μmol). The resulting mixture was mixed and allowed to stand at room temperature for 110 min, then directly purified via RP HPLC (Xbridge prep C18 5 μm OBD, 30 × 100 mm column, 30% → 90% MeCN (1% AcOH) in water (1% AcOH)). The desired product 313 was obtained as a colorless oil (1.8 mg, 1.1 μmol, 26% yield). LCMS (ESI+) C 77 H 127 N 12 O 23 + (M+H + ) calculated value 1587.91. Measured value 1588.05.

[0350] [ka]

[0351] Example 86. Synthesis of 350 A solution of methyltetrazine-NHS ester 349 (19 mg, 0.057 mmol) in DCM (400 μL) was added to amino-PEG (800 μL) dissolved in DCM. 11 -amine (47 mg, 0.086 mmol) was added. After stirring at room temperature for 20 min, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→50% MeOH (0.7 M NH3) in DCM) to give the desired compound 350 as a pink oil (17 mg, 0.022 mmol, 39%). LCMS (ESI+) C 35 H 61 NO 12 + (M+H+ ) calculated value 757.89, actual value 757.46.

[0352] Synthesis of Example 87.351 To a stirred solution of 151 (Fmoc-Gly-Gly-Gly-OH, 10 mg, 0.022 mmol) in anhydrous DMF (500 μL) was added DIPEA (11 μL, 0.067 mmol) and HATU (8.5 mg, 0.022 mmol). After 10 min, 350 (17 mg, 0.022 mmol) dissolved in anhydrous DMF (500 μL) was added. After stirring at room temperature for 18.5 h, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→17% MeOH in DCM) to give the desired compound 351 as a pink oil (26 mg, 0.022 mmol, quantitative). LCMS (ESI+) C 56 H 83 N 10 O 17 + (M+NH4 + ) Calculated value 1168.32 Measured value 1168.67

[0353] Synthesis of Example 88.169 To a solution of 351 (26 mg, 0.022 mmol) in anhydrous DMF (500 μL) was added diethylamine (12 μL, 0.11 mmol). After stirring at room temperature for 1.5 h, the crude mixture was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN in HO (both containing 1% acetic acid)). Product 169 was obtained as a clear pink oil (10.9 mg, 0.011 mmol, 53%). LCMS (ESI+) C 41 H 70 N9O 15 + (M+H + ) Calculated value 929.05 Measured value 929.61.

[0354] [ka]

[0355] Synthesis of Example 89.352 A solution of 349 (methyltetrazine-NHS ester, 10.3 mg, 0.031 mmol) in DCM (200 μL) was added to amino-PEG (200 μL) dissolved in DCM (200 μL). 23 -amine (50 mg, 0.046 mmol) was added. After stirring at room temperature for 50 min, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→60% MeOH (0.7 M NH3) in DCM) to give the desired compound 352 as a pink oil (17.7 mg, 0.013 mmol, 44%). LCMS (ESI+) C 59 H 109 NO 24 + (M+H + ) calculated value 1286.52, actual value 1286.72.

[0356] Synthesis of Example 90.353 To a stirred solution of 151 (5.7 mg, 0.013 mmol) in anhydrous DMF (500 μL) was added DIPEA (7 μL, 0.04 mmol) and HATU (5.3 mg, 0.013 mmol). After 10 min, 352 (17.7 mg, 0.013 mmol) dissolved in anhydrous DMF (500 μL) was added. After stirring at room temperature for 6 h, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→18% MeOH in DCM) to give the desired compound 353 as a pink oil (21 mg, 0.012 mmol, 91%). LCMS (ESI+) C 80 H 131 N 10 O 29 + (M / 2+NH4 + ) Calculated value 857.45 Measured value 857.08

[0357] Example 91. Synthesis of 170 To a solution of 353 (21 mg, 0.012 mmol) in anhydrous DMF (500 μL) was added diethylamine (6.7 μL, 0.06 mmol). After stirring at room temperature for 4 h, the crude mixture was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN in HO (both containing 1% acetic acid)). Product 170 was obtained as a pink oil (11.6 mg, 0.008 mmol, 66%). LCMS (ESI+) C 65 H 118 N9O 27 + (M+H + ) calculated value 1457.68 Measured value 1457.92.

[0358] [ka]

[0359] Synthesis of Example 92.356 To a solution of 354 (tetrafluorophenyl azide-NHS ester, 40 mg, 0.12 mmol) in DCM (1 mL) was added 355 (Boc-NH-PEG-NH, 33 mg, 0.13 mmol) and EtN (50 μL, 0.36 mmol). After stirring at room temperature in the dark for 30 min, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→7% MeOH in DCM) to give the desired compound 356 as a clear oil (47 mg, 0.10 mmol, 84%). LCMS (ESI+) C 18 H 24 F4N5O5 + (M+H + ) calculated value 466.41, actual value 466.23.

[0360] Synthesis of Example 93.357 To a solution of 356 (47 mg, 0.10 mmol) in DCM (2 mL) was added 4.0 M HCl in dioxane (300 μL). After stirring at room temperature in the dark for 17.5 h, the mixture was concentrated to give 357 as a white solid in quantitative yield (36 mg, 0.10 mmol). LCMS (ESI+) C 13 H 16 F4N5O3 + (M+H + ) calculated value 366.29, actual value 366.20.

[0361] Synthesis of Example 94.358 To a stirred solution of 151 (Fmoc-Gly-Gly-Gly-OH, 42 mg, 0.10 mmol) in anhydrous DMF (600 μL) was added DIPEA (50 μL, 0.30 mmol) and HATU (39 mg, 0.10 mmol). After 15 min in the dark, 357 (36 mg, 0.10 mmol) dissolved in anhydrous DMF (500 μL) was added. After stirring for 41 h at room temperature in the dark, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→20% MeOH in DCM) to give the desired compound 358 as a clear oil (36 mg, 0.047 mmol, 47%). LCMS (ESI+) C 34 H 35 F4N8O8 + (M+H + ) calculated value 759.68 Measured value 759.38.

[0362] Synthesis of Example 95.171 To a solution of 358 (36 mg, 0.047 mmol) in anhydrous DMF (750 μL) was added diethylamine (24 μL, 0.24 mmol). After stirring for 55 min at room temperature in the dark, the crude mixture was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN in HO (both containing 1% acetic acid)). Product 171 was obtained as a clear oil (18.7 mg, 0.034 mmol, 74%). LCMS (ESI+) C 19 H 25 F4N8O6 + (M+H+ ) calculated value 537.45, actual value 537.29.

[0363] [ka]

[0364] Synthesis of Example 96.359 To a solution of 102 (56 mg, 0.17 mmol) in DCM (8 mL) was added amino-PEG 24 The alcohol (214 mg, 0.199 mmol) and EtN (80 μL, 0.53 mmol) were added. After stirring at room temperature for 20 h, the solvent was reduced in vacuo and the residue was purified by flash silica gel column chromatography (2→30% MeOH in DCM) to give the desired compound 359 as a yellow oil in 95% yield (210 mg, 0.168 mmol). LCMS (ESI+) C 59 H 111 NO 26 Na + (M+Na + ) Calculated value 1273.50 Measured value 1273.07.

[0365] Synthesis of Example 97.360 To a solution of 359 (170 mg, 0.136 mmol) and 4-nitrophenyl chloroformate (44 mg, 0.22 mmol) in DCM (7 mL) was added EtN (63 μL, 0.40 mmol). After stirring at room temperature for 41 h, the solvent was reduced and the residue was purified by flash silica gel column chromatography (0→10% MeOH in DCM) to give the desired compound 360 as a clear oil in 67% yield (129 mg, 0.091 mmol). LCMS (ESI+) C 66 H 114 N2O 30 Na + (M+Na + ) calculated value 1438.59 Measured value 1438.13.

[0366] Synthesis of Example 98.173 To a solution of 360 (16 mg, 0.011 mmol) in anhydrous DMF (800 μL) was added 167 (peptide H-LPETGG-OH, 6.5 mg, 0.011 mmol) and EtN (5 μL, 0.04 mmol). After stirring at room temperature for 95 h, the crude mixture was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN in HO (both containing 1% acetic acid)). Product 173 was obtained as a clear oil (12.6 mg, 0.0068 mmol, 62%). LCMS (ESI+) C 84 H 153 N8O 37 + (M / 2+NH4 + ) calculated value 942.55, actual value 924.26.

[0367] [ka]

[0368] Synthesis of Example 99.174 To a solution of 361 (methyltetrazine-PEG5-NHS ester, 6.1 mg, 0.011 mmol) in anhydrous DMF (230 μL) was added the peptide H-LPETGG-OH (6.5 mg, 0.011 mmol) and EtN (4 μL, 0.028 mmol). After stirring at room temperature for 22 h, the crude mixture was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN in HO (both containing 1% acetic acid)). Product 174 was obtained as a clear pink oil (9.9 mg, 0.01 mmol, 91%). LCMS (ESI+) C 44 H 70 N 11 O 16 + (M+NH4 + ) Calculated value 1009.09 Measured value 1009.61.

[0369] [ka]

[0370] Synthesis of Example 100.362 To a solution of 354 (31 mg, 0.093 mmol) in DCM (1 mL) was added 181 (56 mg, 0.10 mmol) and EtN (40 μL, 0.28 mmol). After stirring for 25 min at room temperature in the dark, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→15% MeOH in DCM) to give the desired compound 362 as a clear oil (55 mg, 0.072 mmol, 77%). LCMS (ESI+) C 31 H 51 F4N4O 13 + (M+H + ) Calculated value 763.75 Measured value 763.08.

[0371] Synthesis of Example 101.363 To a solution of 362 (55 mg, 0.072 mmol) in DCM (2 mL) was added 4-nitrophenyl chloroformate (13 mg, 0.064 mmol) and EtN (30 μL, 0.21 mmol). After stirring for 21 h at room temperature in the dark, the mixture was concentrated in vacuo and purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN (1% AcOH) in water (1% AcOH)). The product 363 was obtained as a yellow oil (13.3 mg, 0.014 mmol, 20%). LCMS (ESI+) C 38 H 54 F4N5O 17 + (M+H + ) calculated value 928.85 Measured value 928.57.

[0372] Example 102. Synthesis of 175 To a solution of 363 (13.3 mg, 0.014 mmol) in anhydrous DMF (300 μL) was added 167 (peptide H-LPETGG-OH, 8.2 mg, 0.014 mmol) and EtN (6 μL, 0.043 mmol). After 26 h in the dark, the crude mixture was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN in HO (both containing 1% acetic acid)). Product 175 was obtained as a clear oil (11.4 mg, 0.0084 mmol, 59%). LCMS (ESI+) C 56 H 89 F4N 10 O 24 + (M+H + ) calculated value 1362.35 Measured value 1362.81.

[0373] [ka]

[0374] Synthesis of Example 103.365 To a stirred solution of 151 (Fmoc-Gly-Gly-Gly-OH, 20 mg, 0.049 mmol) in anhydrous DMF (350 μL) was added DIPEA (25 μL, 0.15 mmol) and HATU (18 mg, 0.049 mmol). After 10 min, anhydrous solution of compound 364 (N-Boc-ethylenediamine, 7.8 mg, 0.049 mmol) was added. After stirring at room temperature for 45 min, the mixture was concentrated in vacuo and purified by silica gel flash column chromatography (0→30% MeOH in DCM) to give the desired compound 365 as a clear oil (12.4 mg, 0.022 mmol, 46%). LCMS (ESI+) C 28 H 36 N5O7 + (M+H + ) calculated value 554.61, measured value 554.46.

[0375] Synthesis of Example 104.366 To a stirred solution of 365 (12.4 mg, 0.022 mmol) in DCM (0.7 mL) was added 4.0 M HCl in dioxane (400 μL). After stirring at room temperature for 1 h, the mixture was concentrated to give 366 as a white solid (11 mg, 0.022 mmol, quantitative). LCMS (ESI+) C 23 H 28 N5O7 + (M+H + ) Calculated value 545.50 Measured value 454.33

[0376] Synthesis of Example 105.176 To a solution of 191 (8 mg, 0.0059 mmol) in anhydrous DMF (300 μL) was added EtN (2.5 μL, 0.017 mmol) and 366 (stock in anhydrous DMF, 110 μL, 3.0 mg, 0.0059 mmol). After stirring at room temperature for 18 h, diethylamine (2 μL) was added. After an additional 2 h, the mixture was purified by RP HPLC (column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN in HO (both containing 1% acetic acid)). The product 176 was obtained as a clear oil (1.3 mg, 0.0009 mmol, 15%). LCMS (ESI+) C 60 H 103 N 10 O 26 S2 + (M+H + ) Calculated value 1444.64 Measured value 1444.75.

[0377] Example 106. Anti-4-1BB PF31 Anti-4-1BB scFv was designed with a C-terminal sortase A recognition sequence followed by a His tag (amino acid sequence identified by SEQ ID NO: 4). Anti-4-1BB scFv was transiently expressed in HEK293 cells followed by IMAC purification by Absolute Antibody Ltd (Oxford, UK). Mass spectral analysis showed one major product (observed mass 28013 Da, predicted mass 28018 Da).

[0378] Example 107. Cloning of SYR-(G4S)3-IL15(PF18) into the pET32a expression vector SYR-(G4S)3-IL15(PF18) (amino acid sequence identified by SEQ ID NO: 5) was designed with an N-terminal (M)SYR sequence, with the methionine cleaved after expression to leave an N-terminal serine and a flexible (G4S)3 spacer between the SYR sequence and IL15. The codon-optimized DNA sequence was inserted between NdeI and XhoI in the pET32A expression vector, thereby removing the sequence encoding the thioredoxin fusion protein, and was obtained from Genscript, Piscataway, USA.

[0379] Example 108. E. coli expression and inclusion body isolation of SYR-(G4S)3-IL15(PF18) Expression of SYR-(G4S)3-IL15 (PF18) was initiated by transformation of the plasmid (pET32a-SYR-(G4S)3-IL15) into BL21 cells (Novagen). Transformed cells were plated on LB agar containing ampicillin and incubated overnight at 37°C. A single colony was picked and used to inoculate 50 mL of TB medium + ampicillin, followed by overnight incubation at 37°C. The overnight culture was then used to inoculate 1000 mL of TB medium + ampicillin. The culture was incubated at 37°C at 160 RPM and induced with 1 mM IPTG (1 mL of a 1 M stock solution) when the OD600 reached 1.5. After >16 hours of induction at 37°C at 160 RPM, the culture was pelleted by centrifugation (5000 x g for 5 minutes). The cell pellet from a 1000 mL culture was lysed in 60 mL of BugBuster™ containing 1500 units of Benzonase and incubated on a roller bank at room temperature for 30 minutes. After lysis, the insoluble fraction was separated from the soluble fraction by centrifugation (15,000 × g for 15 minutes). Half of the insoluble fraction was dissolved in 30 mL of BugBuster™ containing lysozyme (final concentration: 200 μg / mL) and incubated on a roller bank for 10 minutes. The solution was then diluted with six volumes of 1:10 diluted BugBuster™ and centrifuged for 15 minutes at 15,000 × g. The pellet was resuspended in 200 mL of 1:10 diluted BugBuster™ using a homogenizer and centrifuged for 10 minutes at 12,000 × g. This last step was repeated three times.

[0380] Example 109. Refolding of SYR-(G4S)3-IL15(PF18) from isolated inclusion bodies Purified inclusion bodies containing SYR-(G4S)3-IL15(PF18) were dissolved and denatured in 30 mL of 5 M guanidine containing 40 mM cysteamine and 20 mM Tris pH 8.0. The suspension was centrifuged at 16,000 x g for 5 minutes to pellet any remaining cell debris. The supernatant was diluted to 1 mg / mL in 5 M guanidine containing 40 mM cysteamine and 20 mM Tris pH 8.0 and incubated at room temperature on a roller bank for 2 hours. Add the 1 mg / mL solution dropwise (stirring required) to 10 volumes of refolding buffer (50 mM Tris, 10.53 mM NaCl, 0.44 mM KCl, 2.2 mM MgCl, 2.2 mM CaCl, 0.055% PEG-4000, 0.55 M L-arginine, 4 mM cysteamine, 4 mM cystamine, pH 8.0) in a 4°C cold room. Allow the solution to stand at 4°C for at least 24 hours. Dialyze the solution 1x overnight and 2x for 4 hours against 10 mM NaCl and 20 mM Tris pH 8.0 using Spectrum™ Spectra / Por™ 3 RC Dialysis Membrane Tubing 3500 Dalton MWCO. Refolded SYR-(G4S)3-IL15 (PF18) was loaded onto a Q-trap anion exchange column (GE Healthcare) equilibrated on an AKTA Purifier-10 (GE Healthcare). The column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 8.0). The retained protein was eluted with buffer B (20 mM Tris buffer, 1 M NaCl, pH 8.0) using a 30 mL gradient from buffer A to buffer B. Mass spectrometry analysis revealed a mass of 14,122 Da corresponding to PF18 (expected mass: 14,122 Da). Purified SYR-(G4S)3-IL15 (PF18) was buffer exchanged into PBS using a HiPrep™ 26 / 10 desalting column (Cytiva) on an AKTA Purifier-10 (GE Healthcare).

[0381] Example 110. Cloning of SYR-(G4S)3-IL15Ra-linker-IL15(PF26) into pET32a expression vector SYR-(G4S)3-IL15Ra-linker-IL15(PF26) (amino acid sequence identified by SEQ ID NO: 6) was designed with an N-terminal (M)SYR sequence, with the methionine cleaved after expression to leave an N-terminal serine and a flexible (G4S)3 spacer between the SYR sequence and IL15Ra-linker-IL15. The codon-optimized DNA sequence was inserted between NdeI and XhoI in the pET32A expression vector, thereby removing the sequence encoding the thioredoxin fusion protein, and was obtained from Genscript, Piscataway, USA.

[0382] Example 111. E. coli expression and inclusion body isolation of SYR-(G4S)3-IL15Ra-linker-IL15(PF26) Expression of SYR-(G4S)3-IL15Ra-linker-IL15 (PF26) was initiated by transformation of the plasmid (pET32a-SYR-(G4S)3-IL15Ra-linker-IL15) into BL21 cells (Novagen). The next step was to inoculate a 1000 mL culture (TB medium + ampicillin) with BL21 cells. When the OD600 reached 1.5, the culture was induced with 1 mM IPTG (1 mL of a 1 M stock solution). After induction at 160 RPM for more than 16 hours at 37°C, the culture was pelleted by centrifugation (5000 x g for 5 minutes). The cell pellet from the 1000 mL culture was dissolved in 60 mL of Bugbuster™ containing 1500 units of Benzonase and incubated on a roller bank at room temperature for 30 minutes. After lysis, the insoluble fraction was separated from the soluble fraction by centrifugation (15 min, 15,000 x g). Half of the insoluble fraction was dissolved in 30 mL of Bugbuster™ containing lysozyme (final concentration: 200 μg / mL) and incubated on a roller bank for 10 min. The solution was then diluted with 6 volumes of 1:10 diluted Bugbuster™ and centrifuged for 15 min at 15,000 x g. The pellet was resuspended in 200 mL of 1:10 diluted Bugbuster™ using a homogenizer and centrifuged for 10 min at 12,000 x g. This last step was repeated three times.

[0383] Example 112. Refolding of SYR-(G4S)3-IL15Ra-linker-IL15(PF26) from isolated inclusion bodies Purified inclusion bodies containing SYR-(G4S)3-IL15Ra-linker-IL15(PF26) were dissolved and denatured in 30 mL of 5 M guanidine containing 40 mM cysteamine and 20 mM Tris pH 8.0. The suspension was centrifuged at 16,000 x g for 5 minutes to pellet remaining cell debris. The supernatant was diluted to 1 mg / mL in 5 M guanidine containing 40 mM cysteamine and 20 mM Tris pH 8.0 and incubated at room temperature on a roller bank for 2 hours. Add the 1 mg / mL solution dropwise (stirring required) to 10 volumes of refolding buffer (50 mM Tris, 10.53 mM NaCl, 0.44 mM KCl, 2.2 mM MgCl, 2.2 mM CaCl, 0.055% PEG-4000, 0.55 M L-arginine, 4 mM cysteamine, 4 mM cystamine, pH 8.0) in a 4°C cold room. Allow the solution to stand at 4°C for at least 24 hours. Dialyze the solution against 10 mM NaCl and 20 mM Tris pH 8.0 1x overnight and 2x for 4 hours using Spectrum™ Spectra / Pore™ 3 RC Dialysis Membrane Tubing 3500 Dalton MWCO. The refolded SYR-(G4S)3-IL15Ra-linker-IL15 (PF26) was loaded onto a Q-trap anion exchange column (GE Healthcare) equilibrated on an AKTA Purifier-10 (GE Healthcare). The column was first washed with buffer A (20 mM Tris, 10 mM NaCl, pH 8.0). The retained protein was eluted with buffer B (20 mM Tris buffer, 1 M NaCl, pH 8.0) using a 30 mL gradient from buffer A to buffer B. Mass spectrometry analysis revealed a mass of 24,146 Da (expected mass: 24,146 Da) corresponding to PF26. The purified SYR-(G4S)3-IL15Ra-linker-IL15 (PF26) was buffer exchanged into PBS using a HyPrep™ 26 / 10 desalting column (Cytiva) on an AKTA Purifier-10 (GE Healthcare).

[0384] Example 113. Humanized OKT3 200 Humanized OKT3 (hOKT3) with a C-terminal sortase A recognition sequence (C-terminal tag identified by SEQ ID NO: 1) was obtained from Absolute Antibody Ltd (Oxford, UK). Mass spectral analysis showed one major product (observed mass 28836 Da).

[0385] Example 114. C-terminal sortagging of compound GGG-PEG2-BCN (157) to hOKT3 200 using sortase A to obtain hOKT3-PEG2-BCN 201 A bioconjugate according to the present invention was prepared by C-terminal sortagging using Sortase A (identified by SEQ ID NO: 2). To a solution of hOKT3 200 (500 μL, 500 μg, 35 μM in PBS pH 7.4) was added Sortase A (58 μL, 384 μg, 302 μM in TBS pH 7.5 + 10% glycerol), GGG-PEG2-BCN (157, 28 μL, 50 mM in DMSO), CaCl2 (69 μL, 100 mM in MQ), and TBS pH 7.5 (39 μL). The reaction was incubated overnight at 37°C and subsequently purified on a His-trap excel 1 mL column (GE Healthcare) on an AKTA Explorer-100 (GE Healthcare). The column was equilibrated with buffer A (20 mM Tris, 200 mM NaCl, 20 mM imidazole, pH 7.5), and the sample was loaded at 1 mL / min. The flow-through was collected, and mass spectrometry analysis showed one major product (observed mass 27,829 Da) corresponding to 201. The sample was dialyzed against PBS pH 7.4 and concentrated by spin filtration (Amicon Ultra-0.5, Ultracel-10 Membrane, Millipore) to give hOKT3-PEG2-BCN 201 (60 μL, 169 μg, 101 μM in PBS pH 7.4).

[0386] Example 115. C-terminal sortagging of compound GGG-PEG2-BCN (157) to hOKT3 200 using a sortase A pentamutant to obtain hOKT3-PEG2-BCN 201 A bioconjugate according to the present invention was prepared by C-terminal sortagging using a sortase A penta mutant (BPS Bioscience, catalog no. 71046). To a solution of hOKT3 200 (14.3 μL, 14 μg, 35 μM in PBS pH 7.4) was added sortase A penta mutant (0.5 μL, 1 μg, 92 μM in 40 mM Tris pH 8.0, 110 mM NaCl, 2.2 mM KCl, 400 mM imidazole, and 20% glycerol), GGG-PEG2-BCN (157, 2 μL, 20 mM in DMSO:MQ = 2:3), CaCl2 (2 μL, 100 mM in MQ), and TBS pH 7.5 (1.2 μL). The reaction was incubated overnight at 37°C. Mass spectral analysis showed one major product (observed mass 27829 Da) corresponding to hOKT3-PEG2-BCN 201.

[0387] Example 116. hOKT3-PEG 11 - Compound GGG-PEG using Sortase A to obtain BCN 202 11 C-terminal sortagging of -BCN(161) to hOKT3 200 A bioconjugate according to the invention was prepared by C-terminal sortagging using sortase A (identified by SEQ ID NO: 2). A solution of hOKT3 200 (14.3 μL, 14 μg, 35 μM in PBS pH 7.4) was diluted with sortase A (0.9 μL, 12 μg, 582 μM in TBS pH 7.5 + 10% glycerol), GGG-PEG 11 -BCN (161, 2 μL, 20 mM in MQ), CaCl (2 μL, 100 mM in MQ), and TBS pH 7.5 (0.9 μL) were added. The reaction was incubated overnight at 37 °C. Mass spectrometry analysis revealed one major product (observed mass 21951 Da, approximately 85%) corresponding to sortase A, hOKT3-PEG 11- A minor product corresponding to BCN 202 (observed mass 28227 Da, approximately 5%), and two other minor products (observed masses 28051 Da and 28325 Da, each approximately 5%).

[0388] Example 117. hOKT3-PEG 11 Compound GGG-PEG using sortase A penta mutant to obtain -BCN 202 11 C-terminal sortagging of -BCN(161) to hOKT3 200 A bioconjugate according to the invention was prepared by C-terminal sortagging using sortase A penta mutant (BPS Bioscience, Cat. No. 71046). A solution of hOKT3 200 (14.3 μL, 14 μg, 35 μM in PBS pH 7.4) was mixed with sortase A penta mutant (0.5 μL, 1 μg, 92 μM in 40 mM Tris pH 8.0, 110 mM NaCl, 2.2 mM KCl, 400 mM imidazole and 20% glycerol), GGG-PEG 11 -BCN (161, 2 μL, 20 mM in MQ), CaCl (2 μL, 100 mM in MQ), and TBS pH 7.5 (1.2 μL) were added. The reaction was incubated overnight at 37°C. Mass spectrometry analysis confirmed the activity of hOKT3-PEG. 11 - One major product (observed mass 28225 Da, approximately 60%) corresponding to BCN 202, and one minor product (observed mass 28326 Da, approximately 40%).

[0389] Example 118. hOKT3-PEG 23 - Compound GGG-PEG using Sortase A to obtain BCN 203 23 C-terminal sortagging of -BCN(163) to hOKT3 200 A bioconjugate according to the invention was prepared by C-terminal sortagging using sortase A (identified by SEQ ID NO: 2). A solution of hOKT3 200 (14.3 μL, 14 μg, 35 μM in PBS pH 7.4) was diluted with sortase A (0.9 μL, 12 μg, 582 μM in TBS pH 7.5 + 10% glycerol), GGG-PEG23 -BCN (163, 2 μL, 20 mM in MQ), CaCl (2 μL, 100 mM in MQ), and TBS pH 7.5 (0.9 μL) were added. The reaction was incubated overnight at 37° C. Mass spectrometry analysis revealed one major product (observed mass 21951 Da, approximately 70%) corresponding to sortase A, and hOKT3-PEG. 23 - One minor product corresponding to BCN 203 (observed mass 28755 Da, approximately 30%) was shown.

[0390] Example 119. hOKT3-PEG 23 Compound GGG-PEG using sortase A penta mutant to obtain -BCN 203 23 C-terminal sortagging of -BCN(163) to hOKT3 200 A bioconjugate according to the invention was prepared by C-terminal sortagging using sortase A penta mutant (BPS Bioscience, Cat. No. 71046). A solution of hOKT3 200 (14.3 μL, 14 μg, 35 μM in PBS pH 7.4) was mixed with sortase A penta mutant (0.5 μL, 1 μg, 92 μM in 40 mM Tris pH 8.0, 110 mM NaCl, 2.2 mM KCl, 400 mM imidazole and 20% glycerol), GGG-PEG 23 -BCN (163, 2 μL, 20 mM in MQ), CaCl (2 μL, 100 mM in MQ), and TBS pH 7.5 (1.2 μL) were added. The reaction was incubated overnight at 37° C. Mass spectrometry analysis confirmed the activity of hOKT3-PEG. 23 - One major product (observed mass 28754 Da) corresponding to BCN 203 was shown.

[0391] Example 120. C-terminal sortagging of compound GGG-PEG4-tetrazine (154) to hOKT3 200 using sortase A to give hOKT3-PEG4-tetrazine 204 A bioconjugate according to the present invention was prepared by C-terminal sortagging using Sortase A (identified by SEQ ID NO: 2). To a solution of hOKT3 200 (500 μL, 500 μg, 35 μM in PBS pH 7.4) was added Sortase A (58 μL, 384 μg, 302 μM in TBS pH 7.5 + 10% glycerol), GGG-PEG4-tetrazine (154, 35 μL, 40 mM in MQ), CaCl2 (69 μL, 100 mM in MQ), and TBS pH 7.5 (32 μL). The reaction was incubated overnight at 37°C and subsequently purified on a His-trap excel 1 mL column (GE Healthcare) on an AKTA Explorer-100 (GE Healthcare). The column was equilibrated with buffer A (20 mM Tris, 200 mM NaCl, 20 mM imidazole, pH 7.5), and the sample was loaded at 1 mL / min. The flow-through was collected, and mass spectrometry analysis showed one major product (observed mass 27,868 Da) corresponding to 104. The sample was dialyzed against PBS pH 7.4 and concentrated by spin filtration (Amicon Ultra-0.5, Ultracel-10 Membrane, Millipore) to give hOKT3-PEG4-tetrazine 204 (70 μL, 277 μg, 143 μM in PBS pH 7.4).

[0392] Example 121. C-terminal sortagging of compound GGG-PEG4-tetrazine (154) to hOKT3 200 using a sortase A penta mutant to give hOKT3-PEG4-tetrazine 204 A bioconjugate according to the present invention was prepared by C-terminal sortagging using a sortase A penta mutant (BPS Bioscience, catalog no. 71046). To a solution of hOKT3 200 (14.3 μL, 14 μg, 35 μM in PBS pH 7.4) was added sortase A penta mutant (0.5 μL, 1 μg, 92 μM in 40 mM Tris pH 8.0, 110 mM NaCl, 2.2 mM KCl, 400 mM imidazole, and 20% glycerol), GGG-PEG4-tetrazine (154, 2 μL, 20 mM in MQ), CaCl2 (2 μL, 100 mM in MQ), and TBS pH 7.5 (1.2 μL). The reaction was incubated overnight at 37°C. Mass spectral analysis showed one major product (observed mass 27868 Da) corresponding to hOKT3-PEG4-tetrazine 204.

[0393] Example 122. hOKT3-PEG 11 -GGG-PEG using Sortase A to obtain tetrazine PF01 11 -C-terminal sortagging of tetrazine (169) to hOKT3 200 A bioconjugate according to the invention was prepared by C-terminal sortagging using Sortase A (identified by SEQ ID NO: 2). A solution of hOKT3 200 (1908 μL, 5 mg, 91 μM in PBS pH 7.4) was diluted with Sortase A (81 μL, 948 μg, 533 μM in TBS pH 7.5 + 10% glycerol), GGG-PEG 11 -tetrazine (169, 347 μL, 20 mM in MQ), CaCl (347 μL, 100 mM in MQ), and TBS pH 7.5 (789 μL) were added. The reaction was incubated overnight at 37° C. Mass spectrometry analysis confirmed the hOKT3-PEG 11The reaction mixture showed one major product (observed mass 28,258 Da) corresponding to -tetrazine PF01. The reaction mixture was purified on a His-trap excel 1 mL column (GE Healthcare) on an AKTA Explorer-100 (GE Healthcare). The column was equilibrated with Buffer A (20 mM Tris, 200 mM NaCl, 20 mM imidazole, pH 7.5), and the sample was loaded at 1 mL / min. The flow-through was collected and buffer-exchanged into PBS pH 6.5 using a HiPrep 26 / 10 desalting column (GE Healthcare). Further dialysis against PBS pH 6.5 at 4 °C for 3 days removed residual 169.

[0394] Example 123. hOKT3-PEG 23 -GGG-PEG using Sortase A to obtain tetrazine PF02 23 C-terminal sorting of -tetrazine(170) to hOKT3 200 A bioconjugate according to the invention was prepared by C-terminal sortagging using Sortase A (identified by SEQ ID NO: 2). A solution of hOKT3 200 (1908 μL, 5 mg, 91 μM in PBS pH 7.4) was diluted with Sortase A (81 μL, 948 μg, 533 μM in TBS pH 7.5 + 10% glycerol), GGG-PEG 23 -tetrazine (170, 347 μL, 20 mM in MQ), CaCl (347 μL, 100 mM in MQ), and TBS pH 7.5 (789 μL) were added. The reaction was incubated overnight at 37° C. Mass spectrometry analysis confirmed the hOKT3-PEG 23The reaction mixture showed one major product (observed mass 28,787 Da) corresponding to -tetrazine PF02. The reaction mixture was purified on a His-trap excel 1 mL column (GE Healthcare) on an AKTA Explorer-100 (GE Healthcare). The column was equilibrated with Buffer A (20 mM Tris, 200 mM NaCl, 20 mM imidazole, pH 7.5), and the sample was loaded at 1 mL / min. The flow-through was dialyzed against PBS pH 6.5 and subsequently purified on a Superdex75 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 6.5 as the mobile phase.

[0395] Example 124. C-terminal sortagging of GGG-PEG2-aryl azide (171) to hOKT3 200 using Sortase A to give hOKT3-PEG2-aryl azide PF03 A bioconjugate according to the present invention was prepared by C-terminal sortagging using Sortase A (identified by SEQ ID NO: 2). To a solution of hOKT3 200 (2092 μL, 5 mg, 83 μM in PBS pH 7.4) was added Sortase A (95 μL, 950 μg, 456 μM in TBS pH 7.5 + 10% glycerol), GGG-PEG2-aryl azide (171, 347 μL, 20 mM in MQ), CaCl2 (347 μL, 100 mM in MQ), and TBS pH 7.5 (591 μL). The reaction was incubated overnight at 37°C. Mass spectrometry analysis showed one major product (observed mass 27,865 Da) corresponding to hOKT3-PEG2-aryl azide PF03. The reaction mixture was purified on a His-trap excel 1 mL column (GE Healthcare) mounted on an AKTA Purifier-10 (GE Healthcare). The column was equilibrated with Buffer A (20 mM Tris, 200 mM NaCl, 20 mM imidazole, pH 7.5), and the sample was loaded at 1 mL / min. The flow-through was purified on a Superdex75 10 / 300 GL column (GE Healthcare) mounted on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase.

[0396] Example 125. Anti-4-1BB-PEG 11 -GGG-PEG using Sortase A to obtain tetrazine PF08 11 -C-terminal sortagging of tetrazine (169) to anti-4-1BB PF31 A solution containing protein PF31 (1151 μL, 93 μM in TBS pH 7.5) was added to TBS pH 7.5 (512 μL), CaCl (214 μL, 100 mM) and GGG-PEG 114-IBB-tetrazine (169, 220 μL, 20 mM in MQ) and sortase A (50 μL, 533 μM in TBS pH 7.5) were added. The reaction was incubated overnight at 37 °C and subsequently purified on a His-trap excel 1 mL column (GE Healthcare) on an AKTA Explorer-100 (GE Healthcare). The column was equilibrated with Buffer A (20 mM Tris, 200 mM NaCl, 20 mM imidazole, pH 7.5), and the sample was loaded at 1 mL / min. The flow-through was collected, and mass spectrometry analysis showed one major product (observed mass 27,989 Da) corresponding to 4-IBB-tetrazine PF08.

[0397] Example 126. C-terminal sortagging of compound GGG-PEG2-aryl azide (171) to anti-4-1BB-PF31 using sortase A to obtain anti-4-1BB-PF09 A bioconjugate according to the present invention was prepared by C-terminal sortagging using Sortase A (identified by SEQ ID NO: 2). To a solution of anti-4-1BB-PF31 (665 μL, 2 mg, 107 μM in PBS pH 7.4), Sortase A (100 μL, 1 mg, 357 μM in TBS pH 7.5 + 10% glycerol), GGG-PEG2-aryl azide (171, 140 μL, 20 mM in MQ), CaCl2 (140 μL, 100 mM in MQ), and TBS pH 7.5 (355 μL) were added. The reaction was incubated overnight at 37°C and subsequently purified on a His-trap excel 1 mL column (GE Healthcare) on an AKTA Explorer-100 (GE Healthcare). The column was equilibrated with Buffer A (20 mM Tris, 200 mM NaCl, 20 mM imidazole, pH 7.5) and the sample was loaded at 1 mL / min. The flow-through was collected and mass spectral analysis showed one major product (observed mass 27592 Da) corresponding to anti-4-1BB-azide PF09.

[0398] Example 127. Aryl azide-PEG 11Aryl azide-PEG using sortase A to obtain -GGG-IL15Rα-IL15(PF13) 11 N-terminal sortagging of -LPETGG(175) to GGG-IL15Rα-IL15(208) To a solution containing protein 208 (2000 μL, 140 μM in TBS pH 7.5), TBS pH 7.5 (2686 μL), CaCl (559 μL, 100 mM), and 175 (83 μL, 50 mM in DMSO), and sortase A (260 μL, 537 μM in TBS pH 7.5) were added and incubated at 37 °C for 3 h (protected from light). After incubation, sortase A was removed from the solution using Ni-NTA beads (500 μL beads = 1 mL slurry). The solution was incubated with Ni-NTA beads on a roller bank overnight at 4 °C, after which the solution was centrifuged (5 min, 7,000 × g). The supernatant containing the product PF13 was collected by separating the supernatant from the pellet. The reaction mixture was loaded onto a Superdex75 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase and a flow rate of 0.5 mL / min. Mass spectrometry analysis showed a mass of 24,193 Da (expected mass: 24,193 Da), corresponding to PF13.

[0399] Example 128. BCN-PEG 12 BCN-PEG to obtain -SYR-(G4S)3-IL15Rα-IL15(PF14) 12 N-terminal oxime ligation of -aminooxy (XL13) to SYR-(G4S)3-IL15Rα-IL15 (PF26) Prior to labeling of PF26, the N-terminal serine was oxidized using sodium periodate. To a solution containing the protein PF26 (700 μL, 70 μM in PBS pH 7.4), PBS pH 7.4 (286 μL), NaIO (0.98 μL, 100 mM in MQ), and L-methionine (5 μL, 100 mM in MQ) were added and incubated at 4 °C for 5 min. Mass spectrometry analysis revealed masses of 24,114 Da (aldehyde) and 24,130 Da (hydrate), corresponding to the predicted masses of 24,114 Da (aldehyde) and 24,132 Da (hydrate). Excess NaIO and L-methionine were removed using a PD-10 desalting column. The oxidized PF26 was concentrated to a concentration of 50 μM using an Amicon spin filter 0.5, MWCO 10 kDa (Merck-Millipore). To a solution containing oxidized PF26 (416 μL, 50 μM in PBS pH 7.4), XL13 (41.6 μL, 50 mM in DMSO) was added. After overnight incubation at 37 °C, the reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and eluted with PBS. Mass spectrometry analysis indicated a mass of 25,024 Da (expected mass: 25,042 Da), corresponding to PF14.

[0400] Example 129. N-terminal BCN functionalization of IL15Rα-IL15 PF26 to obtain BCN-IL15Rα-IL15 PF15 To IL15Rα-IL15 PF26 (2.9 mg, 50 μM in PBS) were added 2 equivalents of NaIO4 (4.8 μL of 50 mM stock in PBS) and 10 equivalents of L-methionine (12.5 μL of 100 mM stock in PBS). The reaction was incubated at 4 °C for 5 minutes. Mass spectrometry analysis showed oxidation of serine to the corresponding aldehyde and hydrate (observed masses of 24,114 Da and 24,132 Da). The reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and eluted with PBS. To the eluent (2.6 mg, 50 μM in PBS) were added 160 equivalents of N-methylhydroxylamine.HCl (340 μL of 50 mM stock in PBS) and 160 equivalents of p-anisidine (340 μL of 50 mM stock in PBS). The reaction mixture was incubated at 25°C for 3 hours. Mass spectrometry showed a single peak corresponding to N-methyl-imine-oxide-IL15 (observed mass 24143 Da). The reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and eluted with PBS. The eluent (2.47 mg, 50 μM in PBS) contained 25 equivalents of bis-BCN-PEG. 11 105 (51 μL, 50 mM in DMSO) and 150 μL of DMF were added. The reaction was incubated overnight at room temperature. The reaction was purified using a Superdex75 10 / 300 column (Cytiva). Mass spectral analysis showed one major peak corresponding to BCN-IL15Rα-IL15 PF15 (observed mass 25,041 Da).

[0401] Example 130. N-terminal diazotransfer reaction of IL15 PF18 to obtain azido-IL15 PF19 To IL15 PF18 (5 mg, 50 μM in 0.1 M TEA buffer pH 8.0) was added imidazole-1-sulfonyl azide hydrochloride (708 μL, 50 mM in 50 mM NaOH) and incubated overnight at 37° C. The reaction was purified using a HyPrep™ 26 / 10 desalting column (Cytiva). Mass spectral analysis showed one major peak (observed mass 14,147 Da) corresponding to azido-IL15 PF19.

[0402] Example 131. Tetrazine-PEG 12 Tetrazine-PEG using 2PCA to obtain -SYR-(G4S)3-IL15(PF21) 12 N-terminal incorporation of -2PCA(XL10) into SYR-(G4S)3-IL15(PF18) To SYR-(G4S)3-IL15(PF18) (1052 μL, 50 μM in PBS) was added 20 equivalents of tetrazine-PEG12-2PCA (XL10) (112 μL of 50 mM stock in DMSO) and 4359 μL of PBS. The reaction was incubated overnight at 37°C. The sample was concentrated to less than 1 mL using spin filtration (Amicon Ultra-0.5, Ultracel-10 Membrane, Millipore) and loaded onto a Superdex75 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase and a flow rate of 0.5 mL / min. Mass spectral analysis showed a mass of 24121 Da, corresponding to the starting material SYR-(G4S)3-IL15(PF18) (expected mass: 14121 Da) and a mass of 15093 Da, corresponding to the product PF21 (expected mass: 15094 Da).

[0403] Example 132. Conjugation of tri-BCN(150) to hOKT3-PEG2-arylazide PF03 to obtain bis-BCN-hOKT3 PF22 To a solution of hOKT3-PEG2-arylazide PF03 (87 μL, 1 mg, 411 μM in PBS pH 7.4) was added PBS pH 7.4 (559 μL), DMF (49 μL), and compound 150 (22 μL, 40 mM solution in DMF, 25 equivalents). The reaction was incubated overnight at room temperature. Mass spectral analysis showed one major product (observed mass 29,171 Da) corresponding to bis-BCN-hOKT3 PF22. The reaction was purified on a Superdex75 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase.

[0404] Example 133. C-terminal sortagging of GGG-bis-BCN 176 to hOKT3 200 using sortase A to obtain bis-BCN-hOKT3 PF23 A bioconjugate according to the present invention was prepared by C-terminal sortagging using sortase A (identified by SEQ ID NO: 2). To a solution of hOKT3 200 (272 μL, 0.7 mg, 83 μM in PBS pH 7.4) was added sortase A (25 μL, 250 μg, 456 μM in TBS pH 7.5 + 10% glycerol), GGG-bis-BCN (176, 45 μL, 20 mM in DMSO), CaCl (45 μL, 100 mM in MQ), and TBS pH 7.5 (64 μL). The reaction was incubated overnight at 37°C. Mass spectrometry analysis showed one major product (observed mass 28772 Da) corresponding to bis-BCN-hOKT3 PF23. The reaction was purified on a Superdex75 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase.

[0405] Example 134. N-Terminal Incorporation of Tri-BCN (150) into N-SYR-(G4S)-IL15(PF19) Using Strain-Promoted Alkyne-Azide Cycloaddition to Obtain Bis-BCN-SYR-(G4S)-IL15(PF29) To N3-IL15 PF19 (706 μL, 50 μM in PBS) was added 4 equivalents of tri-BCN(150) (3.5 μL of 40 mM stock in DMF) and 67 μL of DMF. The reaction was incubated overnight at room temperature. Mass spectrometry confirmed the formation of bis-BCN-SYR-(GS)3-IL15 PF29 (observed mass 15453 Da, expected mass 15453 Da). The reaction mixture was purified using a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and eluted with PBS. Further washing was performed six times with 400 μL of PBS using spin filtration (Amicon Ultra-0.5, Ultracel-10 Membrane, Millipore) to remove remaining tri-BCN(150).

[0406] Example 135. Enzymatic deglycosylation of trastuzumab with PNGase F Trastuzumab (Herzuma) (20 mg, 12.5 mg / mL in PBS pH 7.4) was incubated with PNGase F (16 μL, 8000 units) at 37°C. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 23787 Da) corresponding to the predicted product.

[0407] Example 136. Enzymatic deglycosylation of rituximab with PNGase F Rituximab (6 mg, 10 mg / mL in PBS pH 7.4) was incubated with PNGase F (6 μL, 3000 units) at 37°C. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 23754 Da) corresponding to the predicted product.

[0408] Example 137. MTGase-catalyzed incorporation of azido-PEG3-amine into deglycosylated trastuzumab to yield bis-azido-trastuzumab trast-v3 To a solution of deglycosylated trastuzumab (806 μL, 10 mg, 12.4 mg / mL in PBS pH 7.4), PBS pH 7.4 (3544 μL), azido-PEG3-amine (commercially available from BroadPharm, 500 μL, 10 mM solution in MQ, 75 equivalents relative to IgG), and recombinant microbial transglutaminase (commercially available from Zedira, 150 μL, 15 U, 0.1 U / μL) were added. The reaction was incubated overnight at 37°C. Mass spectral analysis of an IdeS-digested sample showed one major product (observed mass 23,988 Da) corresponding to bis-azido-trastuzumab trast-v3. The reaction was purified using a protA column (5 mL, MabSelect Sure, GE Healthcare) on an AKTA Explorer-100 (GE Healthcare) and subsequently dialyzed against PBS pH 7.4.

[0409] Example 138. MTGase-catalyzed incorporation of azido-PEG3-amine into deglycosylated rituximab to yield bis-azido-rituximab rit-v3 To a solution of deglycosylated rituximab (90 μL, 1.8 mg, 20.2 mg / mL in PBS pH 7.4), PBS pH 7.4 (693 μL), azido-PEG3-amine (commercially available from BroadPharm, 90 μL, 10 mM solution in MQ, 75 equivalents relative to IgG), and recombinant microbial transglutaminase (commercially available from Zedira, 27 μL, 2.7 U, 0.1 U / μL) were added. The reaction was incubated overnight at 37°C. Mass spectrometry analysis of the IdeS digested sample showed one major product (observed mass 23,956 Da) corresponding to bis-azido-rituximab rit-v3. The reaction was buffer exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL, MWCO 10 kDa, Merck Millipore).

[0410] Example 139. Conjugation of 201 with trastuzumab (6-N3-GalNAc) 2205 to obtain conjugate 206 A bioconjugate according to the present invention was prepared by conjugation of BCN-modified hOKT3 201 to azide-modified trastuzumab 205. hOKT3-PEG2-BCN 201 (9.9 μL, 28 μg, 101 μM in PBS pH 7.4) was added to a solution of trastuzumab-(6-N3-GalNAc)2 prepared according to WO2016170186 (205, 2 μL, 75 μg, 250 μM in PBS pH 7.4). The reaction was incubated overnight at room temperature. Mass spectral analysis of a Fabricator™ digestion sample showed two major products (observed masses of 24,368 Da and 52,196 Da, approximately 50% each) corresponding to the azide-modified Fc / 2 fragment and conjugate 206, respectively.

[0411] Example 140. Cloning of His6-SSGENLYFQ-GGG-IL15Rα-IL15 into the pET32a expression vector The IL15Rα-IL15 fusion protein 207 was designed with an N-terminal His tag (HHHHHH), a TEV protease recognition sequence (SSGENLYFQ), and an N-terminal sortase A recognition sequence (GGG). The pET32A vector containing the DNA sequence encoding His6-SSGENLYFQ-GGG-IL15Rα-IL15 (SEQ ID NO: 3) between base pairs 158 and 692, thereby removing the thioredoxin coding sequence, was obtained from Genscript.

[0412] Example 141. Escherichia coli expression and inclusion body isolation of His6-SSGENLYFQ-GGG-IL15Rα-IL15(207) Expression of His6-SSGENLYFQ-GGG-IL15Rα-IL15 207 was initiated by transformation of the plasmid (pET32a-IL15Rα-IL15) into BL21 cells (Novagen). The next step was to inoculate a 500 mL culture (LB medium + ampicillin) with BL21 cells. When the OD600 reached 0.7, the culture was induced with 1 mM IPTG (500 μL of a 1 M stock solution). After 4 hours of induction at 37°C, the culture was pelleted by centrifugation. The cell pellet from the 500 mL culture was dissolved in 25 mL of BugBuster™ containing 625 units of benzonase and incubated for 20 minutes at room temperature on a roller bank. After lysis, the insoluble fraction was separated from the soluble fraction by centrifugation (20 minutes, 12,000 × g, 4°C). The insoluble fraction was dissolved in 25 mL of Bugbuster™ containing lysozyme (final concentration: 200 μg / mL) and incubated on a roller bank for 5 minutes. The solution was then diluted with 6 volumes of 1:10 diluted Bugbuster™ and centrifuged at 9000 × g for 15 minutes at 4 °C. The pellet was resuspended in 250 mL of 1:10 diluted Bugbuster™ using a homogenizer and centrifuged at 9000 × g for 15 minutes at 4 °C. The last step was repeated three times.

[0413] Example 142. Refolding of His6-SSGENLYFQ-GGG-IL15Rα-IL15 207 from isolated inclusion bodies Purified inclusion bodies containing His6-SSGENLYFQ-GGG-IL15Rα-IL15 207 were sulfonated overnight at 4°C in 25 mL of denaturation buffer (5 M guanidine, 0.3 M sodium sulfite) and 2.5 mL of 50 mM disodium 2-nitro-5-sulfobenzoate. The solution was diluted with 10 volumes of cold Milli-Q and centrifuged (8,000 × g for 10 min). The pellet was dissolved in 125 mL of cold Milli-Q using a homogenizer and centrifuged (80,000 × g for 10 min). The last step was repeated three times. Purified His6-SSGENLYFQ-GGG-IL15Rα-IL15 207 was denatured in 5 M guanidine and diluted to a protein concentration of 1 mg / mL. Using a 0.8 mm diameter syringe, the denatured protein was added dropwise to 10 volumes of refolding buffer (50 mM Tris, 10.53 mM NaCl, 0.44 mM KCl, 2.2 mM MgCl, 2.2 mM CaCl, 0.055% PEG-4000, 0.55 M L-arginine, 8 mM cysteamine, 4 mM cystamine, pH 8.0) on ice and incubated at 4 °C for 48 h (no stirring required). The refolded His6-SSGENLYFQ-GGG-IL15Rα-IL15 207 was loaded onto a 20 mL HisTrap excel column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). The column was first washed with buffer A (5 mM Tris buffer, 20 mM imidazole, 500 mM NaCl, pH 7.5). The retained protein was eluted with buffer B (20 mM Tris buffer, 500 mM imidazole, 500 mM NaCl, pH 7.5) in a 25 mL gradient from buffer A to buffer B. Fractions were analyzed by SDS-PAGE on polyacrylamide gels (16%). Fractions containing the purified target protein were combined and buffer exchanged against TBS (20 mM Tris pH 7.5 and 150 mM NaCl) by dialysis overnight at 4°C. The purified protein was concentrated to at least 2 mg / mL using an Amicon Ultra-0.5, MWCO 3 kDa (Merck-Millipore).Mass spectral analysis indicated a weight of 25044 Da (expected: 25044 Da). The product was stored at -80°C before further use.

[0414] Example 143. TEV cleavage of His6-SSGENLYFQ-GGG-IL15Rα-IL15 207 to obtain GGG-IL15Rα-IL15 208 To a solution of His6-SSGENLYFQ-GGG-IL15Rα-IL15 (207, 330 μL, 2.3 mg / mL in TBS pH 7.5), TEV protease (50.5 μL, 10 units / μL in 50 mM Tris-HCl, 250 mM NaCl, 1 mM TCEP, 1 mM EDTA, 50% glycerol, pH 7.5, New England Biolabs) was added. The reaction was incubated at 30°C for 1 hour. After TEV cleavage, the solution was purified using size-exclusion chromatography. The reaction mixture was loaded onto a Superdex75 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using TBS pH 7.5 as the mobile phase and a flow rate of 0.5 mL / min. GGG-IL15Rα-IL15 208 eluted with a retention time of 12 mL. The purified protein was concentrated to at least 2 mg / mL using an Amicon Ultra-0.5, MWCO 3 kDa (Merck Millipore). The product was analyzed by mass spectrometry (observed mass: 22965 Da, expected mass: 22964 Da) and corresponded to GGG-IL15Rα-IL15 208. The product was stored at -80°C before further use.

[0415] Example 144. BCN-PEG 12 -BCN-PEG using sortase A to obtain IL15Rα-IL15(209) 12 Incorporation of -LPETGG(168) into GGG-IL15Rα-IL15 208 A solution of GGG-IL15Rα-IL15 (208, 219 μL, 91.4 μM in TBS pH 7.5) was diluted with TBS pH 7.5 (321 μL), CaCl2 (40.0 μL, 100 mM) and BCN-PEG12 -LPETGG (168, 120 μL, 5 mM in DMSO) was added and incubated at 37°C for 1 hour. After incorporation of 168 was complete, sortase A was removed from the solution using Ni-NTA beads in a volume equal to the reaction volume (800 μL). The solution was incubated on a rotating wheel / or table shaker for 1 hour, after which the solution was centrifuged (2 minutes, 13,000 rpm) and the supernatant discarded. BCN-PEG was purified by incubating the beads with 800 μL of wash buffer (40 mM imidazole, 20 mM Tris, 0.5 M NaCl) for 5 minutes on a table shaker at 800 rpm. 12 The IL15Rα-IL15(209) was recovered from the beads. The beads were centrifuged (2 min, 13,000 × rpm), and the supernatant containing 209 was separated and buffer exchanged into TBS by overnight dialysis at 4 °C. Finally, the solution was concentrated to 0.5–1 mg / mL using an Amicon spin filter 0.5, MWCO 3 kDa (Merck-Millipore). Mass spectrometry analysis was performed on BCN-PEG. 12 -IL15Rα-IL15(209) showed a weight of 24155 Da (predicted mass: 24152).

[0416] Example 145. BCN-PEG to obtain conjugate 210 12 Conjugation of IL15Rα-IL15(209) to trastuzumab (6-N3-GalNAc) 2205 A bioconjugate according to the present invention was prepared by conjugation of 209 to azide-modified trastuzumab (205, trastuzumab(6-N3-GalNAc)2, prepared according to WO 2016170186) in a 2:1 molar ratio. Thus, BCN-PEG 12To a solution of -IL15Rα-IL15 (209, 20 μL, 20 μM in TBS pH 7.4), trastuzumab (6-N3-GalNAc)2 (205, 1.2 μL, 82 μM in PBS pH 7.4) was added and incubated overnight at 37°C. Mass spectral analysis of the IdeS-digested sample showed a mass of 48,526 Da (expected mass: 48,518 Da), corresponding to the Fc / 2 fragment of conjugate 210.

[0417] Example 146. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker 105 to give 211 Compound 105 (2.5 μL, 0.8 mM solution in DMF, 2 equivalents relative to IgG) was added to a solution of trastuzumab-(6-azidoGalNAc)2 (7.5 μL, 150 μg, 17.56 mg / mL in PBS pH 7.4; also referred to as trast-v1a), prepared according to WO 2016170186. The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck-Millipore). Mass spectrometry analysis of the IdeS-digested sample showed one major product (calculated mass 49,625 Da, observed mass 49,626 Da) corresponding to the intramolecularly crosslinked trastuzumab derivative 211. HPLC-SEC showed less than 4% aggregation, thus ruling out intermolecular crosslinking.

[0418] Example 147. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker 107 to give 212 To a solution of trastuzumab-(6-azido-GalNAc)2 (7.5 μL, 150 μg, 17.56 mg / mL in PBS pH 7.4) was added compound 107 (2.5 μL, 4 mM solution in DMF, 10 equivalents relative to IgG). The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck-Millipore). Mass spectrometry analysis of the IdeS digested sample showed a product corresponding to the intramolecularly crosslinked trastuzumab derivative 212 (calculated mass 50,153 Da, observed mass 50,158 Da). HPLC-SEC showed less than 4% aggregation, thus excluding intermolecular crosslinking.

[0419] Example 148. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker 117 to give 213 To a solution of trastuzumab-(6-azidoGalNAc)2 (7.5 μL, 150 μg, 17.56 mg / mL in PBS pH 7.4) was added compound 117 (2.5 μL, 0.8 mM solution in DMF, 2 equivalents relative to IgG). The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Mass spectral analysis of the IdeS digested sample showed one major product (calculated mass 49,580 Da, observed mass 49,626 Da) corresponding to the intramolecularly crosslinked trastuzumab derivative 213. HPLC-SEC showed less than 4% aggregation, thus ruling out intermolecular crosslinking.

[0420] Example 149. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker 118 to give 214 To a solution of trastuzumab-(6-azidoGalNAc)2 (7.5 μL, 150 μg, 17.56 mg / mL in PBS pH 7.4) was added compound 118 (2.5 μL, 4 mM solution in DMF, 10 equivalents relative to IgG). The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Mass spectrometry analysis of the IdeS digested sample showed a product (calculated mass 49,358 Da, observed mass 49,361 Da) corresponding to the intramolecularly crosslinked trastuzumab derivative 214. HPLC-SEC showed less than 4% aggregation, thus excluding intermolecular crosslinking.

[0421] Example 150. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker 124 to give 215 To a solution of trastuzumab-(6-azidoGalNAc)2 (7.5 μL, 150 μg, 17.56 mg / mL in PBS pH 7.4) was added compound 124 (2.5 μL, 4 mM solution in DMF, 10 equivalents relative to IgG). The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Mass spectrometry analysis of the IdeS digested sample showed a product corresponding to the intramolecularly crosslinked trastuzumab derivative 215 (calculated mass 49,406 Da, observed mass 49,409 Da). HPLC-SEC showed less than 4% aggregation, thus excluding intermolecular crosslinking.

[0422] Example 151. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker 125 to give 216 To a solution of trastuzumab-(6-azidoGalNAc)2 (7.5 μL, 150 μg, 17.56 mg / mL in PBS pH 7.4) was added compound 125 (2.5 μL, 0.8 mM solution in DMF, 2 equivalents relative to IgG). The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Mass spectral analysis of the IdeS digested sample showed one major product (calculated mass 49,184 Da, observed mass 49,184 Da) corresponding to the intramolecularly crosslinked trastuzumab derivative 216. HPLC-SEC showed less than 4% aggregation, thus ruling out intermolecular crosslinking.

[0423] Example 152. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker 145 to give 217 To a solution of trastuzumab-(6-azidoGalNAc)2 (320 μL, 2 mg, 5.56 mg / mL in PBS pH 7.4) was added compound 145 (80 μL, 1.66 mM solution in DMF, 10 equivalents relative to IgG). The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Mass spectral analysis of the IdeS digested sample showed one major product (calculated mass 49,796 Da, observed mass 49,807 Da) corresponding to the intramolecularly crosslinked trastuzumab derivative 217. HPLC-SEC showed less than 4% aggregation, thus excluding intermolecular crosslinking.

[0424] Example 153. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker-payload construct 137 to obtain DAR1 ADC 218 To a solution of trastuzumab-(6-azidoGalNAc)2 (37.5 μL, 250 μg, 6.67 mg / mL in PBS pH 7.4) was added compound 137 (12.5 μL, 0.67 mM solution in DMF, 5 equivalents relative to IgG). The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Mass spectral analysis of the IdeS digested sample showed one major product (calculated mass 50,464 Da, observed mass 50,474 Da) corresponding to the conjugate ADC 218 obtained via intramolecular crosslinking. HPLC-SEC showed less than 4% aggregation, thus excluding intermolecular crosslinking. RP-HPLC revealed a chromatographic separation of Fc / 2(t r 6.099), Fc-toxin (t r 8.275, corresponding to 82.4% of the total Fc / 2 fragments) and Fab (t r 9.320) fragment was shown.

[0425] Example 154. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker-payload construct 131 to obtain DAR1 ADC 219 To a solution of trastuzumab-(6-azidoGalNAc)2 (37.5 μL, 250 μg, 6.67 mg / mL in PBS pH 7.4) was added compound 131 (12.5 μL, 0.67 mM solution in DMF, 5 equivalents relative to IgG). The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Mass spectral analysis of the IdeS digested sample showed one major product (calculated mass 50,638 Da, observed mass 50,649 Da) corresponding to ADC 219 obtained via intramolecular crosslinking. HPLC-SEC showed less than 4% aggregation, thus ruling out intermolecular crosslinking. RP-HPLC revealed a single major product corresponding to Fc / 2 (t r 6.082), Fc-toxin (t r 9.327, corresponding to 76.7% of the total Fc / 2 fragments) and Fab (t r9.347) fragment was shown.

[0426] Example 155. Intramolecular crosslinking of trastuzumab-(azide)2 with bivalent linker-payload construct 139 to obtain DAR1 ADC 220 To a solution of trastuzumab-(6-azidoGalNAc)2 (37.5 μL, 250 μg, 6.67 mg / mL in PBS pH 7.4) was added compound 139 (12.5 μL, 0.67 mM solution in DMF, 5 equivalents relative to IgG). The reaction was incubated at room temperature for 1 day and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Mass spectral analysis of the IdeS digested sample showed one major product (calculated mass 50392 Da, observed mass 50402 Da) corresponding to ADC 220 obtained via intramolecular crosslinking. HPLC-SEC showed less than 4% aggregation, thus excluding intermolecular crosslinking. RP-HPLC revealed a single major product corresponding to Fc / 2 (t r 6.062), Fc-toxin (t r 8.548, corresponding to 89.5% of the total Fc / 2 fragments) and Fab (t r 9.295) fragment was shown.

[0427] Example 156.2: Intramolecular cross-linking of trastuzumab derivative 217 (containing a single BCN) with tetrazine-modified anti-CD3 immune cell engager 204 to obtain T cell engager 221 with a single molecule format To a solution of 217 (8 μL, 141 μg, 17.7 mg / mL in PBS pH 7.4) was added hOKT-PEG4-tetrazine (204, 13.15 μL, 280 μg, 21.45 mg / mL in PBS pH 7.4, 2 equivalents relative to IgG). Mass spectral analysis of the IdeS-digested sample showed one major product (calculated mass 77,664 Da, observed mass 77,647 Da) corresponding to the conjugate Fc-PEG4-hOKT3 (221).

[0428] Example 157. Intramolecular crosslinking of bis-azido-rituximab rit-v1a with trivalent linker 145 to obtain BCN-rituximab rit-v1a-145 To a solution of bis-azido-rituximab rit-v1a (494 μL, 30 mg, 60.7 mg / mL in PBS pH 7.4) prepared according to WO 2016170186, PBS pH 7.4 (2506 μL), propylene glycol (2980 μL), and trivalent linker 145 (20 μL, 40 mM solution in DMF, 4.0 equivalents relative to IgG) were added. The reaction was incubated overnight at room temperature and subsequently purified on a Superdex 200 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase. Reducing SDS-PAGE showed one major HC product corresponding to the cross-linked heavy chain (see Figure 16, right panel, lane 3), indicating the formation of rit-v1a-145. Furthermore, non-reducing SDS-PAGE showed one major band of approximately the same height as rit-v1a (see Figure 16, left panel, lane 3), demonstrating that only intramolecular cross-linking occurred.

[0429] Example 158. Intramolecular crosslinking of bis-azido-B12 B12-v1a with trivalent linker 145 to obtain BCN-B12 B12-v1a-145 To a solution of bis-azido-B12 B12-v1a (415 μL, 4 mg, 9.6 mg / mL in PBS pH 7.4) prepared according to WO2016170186, propylene glycol (412 μL) and trivalent linker 145 (2.7 μL, 40 mM solution in DMF, 4.0 equivalents relative to IgG) were added. The reaction was incubated overnight at room temperature and subsequently purified on a Superdex200 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase. RP-HPLC analysis of the IdeS-digested sample indicates the formation of B12-v1a-145 (see Figure 17).

[0430] Example 159. Intramolecular crosslinking of bis-azido-trastuzumab trast-v1a with bis-BCN-TCO XL11 to obtain TCO-trastuzumab trast-v1a-XL11 To a solution of bis-azido-trastuzumab trast-v1a (36 μL, 2 mg, 56.1 mg / mL in PBS pH 7.4) prepared according to WO 2016170186, PBS pH 7.4 (164 μL), propylene glycol (195 μL), and bis-BCN-TCO XL11 (5.3 μL, 10 mM solution in DMF, 4.0 equivalents relative to IgG) were added. The reaction was incubated overnight at room temperature and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Reducing SDS-PAGE showed two major HC products corresponding to the unconjugated heavy chain and the cross-linked heavy chain (see Figure 18, right panel, lane 2), indicating partial conversion to trast-v1a-XL11. Furthermore, non-reducing SDS-PAGE showed one major band at the height of trast-v1a (see Figure 18, left panel, lane 2), indicating that only intramolecular cross-linking occurred.

[0431] Example 160. Intramolecular crosslinking of bis-azido-rituximab rit-v1a with bis-BCN-TCO XL11 to obtain TCO-rituximab rit-v1a-XL11 To a solution of bis-azido-rituximab rit-v1a (37 μL, 2 mg, 54.5 mg / mL in PBS pH 7.4), PBS pH 7.4 (163 μL), propylene glycol (195 μL), and bis-BCN-TCO XL11 (5.3 μL, 10 mM solution in DMF, 4.0 equivalents relative to IgG) were added. The reaction was incubated overnight at room temperature and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Reducing SDS-PAGE showed two major HC products corresponding to the unconjugated and cross-linked heavy chains (see Figure 18, right panel, lane 6), indicating partial conversion to rit-v1a-XL11. Furthermore, non-reducing SDS-PAGE showed one major band at the height of rit-v1a (see FIG. 18, left panel, lane 2), indicating that only intramolecular cross-linking occurred.

[0432] Example 161. Intramolecular crosslinking of bis-azido-trastuzumab trast-v3 with bis-BCN-MMAE 137 to obtain DAR1 ADC trast-v3-137 To a solution of trast-v3 (15 μL, 150 μg, 10 mg / mL in PBS pH 7.4) was added bis-BCN-MMAE (137, 15 μL, 0.27 mM solution in PG, 4 equivalents relative to IgG). The reaction was incubated at room temperature for 16 h and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL, MWCO 10 kDa, Merck Millipore). Mass spectral analysis of the IdeS-digested sample showed one major product (observed mass 48,719 Da) corresponding to trast-v3-137 obtained via intramolecular crosslinking.

[0433] Example 162. Intramolecular crosslinking of deglycosylated trastuzumab with bis-BCN-MMAE LD03 Deglycosylated trastuzumab (8.3 μL, 0.15 mg, 18.1 mg / mL in PBS 5.5) was incubated with bis-BCN-MMAE (LD03, 8.3 μL, 1.2 mM in PG) and mushroom tyrosinase (3 μL, 10 mg / mL in phosphate buffer pH 6.0, Sigma Aldrich T3824) at room temperature for 16 hours. See also Dutch Patent Application No. 2026947, incorporated herein by reference. RP-HPLC analysis of the DTT-treated ADC showed 35% conversion to trast-v4-LD03 (see Figure 19).

[0434] Example 163. Intramolecular crosslinking of bis-azido-trastuzumab trast-v3 with bis-BCN-MMAE LD03 to obtain DAR1 ADC trast-v3-LD03 To a solution of trast-v3 (22.5 μL, 5 mg, 6.7 mg / mL in PBS pH 7.4) was added bis-BCN-MMAE (LD03, 7.5 μL, 0.53 mM solution in DMF, 4 equivalents relative to IgG). The reaction was incubated at room temperature for 16 h and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL, MWCO 10 kDa, Merck Millipore). Mass spectral analysis of the IdeS-digested sample showed one major product (observed mass 50,052 Da) corresponding to trast-v3-LD03 obtained via intramolecular crosslinking.

[0435] Example 164. Intramolecular crosslinking of bis-azido-rituximab rit-v3 with bis-BCN-MMAE LD03 to obtain DAR1 ADC rit-v3-LD03 To a solution of rit-v3 (22.5 μL, 5 mg, 6.7 mg / mL in PBS pH 7.4) was added bis-BCN-MMAE (LD03, 7.5 μL, 0.53 mM solution in DMF, 4 equivalents relative to IgG). The reaction was incubated at room temperature for 16 h and subsequently buffer-exchanged into PBS pH 7.4 using a centrifugal filter (Amicon Ultra-0.5 mL, MWCO 10 kDa, Merck Millipore). Mass spectrometry analysis of the IdeS digested sample showed one major product (mass 49989 Da) corresponding to rit-v3-LD03 obtained via intramolecular crosslinking.

[0436] Example 165. Intramolecular crosslinking of bis-BCN-IL15Rα-IL15 PF27 to trast-v3 via strain-promoted alkyne-azide cycloaddition (SPAAC) (P:A ratio 1:1) trast-v3 (2.57 μL, 0.05 mg, 19.5 mg / mL in PBS) was incubated with bis-BCN-IL15Rα-IL15 (PF27, 5.6 μL, 3 equivalents of bis-BCN-labeled IL15Rα-IL15, 7.6 mg / mL in PBS) for 16 h at room temperature. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 73,432 Da) corresponding to the predicted product trast-v3-PF27.

[0437] Example 166. Intramolecular crosslinking of hOKT3-bis-BCN PF22 to trast-v3 via SPAAC (P:A ratio 1:1) trast-v3 (2.57 μL, 0.05 mg, 19.5 mg / mL in PBS) was incubated with hOKT3-bis-BCN PF22 (5.15 μL, 3 equiv., 5.7 mg / mL in PBS) at room temperature for 16 h. Mass spectral analysis of the sample after IdeS treatment showed one major Fc / 2 product (observed mass 77,150 Da) corresponding to the predicted product trast-v3-PF22.

[0438] Example 167.2: Conjugation of hOKT3-PEG4-tetrazine 204 to BCN-rituximab rit-v1a-145 to obtain T cell engager rit-v1a-145-204 in a single molecule format To a solution of rit-v1a-145 (287 μL, 6.6 mg, 154 μM in PBS pH 7.4) was added hOKT3-PEG4-tetrazine 204 (247 μL, 1.9 mg, 269 μM in PBS pH 6.5, 1.5 equivalents relative to IgG). The reaction was incubated overnight at room temperature and subsequently purified on a Superdex 200 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase. Non-reducing SDS-PAGE analysis showed a single major product consisting of the antibody conjugated to a single hOKT3 (see Figure 16, left panel, lane 5), thereby confirming the formation of rit-v1a-145-204. Furthermore, reducing SDS-PAGE confirms one major HC product corresponding to two heavy chains conjugated to a single hOKT3 (see Figure 16, right panel, lane 5).

[0439] Example 168.2: hOKT3-PEG to obtain T cell engager rit-v1a-145-PF01 in a single molecule format 11 -Conjugation of tetrazine PF01 to BCN-rituximab rit-v1a-145 A solution of rit-v1a-145 (247 μL, 6.3 mg, 171 μM in PBS pH 7.4) was added to hOKT3-PEG 11rit-v1a-145-PF01 (304 μL, 2.0 mg, 230 μM in PBS pH 6.5, 1.7 equivalents relative to IgG) was added. The reaction was incubated overnight at room temperature and subsequently purified on a Superdex200 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase. Non-reducing SDS-PAGE analysis showed one major product consisting of the antibody conjugated to a single hOKT3 (see Figure 16, left panel, lane 6), thereby confirming the formation of rit-v1a-145-PF01. Furthermore, reducing SDS-PAGE confirmed one major HC product corresponding to two heavy chains conjugated to a single hOKT3 (see Figure 16, right panel, lane 6).

[0440] Example 169.2: hOKT3-PEG to obtain T cell engager B12-v1a-145-PF01 in a single molecule format 11 -Conjugation of tetrazine PF01 to BCN-B12 B12-v1a-145 To a solution of B12-v1a-145 (38 μL, 1.0 mg, 178 μM in PBS pH 7.4), hOKT3-PEG 11 -Tetrazine PF01 (44 μL, 0.3 mg, 230 μM in PBS pH 6.5, 1.5 equivalents relative to IgG) was added. The reaction was incubated overnight at room temperature and subsequently purified on a Superdex200 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase. Non-reducing SDS-PAGE analysis showed one major product consisting of the antibody conjugated to a single hOKT3 (see Figure 20, lane 4), thereby confirming the formation of B12-v1a-145-PF01.

[0441] Example 170. Conjugation of hOKT3-PEG4-tetrazine 204 to TCO-trastuzumab trast-v1a-XL11 to obtain T cell engager trast-v1a-XL11-204 in a single molecule format To a solution of TCO-trastuzumab trast-v1a-XL11 (5.7 μL, 100 μg, 117 μM in PBS pH 7.4), hOKT3-PEG4-tetrazine 204 (5 μL, 38 μg, 269 μM in PBS pH 6.5, 2.0 equivalents relative to IgG) was added. The reaction was incubated overnight at room temperature. Non-reducing SDS-PAGE analysis showed two major products corresponding to unconjugated antibody and antibody conjugated to a single hOKT3 (see Figure 22, left panel, lane 3), thereby confirming the formation of trast-v1a-XL11-204. Furthermore, reducing SDS-PAGE confirmed that OKT3 was conjugated to a cross-linked heavy chain containing a TCO-reactive handle (see Figure 22, right panel, lane 3).

[0442] Example 171.2: Conjugation of hOKT3-PEG4-tetrazine 204 to TCO-rituximab rit-v1a-XL11 to obtain T cell engager rit-v1a-XL11-204 in a single molecule format To a solution of TCO-rituximab rit-v1a-XL11 (56.3 μL, 100 μg, 106 μM in PBS pH 7.4), hOKT3-PEG4-tetrazine 204 (5 μL, 38 μg, 269 μM in PBS pH 6.5, 2.0 equivalents relative to IgG) was added. The reaction was incubated overnight at room temperature. Non-reducing SDS-PAGE analysis showed two major products corresponding to unconjugated antibody and antibody conjugated to a single hOKT3 (see Figure 22, left panel, lane 7), thereby confirming the formation of rit-v1a-XL11-204. Furthermore, reducing SDS-PAGE confirmed that OKT3 was conjugated to a cross-linked heavy chain containing a TCO-reactive handle (see Figure 22, right panel, lane 7).

[0443] Example 172.2: hOKT3-PEG to obtain T cell engager rit-v1a-145-PF02 in a single molecule format 23 -Conjugation of tetrazine PF02 to BCN-rituximab rit-v1a-145 A solution of rit-v1a-145 (247 μL, 6.3 mg, 171 μM in PBS pH 7.4) was added to hOKT3-PEG 23 t-tetrazine PF02 (262 μL, 2.0 mg, 267 μM in PBS pH 6.5, 1.7 equivalents relative to IgG) was added. The reaction was incubated overnight at room temperature and subsequently purified on a Superdex200 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase. Non-reducing SDS-PAGE analysis showed one major product consisting of the antibody conjugated to a single hOKT3 (see Figure 18, left panel, lane 7), thereby confirming the formation of rit-v1a-145-PF02. Furthermore, reducing SDS-PAGE confirmed one major HC product corresponding to two heavy chains conjugated to a single hOKT3 (see Figure 18, right panel, lane 7).

[0444] Example 173.2: Conjugation of hOKT3-PEG2-arylazide PF03 to BCN-trastuzumab trast-v1a-145 to obtain T cell engager trast-v1a-145-PF03 in a single molecule format To a solution of trast-v1a-145 (2.9 μL, 150 μg, 347 μM in PBS pH 7.4) was added hOKT3-PEG2-arylazide PF03 (4.9 μL, 56 μg, 411 μM in PBS pH 7.4, 2.0 equivalents relative to IgG). The reaction was incubated overnight at room temperature. Mass spectral analysis of the reduced sample showed a single major heavy chain product (observed mass 128,388 Da) corresponding to trast-v1a-145-PF03.

[0445] Example 174.2: Conjugation of hOKT3-PEG2-arylazide PF03 to BCN-rituximab rit-v1a-145 to obtain the T cell engager rit-v1a-145-PF03 in a single molecule format To a solution of rit-v1a-145 (30 μL, 1.5 mg, 337 μM in PBS pH 7.4) was added hOKT3-PEG2-arylazide PF03 (49 μL, 0.6 mg, 411 μM in PBS pH 7.4, 2.0 equivalents relative to IgG). The reaction was incubated overnight at room temperature and subsequently purified on a Superdex 200 10 / 300 GL column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare) using PBS pH 7.4 as the mobile phase. Mass spectral analysis of the reduced sample showed a single major heavy chain product (observed mass 128,211 Da) corresponding to rit-v1a-145-PF03.

[0446] Example 175.2: Conjugation of bis-BCN-hOKT3 PF22 to bis-azido-trastuzumab trast-v1a to obtain T cell engager trast-v1a-PF22 with a single molecule format To a solution of trast-v1a (1.8 μL, 100 μg, 374 μM in PBS pH 7.4) was added PBS pH 7.4 (4.5 μL) and bis-BCN-hOKT3 PF22 (13.7 μL, 78 μg, 194 μM in PBS pH 7.4, 4.0 equivalents relative to IgG). The reaction was incubated overnight at room temperature. Non-reducing SDS-PAGE analysis showed one major product consisting of a single hOKT3-conjugated antibody (see Figure 21, lane 5), thereby confirming the formation of trast-v1a-PF22.

[0447] Example 176.2: Conjugation of bis-BCN-hOKT3 PF22 to bis-azido-rituximab rit-v1a to obtain T cell engager rit-v1a-145-PF22 in a single molecule format To a solution of rit-v1a (1.8 μL, 100 μg, 363 μM in PBS pH 7.4) was added PBS pH 7.4 (7.9 μL) and bis-BCN-hOKT3 PF22 (10.3 μL, 58 μg, 194 μM in PBS pH 7.4, 3.0 equivalents relative to IgG). The reaction was incubated overnight at room temperature. Non-reducing SDS-PAGE analysis showed one major product consisting of a single hOKT3-conjugated antibody (see Figure 21, lane 4), thereby confirming the formation of rit-v1a-PF22.

[0448] Example 177.2: Conjugation of bis-BCN-hOKT3 PF23 to bis-azido-trastuzumab trast-v1a to obtain T cell engager trast-v1a-PF23 with a single molecule format To a solution of trast-v1a (1.8 μL, 100 μg, 374 μM in PBS pH 7.4) was added PBS pH 7.4 (9.9 μL) and bis-BCN-hOKT3 PF23 (8.4 μL, 58 μg, 239 μM in PBS pH 7.4, 3.0 equivalents relative to IgG). The reaction was incubated overnight at 37°C. Non-reducing SDS-PAGE analysis showed two major products consisting of unconjugated trastuzumab and trastuzumab conjugated to bis-BCN-hOKT3 PF23 (see Figure 22, lane 2), thereby confirming the partial formation of trast-v1a-PF23.

[0449] Example 178. Conjugation of bis-BCN-hOKT3 PF23 to bis-azido-rituximab rit-v1a to obtain T cell engager rit-v1a-PF23 with a single molecule format To a solution of rit-v1a (1.8 μL, 100 μg, 363 μM in PBS pH 7.4) was added PBS pH 7.4 (13.6 μL) and bis-BCN-hOKT3 PF23 (4.3 μL, 30 μg, 239 μM in PBS pH 7.4, 1.5 equivalents relative to IgG). The reaction was incubated overnight at 37°C. Non-reducing SDS-PAGE analysis showed two major products consisting of unconjugated rituximab and rituximab conjugated once to bis-BCN-hOKT3 PF23 (see Figure 23, lane 5), thereby confirming the partial formation of rit-v1a-PF23.

[0450] Example 179. 4-1BB-PEG to obtain T cell engager rit-vla-145-PF08 in a single molecule format 11 -Conjugation of tetrazine PF08 to BCN-rituximab rit-v1a-145 A solution of rit-v1a-145 (35 μL, 0.9 mg, 170 μM in PBS pH 7.4) was added to 4-1BB-PEG 11 -Tetrazine PF08 (40 μL, 248 μg, 222 μM in PBS pH 7.4, 1.5 equivalents relative to IgG) was added. The reaction was incubated overnight at room temperature. Non-reducing SDS-PAGE analysis confirmed that 4-1BB-PEG 23 -BCN PF08 (see lane 3, Figure 20), thereby confirming the partial formation of rit-v1a-145-PF08.

[0451] Example 180. 4-1BB-PEG to obtain T cell engager B12-v1a-145-PF08 with a 2:1 molecular format 11 -Conjugation of tetrazine PF08 to BCN-B12 B12-v1a-145 To a solution of B12-v1a-145 (34 μL, 0.9 mg, 178 μM in PBS pH 7.4), 4-1BB-PEG 11-Tetrazine PF08 (40 μL, 248 μg, 222 μM in PBS pH 7.4, 1.5 equivalents relative to IgG) was added. The reaction was incubated overnight at room temperature. Non-reducing SDS-PAGE analysis confirmed that 4-1BB-PEG 23 -BCN PF08 showed one major product consisting of B12 conjugated to PF08 (see FIG. 20, lane 5), thereby confirming the partial formation of B12-v1a-145-PF08.

[0452] Example 181.2: Conjugation of 4-1BB-PEG2-arylazide PF09 to BCN-trastuzumab trast-v1a-145 to obtain T cell engager trast-v1a-145-PF09 in a single molecule format To a solution of trast-v1a-145 (1.9 μL, 100 μg, 347 μM in PBS pH 7.4) was added 4-1BB-PEG2-arylazide PF09 (5.9 μL, 37 μg, 225 μM in PBS pH 7.4, 2.0 equivalents relative to IgG). The reaction was incubated overnight at room temperature. Non-reducing SDS-PAGE analysis showed one major product consisting of trastuzumab conjugated to a single 4-1BB-PEG2-arylazide PF09 (see Figure 24, lane 4), thereby confirming the formation of trast-v1a-145-PF09.

[0453] Example 182.2: Conjugation of 4-1BB-PEG2-arylazide PF09 to BCN-rituximab rit-v1a-145 to obtain T cell engager rit-v1a-145-PF09 in a single molecule format To a solution of rit-v1a-145 (2.0 μL, 100 μg, 337 μM in PBS pH 7.4) was added 4-1BB-PEG2-arylazide PF09 (5.9 μL, 37 μg, 225 μM in PBS pH 7.4, 2.0 equivalents relative to IgG). The reaction was incubated overnight at room temperature. Non-reducing SDS-PAGE analysis showed one major product consisting of rituximab conjugated to a single 4-1BB-PEG2-arylazide PF09 (see Figure 24, lane 2), thereby confirming the formation of rit-v1a-145-PF09.

[0454] Example 183.2: Conjugation of tetrazine-PEG3-GGG-IL15Rα-IL15(PF12) to BCN-trastuzumab trast-v1a-145 to obtain the T cell engager trast-v1a-145-PF12 in a single molecule format Trast-v1a-145 (75 μL, 1.575 mg, 21 mg / mL in PBS) was incubated with PF12 (80 μL, 2 equivalents, 6.5 mg / mL in PBS) for 16 hours at 37° C. Analysis by non-reducing SDS-PAGE confirmed the formation of Trast-v1a-145-PF12 (see Figure 25, lane 5).

[0455] Example 184.2: Conjugation of arylazide-PEG11-GGG-IL15Rα-IL15 (PF13) to BCN-trastuzumab trast-v1a-145 to obtain the T cell engager trast-v1a-145-PF13 in a single molecule format trast-v1a-145 (280 μL, 5.2 mg, 18.6 mg / mL in PBS) was incubated with PF13 (477 μL, 1.5 equivalents, 2.6 mg / mL in PBS) for 16 h at 37°C. Mass spectral analysis of the sample after IdeS treatment showed one major product of 73,991 Da (expected mass: 73,989 Da), corresponding to the cross-linked Fc fragment conjugated to PF13, thereby confirming the formation of trast-v1a-145-PF13.

[0456] Example 185.2: Conjugation of Arylazido-PEG11-GGG-IL15Rα-IL15 (PF13) to BCN-Rituximab Rit-v1a-145 to Obtain the T Cell Engager Rit-v1a-145-PF13 with a Single Molecule Format Rit-v1a-145 (0.5 μL, 0.025 mg, 50.6 mg / mL in PBS) was incubated with PF13 (6.6 μL, 4 equivalents, 2.6 mg / mL in PBS) at room temperature for 16 h. Mass spectral analysis of the sample after IdeS treatment showed one major product of 73,927 Da (expected mass: 73,925 Da), corresponding to the cross-linked Fc fragment conjugated to PF13, thereby confirming the formation of rit-v1a-145-PF13.

[0457] Example 186. Conjugation of bis-BCN-SYR-(G4S)3-IL15Rα-IL15(PF27) to bis-azido-trastuzumab trast-v1a to obtain the T cell engager trast-v1a-145-PF27 with a single molecule format Trast-v1a (1.78 μL, 0.099 mg, 56.1 mg / mL in PBS) was incubated with PF27 (18.4 μL, 4 equivalents, 7.62 mg / mL in PBS) and 2.87 μL of PBS for 16 h at 37° C. Mass spectral analysis of the sample after IdeS treatment showed one major product of 74,193 Da (expected mass: 74,178 Da), corresponding to the cross-linked Fc fragment conjugated to PF27, thereby confirming the formation of trast-v1a-145-PF27.

[0458] Example 187.2: Conjugation of Bis-BCN-SYR-(G4S)3-IL15Rα-IL15(PF27) to Bis-Azido-Rituximab Rit-v1a to Obtain the T Cell Engager Rit-v1a-145-PF27 in a Single Molecular Format Rit-v1a (1 μL, 0.055 mg, 54.6 mg / mL in PBS) was incubated with PF27 (8.9 μL, 4 equivalents, 6.2 mg / mL in PBS) and 1.6 μL of PBS for 16 h at 37°C. Mass spectral analysis of the sample after IdeS treatment showed one major product of 74118 Da (expected mass: 74114 Da), corresponding to the cross-linked Fc fragment conjugated to PF27, thereby confirming the formation of rit-v1a-145-PF27.

[0459] Example 188.2: Conjugation of azido-IL15Rα-IL15 PF17 to BCN-trastuzumab trast-v1a-145 to obtain T cell engager trast-v1a-145-PF17 in a single molecule format To a solution of trast-v1a-145 (29 μL, 1.5 mg, 347 μM in PBS pH 7.4) was added azido-IL15Rα-IL15 PF17 (97 μL, 1.1 mg, 411 μM in PBS pH 7.4, 4.0 equivalents relative to IgG). The reaction was incubated overnight at 37°C. Non-reducing SDS-PAGE analysis showed one major product consisting of trastuzumab conjugated to a single azido-IL15Rα-IL15 PF17 (see Figure 26, lane 4), thereby confirming the formation of trast-v1a-145-PF17.

[0460] Example 189.2: Conjugation of Azido-IL15Rα-IL15 PF17 to BCN-Rituximab rit-v1a-145 to Obtain T Cell Engager rit-v1a-145-PF17 in a Single Molecule Format To a solution of rit-v1a-145 (3 μL, 150 μg, 337 μM in PBS pH 7.4) was added azido-IL15Rα-IL15 PF17 (9.7 μL, 111 μg, 411 μM in PBS pH 7.4, 4.0 equivalents relative to IgG). The reaction was incubated overnight at 37°C. Non-reducing SDS-PAGE analysis showed one major product consisting of rituximab conjugated to a single azido-IL15Rα-IL15 PF17 (see Figure 26, lane 2), thereby confirming the formation of rit-v1a-145-PF17.

[0461] Example 190. Conjugation of Azido-IL15 PF19 to BCN-Trastuzumab tras-v1a-145 to Obtain T Cell Engager tras-v1a-145-PF19 with a 2:1 Molecular Format trast-v1a-145 (4.0 μL, 0.075 mg, 18.6 mg / mL in PBS) was incubated with PF19 (4.6 μL, 5 equivalents, 7.7 mg / mL in PBS) at room temperature for 16 h. Mass spectral analysis of the sample after IdeS treatment showed one major product of 63,941 Da (expected mass: 63,936 Da), corresponding to the cross-linked Fc fragment conjugated to PF19, thereby confirming the formation of trast-v1a-145-PF19.

[0462] Example 191. Conjugation of Azido-IL15 PF19 to BCN-Rituximab rit-v1a-145 to Obtain the T Cell Engager rit-v1a-145-PF19 in a 2:1 Molecular Format rit-v1a-145 (2.0 μL, 0.112 mg, 50.6 mg / mL in PBS) was incubated with PF19 (5.1 μL, 4 equivalents, 7.7 mg / mL in PBS) at room temperature for 16 h. Mass spectral analysis of the sample after IdeS treatment showed a single major product of 63,882 Da (expected mass: 63,879 Da), corresponding to the cross-linked Fc fragment conjugated to PF19, thereby confirming the formation of rit-v1a-145-PF19.

[0463] Example 192. Conjugation of bis-BCN-SYR-(G4S)3-IL15(PF29) to bis-azido-trastuzumab tras-v1a to obtain T cell engager Tras-v1a-PF29 with a 2:1 molecular format Trast-v1a (1 μL, 0.056 mg, 56.1 mg / mL in PBS) was incubated with PF29 (11 μL, 4 equivalents, 3.6 mg / mL in PBS) for 16 hours at 37° C. Non-reducing SDS-PAGE analysis showed two major products corresponding to unconjugated trastuzumab and trastuzumab conjugated to single bis-BCN-SYR-(G4S)3-IL15 PF29 (see Figure 27, lane 2), thereby confirming partial conversion to Trastuzumab-v1a-PF29.

[0464] Example 193. Conjugation of Bis-BCN-SYR-(G4S)3-IL15(PF29) to Bis-Azido-Rituximab Rit-v1a to Obtain the T Cell Engager Rit-v1a-PF29 with a 2:1 Molecular Format Rit-v1a (1 μL, 0.055 mg, 54.6 mg / mL in PBS) was incubated with PF29 (11 μL, 4 equivalents, 3.6 mg / mL in PBS) for 16 hours at 37° C. Non-reducing SDS-PAGE analysis showed two major products corresponding to unconjugated rituximab and rituximab conjugated to single bis-BCN-SYR-(G4S)3-IL15 PF29 (see Figure 27, lane 4), thereby confirming partial conversion to rit-v1a-PF29.

[0465] Example 194.2: Tetrazine-PEG to obtain T cell engager trast-v1a-145-PF21 in a single molecule format 12 Conjugation of -SYR-(G4S)3-IL15(PF21) to BCN-trastuzumab trast-v1a-145 Trast-v1a (2 μL, 0.042 mg, 21 mg / mL in PBS) was incubated with PF21 (10 μL, 6.7 equivalents, 2.9 mg / mL in PBS) for 16 h at 37° C. Mass spectral analysis of the sample after IdeS treatment showed one major product of 64865 Da (expected mass: 64863 Da), corresponding to the cross-linked Fc fragment conjugated to PF21, thereby confirming the formation of trast-v1a-145-PF21.

[0466] Example 195. CD3 Binding Assay Specific binding to CD3 was assessed using Jurkat E6.1 cells, which express CD3 on the cell surface, and MOLT-4 cells, which do not express CD3 on the cell surface. Both cell lines were cultured at 2 × 10 5 ~1×10 6 Cells were cultured at a concentration of 100,000 cells / ml in RPMI 1640 supplemented with 1% penicillin / streptomycin and 10% fetal bovine serum. Cells were washed with fresh medium before the experiment and seeded in duplicate in a 96-well plate at 100,000 cells per well. A dilution series of six antibodies was prepared in phosphate-bu...

Claims

1. Structure (1): 【Chemistry 1】 (In the formula, Ab is an antibody; a, b and c are 1; L １ , L ２ and L ３ is a linker covalently connecting BM and Z or D; D is a payload, said payload being an active agent selected from the group consisting of cytotoxins, antivirals, antibacterials, peptides and oligonucleotides; BM is a branching moiety, said branching moiety being selected from a carbon atom or a nitrogen atom; Z is a connecting group obtainable by a cycloaddition reaction and contains a triazole, cyclohexene, cyclohexadiene, isoxazoline, isoxazolidine, pyrazoline, piperazine, pyridazine, or dihydropyridazine; L 1 and L 2 are a chain of 5 to 100 skeletal atoms selected from C, N, O and S, L 1 and L 2 are selected from straight or branched C 1 -C 20 alkylene groups, said alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR 3 , R 3 being independently selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 2 -C 24 alkenyl groups, C 2 -C 24 alkynyl groups and C 3 -C 24 cycloalkyl groups, said alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted; L 3 is -(L 4 ) n -(L 5 ) o -(L 6 ) p -(L 7 ) q -, where L 4 , L 5 , L 6 and L 7 are linkers together forming linker L 3 ; n, o, p and q are independently 0 or 1; (a) the linker L 4 is represented by -(W) k1 -(A) d1 -(B) e1 -(A) f1 -(B) g1 -C(O)-; d1=0 or 1; e1=an integer ranging from 1 to 10; f1=0 or 1; g1=an integer ranging from 0 to 10; k1=0 or 1, provided that when k1=1, d1=0; A is structure (23) 【Chemistry 2】 wherein a1=0 or 1, R 13 is selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups, wherein said C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 14 , and R 14 is independently selected from the group consisting of hydrogen and C 1 -C 4 alkyl groups, or R 13 is D connected to N, possibly via a spacer moiety. is a sulfamide group according to B is —CH 2 —CH 2 —O— or —O—CH 2 —CH 2 —, or (B) e1 is —(CH 2 —CH 2 —O) e3 —CH 2 —CH 2 —, where e3 is defined in the same way as e1; W is —OC(O)—, —C(O)O—, —C(O)NH—, —NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)(CH 2 ) 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 ranging from 0 to 10; (b) the linker L 5 is a peptide spacer consisting of a peptide containing 2 to 5 amino acids; (c) the linker L 6 has the structure (25): 【Transformation 3】 wherein R 3 is H, R 4 or C(O)R 4 , R 4 is an optionally substituted C 1 -C 24 (hetero)alkyl group, a C 3 -C 10 (hetero)cycloalkyl group, a C 2 -C 10 (hetero)aryl group, a C 3 -C 10 alkyl(hetero)aryl group and a C 3 -C 10 (hetero)arylalkyl group, optionally interrupted by one or more heteroatoms selected from O, S and NR 5 , and R 5 is independently selected from the group consisting of hydrogen and a C 1 -C 4 alkyl group. is a self-immolative spacer represented by: and (d) linker L 7 is an aminoalkanoic acid spacer according to the structure —N—(C x -alkylene)-C(O)—, where x is an integer ranging from 1 to 10; or linker L 7 is an ethylene glycol spacer according to the structure —N—(CH 2 —CH 2 —O) e6 —(CH 2 ) e7 —C(O)—, where e6 is an integer ranging from 1 to 10 and e7 is an integer ranging from 1 to 3. and having a payload antibody ratio of 1.

2. 2. The antibody-payload conjugate of claim 1, wherein Z is obtainable by a [4+2] cycloaddition or a 1,3-dipolar cycloaddition.

3. Structure (5): 【Chemistry 4】 (In the formula, e is an integer ranging from 0 to 10; Su is a monosaccharide; G is a monosaccharide; GlcNAc is N-acetylglucosamine; Fuc is fucose; d is 0 or 1) 2. The antibody-payload conjugate of claim 1, having the formula:

4. L １ and L ２ and each occurrence of Su, Z, G and e is also the same.

5. (b) linker L ５ Structure (27) 【Transformation 5】 (In the formula, R １７ is CH ３ or CH ２ CH ２ CH ２ NHC(O)NH ２ is) It is represented by: and / or (c) linker L ６ Structure (25) 【Transformation 6】 (In the formula, R ３ is H) represented by The antibody-payload conjugate of claim 1.

6. 2. The antibody-payload conjugate of claim 1, wherein D is a cytotoxin selected from a PBD dimer, an indolinobenzodiazepine dimer (IGN), an enediyne, PNU159,682, a duocarmycin dimer, amanitin, or an auristatin.

7. 7. The antibody-payload conjugate of claim 6, wherein D is a cytotoxin selected from a PBD dimer, an indolinobenzodiazepine dimer (IGN), an enediyne, or PNU 159,682.

8. 1. A method for preparing an antibody-payload conjugate having a hypothetical payload-antibody ratio of 1, comprising: (a) reacting a compound having structure (2) containing at least two reactive groups Q with an antibody having structure (3) that is symmetrically functionalized with two reactive groups F to form: 【Transformation 7】 (In the formula, Ab is an antibody; a, b, and c are each independently 0 or 1; L １ , L ２ and L ３ is a linker covalently connecting BM and Q or V; V is a reactive group Q' or a payload D, said payload D being an active agent selected from the group consisting of cytotoxins, antivirals, antibacterials, peptides and oligonucleotides; BM is a branching moiety, said branching moiety being selected from a carbon atom or a nitrogen atom; Q and F are reactive groups capable of undergoing a cycloaddition reaction that link them to form a connecting group Z, said connecting group Z being selected from triazole, cyclohexene, cyclohexadiene, isoxazoline, isoxazolidine, pyrazoline, piperazine, pyridazine or dihydropyridazine. Structure (1'): 【Transformation 8】 wherein Z is a connecting group obtained by cycloaddition of Q and F: The functionalized antibody according to structure (1') is an antibody-payload conjugate when V is a payload D; or the functionalized antibody according to structure (1') is further reacted according to step (b) when V is a reactive group Q'. obtaining a functionalized antibody by (b) if V=Q′, reacting the reactive group Q′ with a payload containing a reactive group F′ to obtain an antibody-payload conjugate where V is payload D; Including, L 1 and L 2 are selected from linear or branched C 1 -C 20 alkylene groups, said alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR 3 , and R 3 is independently selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 2 -C 24 alkenyl groups, C 2 -C 24 alkynyl groups and C 3 -C 24 cycloalkyl groups, said alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted; L 3 is -(L 4 ) n -(L 5 ) o -(L 6 ) p -(L 7 ) q -, where L 4 , L 5 , L 6 and L 7 are linkers together forming linker L 3 ; n, o, p and q are independently 0 or 1; (i) the linker L 4 is represented by -(W) k1 -(A) d1 -(B) e1 -(A) f1 -(B) g1 -C(O)-; d1=0 or 1; e1=an integer ranging from 1 to 10; f1=0 or 1; g1=an integer ranging from 0 to 10; k1=0 or 1, provided that when k1=1, d1=0; A is structure (23) 【Chemistry 9】 wherein a1=0 or 1, R 13 is selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups, wherein said C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 14 , and R 14 is independently selected from the group consisting of hydrogen and C 1 -C 4 alkyl groups, or R 13 is D connected to N, possibly via a spacer moiety. is a sulfamide group according to B is —CH 2 —CH 2 —O— or —O—CH 2 —CH 2 —, or (B) e1 is —(CH 2 —CH 2 —O) e3 —CH 2 —CH 2 —, where e3 is defined in the same way as e1; W is —OC(O)—, —C(O)O—, —C(O)NH—, —NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)(CH 2 ) 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 ranging from 0 to 10; (ii) the linker L 5 is a peptide spacer consisting of a peptide containing 2 to 5 amino acids; (iii) the linker L 6 has the structure (25): 【Chemistry 10】 wherein R 3 is H, R 4 or C(O)R 4 , R 4 is an optionally substituted C 1 -C 24 (hetero)alkyl group, a C 3 -C 10 (hetero)cycloalkyl group, a C 2 -C 10 (hetero)aryl group, a C 3 -C 10 alkyl(hetero)aryl group and a C 3 -C 10 (hetero)arylalkyl group, optionally interrupted by one or more heteroatoms selected from O, S and NR 5 , and R 5 is independently selected from the group consisting of hydrogen and a C 1 -C 4 alkyl group. is a self-immolative spacer represented by: and (iv) the linker L 7 is an aminoalkanoic acid spacer according to the structure —N—(C x -alkylene)-C(O)—, where x is an integer ranging from 1 to 10; or the linker L 7 is an ethylene glycol spacer according to the structure —N—(CH 2 —CH 2 —O) e6 —(CH 2 ) e7 —C(O)—, where e6 is an integer ranging from 1 to 10 and e7 is an integer ranging from 1 to 3. method.

9. The method of claim 8, wherein the cycloaddition reaction is a [4+2] cycloaddition or a 1,3-dipolar cycloaddition.

10. 10. The method of claim 9, wherein Q comprises a terminal alkyne group or a cyclooctyne group.

11. 11. The method of claim 10, wherein Q comprises bicyclononyne (BCN), azadibenzocyclooctyne (DIBAC / DBCO), dibenzocyclooctyne (DIBO), or sulfonylated dibenzocyclooctyne (s-DIBO).

12. 9. The method of claim 8, wherein in step (a), a functionalized antibody according to structure (1') is obtained, V is the payload D, and step (b) is not performed.

13. 9. The method of claim 8, wherein in step (a), a functionalized antibody according to structure (1') is obtained, V is a reactive group Q', and step (b) is carried out.

14. Structure (2): 【Chemistry 11】 (In the formula, a, b and c are 1; L １ , L ２ and L ３ is a linker covalently connecting BM and Q or D; D is a payload, said payload being selected from the group consisting of a cytotoxin, an antiviral, an antibacterial, a peptide, and an oligonucleotide; BM is a branching moiety, said branching moiety being selected from a carbon atom or a nitrogen atom; Q comprises a cyclooctyne group; L 1 and L 2 are selected from linear or branched C 1 -C 20 alkylene groups, said alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR 3 , and R 3 is independently selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 2 -C 24 alkenyl groups, C 2 -C 24 alkynyl groups and C 3 -C 24 cycloalkyl groups, said alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted; L 3 is -(L 4 ) n -(L 5 ) o -(L 6 ) p -(L 7 ) q -, where L 4 , L 5 , L 6 and L 7 are linkers together forming linker L 3 ; n, o, p and q are independently 0 or 1; (a) the linker L 4 is represented by -(W) k1 -(A) d1 -(B) e1 -(A) f1 -(B) g1 -C(O)-; d1=0 or 1; e1=an integer ranging from 1 to 10; f1=0 or 1; g1=an integer ranging from 0 to 10; k1=0 or 1, provided that when k1=1, d1=0; A is structure (23) 【Chemistry 12】 wherein a1=0 or 1, R 13 is selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups, wherein said C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 14 , and R 14 is independently selected from the group consisting of hydrogen and C 1 -C 4 alkyl groups, or R 13 is D connected to N, possibly via a spacer moiety. is a sulfamide group according to B is —CH 2 —CH 2 —O— or —O—CH 2 —CH 2 —, or (B) e1 is —(CH 2 —CH 2 —O) e3 —CH 2 —CH 2 —, where e3 is defined in the same way as e1; W is —OC(O)—, —C(O)O—, —C(O)NH—, —NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)(CH 2 ) 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 ranging from 0 to 10; (b) the linker L 5 is a peptide spacer consisting of a peptide containing 2 to 5 amino acids; (c) the linker L 6 has the structure (25): 【Chemistry 13】 wherein R 3 is H, R 4 or C(O)R 4 , R 4 is an optionally substituted C 1 -C 24 (hetero)alkyl group, a C 3 -C 10 (hetero)cycloalkyl group, a C 2 -C 10 (hetero)aryl group, a C 3 -C 10 alkyl(hetero)aryl group and a C 3 -C 10 (hetero)arylalkyl group, optionally interrupted by one or more heteroatoms selected from O, S and NR 5 , and R 5 is independently selected from the group consisting of hydrogen and a C 1 -C 4 alkyl group. is a self-immolative spacer represented by: and (d) linker L 7 is an aminoalkanoic acid spacer according to the structure —N—(C x -alkylene)-C(O)—, where x is an integer ranging from 1 to 10; or linker L 7 is an ethylene glycol spacer according to the structure —N—(CH 2 —CH 2 —O) e6 —(CH 2 ) e7 —C(O)—, where e6 is an integer ranging from 1 to 10 and e7 is an integer ranging from 1 to 3. A compound having the formula:

15. 15. The compound of claim 14, wherein Q is bicyclononyne (BCN), azadibenzocyclooctyne (DIBAC / DBCO), dibenzocyclooctyne (DIBO), or sulfonylated dibenzocyclooctyne (s-DIBO).

16. L １ and L ２ The compound of claim 14, wherein:

17. A pharmaceutical composition comprising the antibody-payload conjugate of claim 1 and a pharmaceutically acceptable carrier.

18. 10. The antibody-payload conjugate of claim 1 for use in treating a subject in need thereof.

19. The antibody-payload conjugate of claim 1 for use in the treatment of cancer.

20. An antibody-payload conjugate as described in claim 18 or 19, wherein the conjugate does not bind to the Fc gamma receptor CD16.

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