Patent Application
20250090679
The content of the electronically submitted Sequence Listing (Name: “P6109508PCT.xml”; Size: 43,000 bytes; Date of Creation: Mar. 23, 2023) submitted in this application is incorporated herein by reference in its entirety.
The present invention is in the field of bioconjugation. More specifically, the present invention relates to antibody-drug conjugates for targeted treatment of patients with cancer, in particular Trop-2-expressing tumours.
A promising approach for targeted treatment of tumours entails the conjugation of a multitude (2 to 8) of highly toxic payloads to a monoclonal antibody, thereby generating an antibody-drug conjugate (ADCs). ADCs are well known in the art, as for example described by Chari et al., Angew. Chem. Int. Ed. 2014, 53, 3796 and Beck et al., Nat. Rev. Drug Discov. 2017, 16, 315-37. Mechanistically, the antibody is designed to bind with high specificity to tumour-associated receptor that is overexpressed versus healthy tissue. The ADC is thought to internalize into the tumour cell after binding to the receptor, then to release the toxic payload upon degradation of the antibody and/or the linker in the lysosome.
Current ADCs are commonly prepared by various conjugation technologies (summarized in
Conjugation through the glycan by an oxidation-ligation sequence is known in the art and has for example been described by Hamann et al. (Bioconjugate Chem. 2002, 13, 47-58). Chemoenzymatic conjugation through the glycan is known in the art and has been described for the use of sialyltransferase by Boons et al, Angew. Chem. Int. Ed. 2014, 53, 7179, and for the use of a mutant galactosyltransferase by Zhu et al, mAbs 2014, 6, 1 and Cook et al, Bioconjugate Chem. 2016, 27, 1789.
Chemoenzymatic conjugation through the glycan including first trimming of the glycan is known in the art and has been described by van Geel et al, Bioconjugate Chem. 2015, 26, 2233 and is schematically depicted in
Various cyclooctynes for application in metal-free click chemistry are known in the art (
A payload for an ADCs is typically a highly cytotoxic molecule, with IC50-value in low nanomolar or picomolar range, in particular low to medium molecular weight compounds (e.g. about 200 to about 2500 Da). Examples of suitable cytotoxin classes for ADCs include anthracyclines, camptothecins, taxanes, tubulysins, enediynes, inhibitory peptides, amanitins, duocarmycins, maytansinoids, auristatins, eribulins, hemiasterlins, BCL-XL inhibitors, KSP inhibitors, TLR agonists, indolinobenzodiazepine dimers or pyrrolobenzodiazepine dimers (PBDs) and analogues or prodrugs thereof. A representative set of cytotoxic molecules, and/or synthetic derivatives or prodrugs thereof, with suitable attachment point for conjugation to a monoclonal antibody, is depicted in
Specific examples of anthracyclins suitable of application in ADCs include (but are not limited to) doxorubicin, daunorubicin, nemorubicin and PNU-159,682.
Specific examples of camptothecins suitable for application in ADCs include (but are not limited to)SN-38, exatecan, exatecan-S, topotecan, silatecan, cositecan, lurtotecan, gimatecan, belotecan, rubitecan, AMDCPT, G-AMDCPT and other synthetic camptothecins the structures of which are depicted in
Specific examples of enediynes suitable for application in ADCs include (but are not limited to) calicheamicin, esperamicins, shishijimicins and namenamicins and other enediynes as summarized by Galm et al., Chem. Rev. 2005, 105, 739-758.
Specific examples of auristatins suitable for application in ADCs include (but are not limited to) MMAD, MMAE, MMAF and PF-06380101 and other auristatins as summarized by Maderna et al., Mol. Pharmaceutics 2015, 12, 1798-1812.
Proteins that have roles in breast cancer growth, differentiation, invasion and/or metastasis can influence the biological progress of tumours and can thus provide important prognostic information. One such candidate is Trop-2 (GA733-1, EGP-1), a 45 kDa monomeric trans-membrane glycoprotein that belongs to the TACSTD gene family, specifically TACSTD2, which is expressed in human epithelial cells at diverse stages of differentiation. Over-expression of Trop-2 has been demonstrated to be necessary and sufficient to stimulate tumour growth and has been linked to an overall poor prognosis. Expression of Trop-2 is associated with poor prognosis of several human cancers, including oral, pancreatic, gastric, ovarian, colorectal, breast and lung tumours. For example, Trop-2 overexpression was observed in 55% of pancreatic cancer patients studied, with a positive correlation with metastasis, tumour grade, and poor progression-free survival of patients who underwent surgery with curative intent. Likewise, in gastric cancer, 56% of patients may exhibit Trop-2 overexpression on their tumours, which again correlated with shorter disease-free survival and a poorer prognosis in those patients with lymph node involvement of Trop-2-positive tumour cells.
Given these characteristics and the fact that Trop-2 is linked to so many intractable cancers, Trop-2 is an attractive target for therapeutic intervention. Nevertheless, it has to be taken into consideration that Trop-2 is also expressed in some normal tissues, although usually at much lower intensities when compared to those in neoplastic tissue, and often in regions of the tissues with restricted vascular access.
Several monoclonal antibodies against Trop-2 have been established. Some anti-Trop-2 monoclonal antibodies such as 77220 are commercially available as reagents. Some of these established anti-Trop-2 monoclonal antibodies are being under investigation for treating cancers. Most of the anti-Trop-2 monoclonal antibodies at present available on the market, e.g. T16, have been generated using myeloma cell lines such as NS-1 or SP2-1 as fusion partners, which retains the expression of the parental immunoglobulin light chain. This caused these antibodies to be actually heterogeneous mixtures of antibodies with one, both or neither light chain directly participating in Trop-2 recognition.
WO 1997/14796 describes a monoclonal antibody, BR110, which is known to bind to Trop-2 on the cell surface and internalize within the cells. Patent applications WO 2003/074566, US 2004/001825, US 2007/212350 and US 2008/131363 and U.S. Pat. No. 10,179,171 B2 and EP3483183B1 teach RS7 antibodies and their uses in the treatment and diagnosis of tumours. These applications further relate to humanized, human and chimeric RS7 antigen binding proteins (hRS7, sacituzumab), and the use of such binding proteins in diagnosis and therapy. Anti-Trop-2 monoclonal antibody AR47A6.4.2 is disclosed in WO 2007/095748 and AR52A301.5 is disclosed in WO 2007/095749, both of which are antibodies that specifically injure Trop-2-expressing cancer cell as a target cell to exhibit effects. Patent application WO 2008/144891 teaches a humanized version of AR47A6.4.2 as anti-Trop-2 monoclonal antibody for the treatment of tumours. Patent application WO 2011/155579 (Sapporo) teaches a humanized monoclonal antibody AR47A6.4.2 or an antibody fragment thereof, which binds to the extracellular region of human Trop-2 with high affinity and exhibits high ADCC activity and high antitumor activity. Patent application WO2013/077458 and U.S. Pat. No. 9,427,464B2 (LivTech/Chiome) teaches anti-human Trop-2 antibodies with antitumor activity, in particular humanized antibodies including Huk5-70-2, especially having anti-tumour activity in vivo. Patent application WO 2013/068946 and U.S. Pat. No. 8,871,908B2 (Rinat/Pfizer) teaches antibody 7E6 and humanized h7E6 that specifically bind to trophoblast cell-surface antigen-2 (Trop-2). The invention further relates to therapeutic methods for use of these antibody conjugates for the treatment of a condition associated with Trop-2 expression (e.g., cancer), such as colon, esophageal, gastric, head and neck, lung, ovarian, or pancreatic cancer. U.S. Pat. No. 8,715,662 (Oncoxx) teaches antibody 2G10 that specifically binds to TROP-2.
ADCs targeting Trop-2 are known in the art and are at various stages of clinical development. IMMU-132 is an ADC derived from humanized antibody hRS7/sacituzumab, conjugated to campthothecin analogue SN-38 through an acid-cleavable linker CL2A, as disclosed in WO 2015/012904, and is currently in late-stage clinical development for treatment of a range of clinical indication, including (triple negative) breast cancer, small-cell lung cancer and pancreatic cancer. DS-1062a (datopotamab-deruxtecan) is an ADC derived from humanized antibody hTINA/datopotamab, conjugated to campthothecin analogue DXd through a protease-sensitive GGFG cleavable linker as disclosed in WO 2015/098099, U.S. Pat. No. 11,008,398B2 and EP3088419B1, is under clinical evaluation for treatment of solid tumours. Thirdly, PF-06664178 is an ADC derived from monoclonal antibody RN926, site-specifically conjugated to auristatin analogue PF-06380101 under the action of microbial transglutaminase. PF-06664178 has been evaluated in a phase I clinical study in patients with advanced or metastatic solid tumours, however the ADC showed toxicity at high dose levels with only modest antitumor activity, so development was discontinued.
The inventors have developed antibody-conjugates that are highly suitable for targeting Trop-2-expressing cells, in particular tumours. Thus, the antibody-conjugates according to the invention are highly suitable for treating Trop-2-positive cancer, especially breast cancer, colorectal cancer, pancreatic cancer, lung cancer, bladder cancer, head & neck cancer, ovarian cancer or esophageal cancer.
In a first aspect, the present invention concerns an antibody-conjugate. Related thereto, in a second aspect, the invention concerns a process for preparing the antibody-conjugate according to the invention. In a third aspect, the invention concerns a method for targeting Trop-2-expressing cells. Related thereto are the first medical use of the antibody-conjugate according to the invention, as well as the second medical use for the treatment of cancer. In a last aspect, the invention concerns the use of a mode of conjugation for increasing the therapeutic index of an antibody-conjugate in the treatment of Trop-2-expressing tumours.
The verb “to comprise”, and its conjugations, as used in this description and in the claims is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
A linker is herein defined as a moiety that connects (covalently links) two or more elements of a compound. A linker may comprise one or more spacer moieties. A spacer-moiety is herein defined as a moiety that spaces (i.e. provides distance between) and covalently links together two (or more) parts of a linker. The linker may be part of e.g. a linker-construct, a linker-conjugate, a linker-payload (e.g. linker-drug) or an antibody-conjugate, as defined below.
A “hydrophilic group” or “polar linker” is herein defined as any molecular structure containing one or more polar functional groups that imparts improved polarity, and therefore improved aqueous solubility, to the molecule it is attached to. Preferred hydrophilic groups are selected from a carboxylic acid group, an alcohol group, an ether group, a polyethylene glycol group, an amino group, an ammonium group, a sulfonate group, a phosphate group, an acyl sulfamide group or a carbamoyl sulfamide group. In addition to higher solubility other effects of the hydrophilic group include improved click conjugation efficiency, and, once incorporated into an antibody-drug conjugate: less aggregation, improved pharmacokinetics resulting in higher efficacy and in vivo tolerability.
The term “salt thereof” means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient. For example, in a salt of a compound the compound may be protonated by 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. The term “pharmaceutically accepted” salt means a salt that is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from 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 counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.
The term “enediyne” or “enediyne antibiotic” or “enediyne-containing cytotoxin” refers to any cytotoxin characterized by the presence of a 3-ene-1,5-diyne structural feature as part of a cyclic molecule as known in the art and include neocarzinostatin (NCS), C-1027, kedarcidin (KED), maduropeptin (MDP), N1999A2, the sporolides (SPO), the cyanosporasides (CYA and CYN), and the fijiolides, calicheamicins (CAL), the esperamicins (ESP), dynemicin (DYN), namenamicin, shishijimicin, and uncialamycin (UCM).
The term “alkylaminosugar” as used herein means a tetrahydropyranyl moiety connected to an alcohol function via its 2-position, thereby forming an acetal function, and further substituted by (at least) one N-alkylamino group in position 3, 4 or 5. “N-alkylamino group” in this context refers to an amino group having one methyl, ethyl or 2-propyl group.
The term “click probe” refers to a functional moiety that is capable of undergoing a click reaction, i.e. two compatible click probes mutually undergo a click reaction such that they are covalently linked in the product. Compatible probes for click reactions are known in the art, and preferably include (cyclic) alkynes and azides. In the context of the present invention, click probe Q in the compound according to the invention is capable of reacting with click probe F on the (modified) protein, such that upon the occurrence of a click reaction, a conjugate is formed wherein the protein is conjugated to the compound according to the invention. Herein, F and Q are compatible click probes.
An “acylsulfamide moiety” is herein defined as a sulfamide moiety (H2NSO2NH2) that is N-acylated or N-carbamoylated on one end of the molecule and N-alkylated (mono or bis) at the other end of the molecule. In the context of the present invention, especially in the examples, this group is also referred to as “HS”.
A “domain” may be any region of a protein, generally defined on the basis of sequence homologies and often related to a specific structural or functional entity. The term domain is used in this document to designate either individual Ig-like domains, such as “V-domain” or for groups of consecutive domains, such as “C2 type1-2 domain”.
A “coding sequence” or a sequence “encoding” an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.
The term “gene” means a DNA sequence that codes for, or corresponds to, a particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.
The term “glycoprotein” is herein used in its normal scientific meaning and refers to a protein comprising one or more monosaccharide or oligosaccharide chains (“glycans”) covalently bonded to the protein. A glycan may be attached to a hydroxyl group on the protein (O-linked-glycan), e.g. to the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline, or to an amide function on the protein (N-glycoprotein), e.g. asparagine or arginine, or to a carbon on the protein (C-glycoprotein), e.g. tryptophan. A glycoprotein may comprise more than one glycan, may comprise a combination of one or more monosaccharide and one or more oligosaccharide glycans, and may comprise a combination of N-linked, O-linked and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and therefore qualify as glycoprotein. Examples of glycoproteins include PSMA (prostate-specific membrane antigen), CAL (Candida antartica lipase), gp41, gp120, EPO (erythropoietin), antifreeze protein and antibodies.
The term “glycan” is herein used in its normal scientific meaning and refers to a monosaccharide or oligosaccharide chain that is linked to a protein. The term glycan thus refers to the carbohydrate-part of a glycoprotein. The glycan is attached to a protein via the C-1 carbon of one sugar, which may be without further substitution (monosaccharide) or may be further substituted at one or more of its hydroxyl groups (oligosaccharide). A naturally occurring glycan typically comprises 1 to about 10 saccharide moieties. However, when a longer saccharide chain is linked to a protein, said saccharide chain is herein also considered a glycan. A glycan of a glycoprotein may be a monosaccharide. Typically, a monosaccharide glycan of a glycoprotein consists of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently attached to the protein. A glycan may also be an oligosaccharide. An oligosaccharide chain of a glycoprotein may be linear or branched. In an oligosaccharide, the sugar that is directly attached to the protein is called the core sugar. In an oligosaccharide, a sugar that is not directly attached to the protein and is attached to at least two other sugars is called an internal sugar. In an oligosaccharide, a sugar that is not directly attached to the protein but to a single other sugar, i.e. carrying no further sugar substituents at one or more of its other hydroxyl groups, is called the terminal sugar. For the avoidance of doubt, there may exist multiple terminal sugars in an oligosaccharide of a glycoprotein, but only one core sugar. A glycan may be an O-linked glycan, an N-linked glycan or a C-linked glycan. In an O-linked glycan a monosaccharide or oligosaccharide glycan is bonded to an O-atom in an amino acid of the protein, typically via a hydroxyl group of serine (Ser) orthreonine (Thr). In an N-linked glycan a monosaccharide or oligosaccharide glycan is bonded to the protein via an N-atom in an amino acid of the protein, typically via an amide nitrogen in the side chain of asparagine (Asn) or arginine (Arg). In a C-linked glycan a monosaccharide or oligosaccharide glycan is bonded to a C-atom in an amino acid of the protein, typically to a C-atom of tryptophan (Trp).
The term “antibody” (AB) is herein used in its normal scientific meaning. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. An antibody is an example of a glycoprotein. The term antibody herein is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g. bispecific antibodies), antibody fragments, and double and single chain antibodies. The term “antibody” is herein also meant to include human antibodies, humanized antibodies, chimeric antibodies and antibodies specifically binding cancer antigen. The term “antibody” is meant to include whole antibodies, but also fragments of an antibody, for example an antibody Fab fragment, F(ab′)2, Fv fragment or Fc fragment from a cleaved antibody, a scFv-Fc fragment, a minibody, a diabody or a scFv. Furthermore, the term includes genetically engineered antibodies and derivatives of an antibody. Antibodies, fragments of antibodies and genetically engineered antibodies may be obtained by methods that are known in the art.
An antibody may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (I) and kappa (κ). The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass, or allotype (e.g. human G1m1, G1m2, G m3, non-G1 m1 [that, is any allotype other than G1 m1], G1m17, G2m23, G3m21, G3m28, G3m1.1, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2 m1, A2m2, Km1, Km2 and Km3) of immunoglobulin molecule. Preferred allotypes for administration include a non-G1 m1 allotype (nG1 m1), such as G1m17,1, G1m3, G1m3.1, G1m3.2 or G1m3.1.2. More preferably, the allotype is selected from the group consisting of the G1m17,1 or G1m3 allotype. The antibody may be engineered in the Fc-domain to enhance or nihilate binding to Fc-gamma receptors, as summarized by Saunders et al. Front. Immunol. 2019, 10, doi: 10.3389/fimmu.2019.01296 and Ward et al., Mol. Immunol. 2015, 67, 131-141. For example, the combination of Leu234Ala and Leu235Ala (commonly called LALA mutations) eliminate FcγRlla binding. Elimination of binding to Fc-gamma receptors can also be achieved by mutation of the N297 amino acid to any other amino acid except asparagine, by mutation of the T299 amino acid to any other amino acid except threonine or serine, or by enzymatic deglycosylation or trimming of the fully glycosylated antibody with for example PNGase F or an endoglycosidase. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin. Each chain contains distinct sequence domains.
A percentage of “sequence identity” may be determined by comparing the two sequences, optimally aligned over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. A sequence “at least 85% identical to a reference sequence” is a sequence having, on its entire length, 85%, or more, for instance 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of the reference sequence.
The term “CDR” refers to complementarity-determining region: the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs therefore refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. A conventional antibody antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. “CDR”
The term “monoclonal antibody” or “mAb” as used herein refers to an antibody molecule of a single amino acid sequence, which is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e. produced by protein engineering.
The term “chimeric antibody” refers to an engineered antibody which, in its broadest sense, contains one or more regions from one antibody and one or more regions from one or more other antibodies. In an embodiment, a chimeric antibody comprises a VH domain and a VL domain of an antibody derived from a non-human animal, in association with a CH domain and a CL domain of another antibody, in an embodiment, a human antibody. As the non-human animal, any animal such as mouse, rat, hamster, rabbit or the like can be used. A chimeric antibody may also denote a multispecific antibody having specificity for at least two different antigens.
The term “humanised antibody” refers to an antibody which is wholly or partially of non-human origin and which has been modified to replace certain amino acids, for instance in the framework regions of the VH and VL domains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most of the time human CH and CL domains. “Fragments” of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab′)2, Fab′, dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.
In a first aspect, the invention concerns antibody-conjugates of general structure (1)
AB-[(L6)b-{Z-L-D}x]y (1)
wherein:
Also contemplated within the present invention are salts, preferably pharmaceutically acceptable salts, of the antibody-conjugate according to structure (1).
In a second aspect, the invention concerns a process for preparing the antibody-conjugate according to the invention, comprising reacting the compound according to general structure (2) with an antibody (3). The compound according to general structure (2) comprises a reactive moiety Q and the antibody a reactive moiety F which is capable of reacting with Q in a conjugation reaction, wherein Q and F react to form connecting group Z. In this reaction, a conjugate according to general structure (1) is formed. The process according to this aspect this concerns the following bioconjugation reaction:
Here below, the antibody-conjugate according to structure (1) is first defined. The structural features of the antibody-conjugate according to structure (1) also apply to the compound according to structure (2) and the antibody according to structure (3), as those are unchanged in the conjugation reaction except for reactive moieties F and Q, which are transformed into connecting group Z upon reaction of the compound according to structure (2) with an antibody according to structure (3).
In a third aspect, the invention concerns the application antibody-conjugate according to structure (1), for targeting Trop-2-expressing cells. Related thereto, the invention concerns the first medical use and second medical use of the antibody-conjugate according to structure (1).
As will be understood by the skilled person, the definition of the chemical moieties, as well as their preferred embodiments, apply to all aspects of the invention.
In a first aspect, the invention concerns antibody-conjugates of general structure (1):
AB-[(L6)b-{Z-L-D}x]y (1)
wherein:
The antibody-conjugate according to the invention contains an antibody that is capable of targeting Trop-2-expressing cells, in particular tumour cells. Trop-2 is a known target for cancer treatment. The term “expressing” is used as common in the art, and refers to overexpression of the target with respect to the expression in healthy tissue. Antibodies capable of targeting Trop-2-expressing tumours may also be referred to as anti-Trop-2 antibodies, Trop-2-targeting antibodies or Trop-2-binding antibodies. Anti-Trop-2antibodies selectively bind to Trop-2-expressing cells.
Anti-Trop-2 antibodies are known in the art, and any suitable one can be used in the context of the present invention. Preferred antibodies are selected from the list consisting of Huk5-70-2, hRS7, hTINA, AR47A6.4.2, RN926. Most preferably, the antibody is hRS7.
Herein, the Fc regions of these antibodies may have one or more mutations, such as 0-10 mutations or 0-5 mutations. Especially preferred are mutations that change binding to the FcRn receptor to modulate half-life of the antibody. For example, inclusion of mutations Met252Tyr, Ser254Thr, and Thr256Glu, often called YTE, in the IgG1 Fc results in a ˜11-fold higher binding of the antibody to human FcRn, thereby increasing the circulation half-life with a factor ˜3.5. Therefore, in one embodiment, the antibody has an apparent human FcRn binding affinity KD,app of below 2.5×10−6 M, preferably in the range of 0.05-0.99×10−6 M, more preferably in the range of 0.1-0.49×10−6 M, most preferably in the range of 0.2-0.4×10−6 M. The apparent binding affinity KD,app may be determined according to Mackness et al. MABS, 2019, 11(7), 1276-1288. In a preferred embodiment, the antibody AB is the YTE mutant of the preferred antibodies defined here above or below.
hRS7 is also known as sacituzumab, and may also be defined as containing a light chain sequence according to SEQ ID No. 3 and a heavy chain sequence according to SEQ ID No. 4, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably with exatecan as payload D
hTINA is also known as datopotamab, and may also be defined as containing a light chain sequence according to SEQ ID No. 7 and a heavy chain sequence according to SEQ ID No. 8, wherein the sequence identity is at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100%. In a particularly preferred embodiment, the antibody according to this embodiment is combined with a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan and DXd, more preferably with a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably with exatecan as payload D
Alternatively, the antibody is defined by its VL and VH domains, which together form the variable domain that binds to the antigen. Thus, in a preferred embodiment, the antibody contains a VL domain selected from the group consisting of SEQ ID No. 1, 5, 9, 13, 19, 21 and 23, and a VH domain selected from the group consisting of SEQ ID No. 2, 6, 10, 14, 15, 20, 22 and 24, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a further preferred embodiment, the antibody contains a VL domain of SEQ ID No. 1 or 5, and a VH domain of SEQ ID No. 2 or 6, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a especially preferred embodiment, the antibody contains a VL domain of SEQ ID No. 1, and a VH domain of SEQ ID No. 2, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%.
The aforementioned sequence identities refer to the complete sequence of the VL and VH domains. While the entire sequences of these domains allow for some variation in the sequence without jeopardizing the binding to Trop-2, it is preferred that the complementarity-determining regions (CDRs) have a higher sequence identity, to ensure that the binding to Trop-2 is not significantly jeopardized. The location of the CDRs is given in the tables below. Thus, it is preferred that the antibody contains a VL domain selected from the group consisting of SEQ ID No. 1, 5, 9, 13, 19, 21 and 23, and a VH domain selected from the group consisting of SEQ ID No. 2, 6, 10, 14, 15, 20, 22 and 24, preferably a VL domain of SEQ ID No. 1 or 5, and a VH domain of SEQ ID No. 2 or 6, more preferably a VL domain of SEQ ID No. 1, and a VH domain of SEQ ID No. 2, wherein the sequence identity of the CDR is at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100%. The skilled person understands that the VL and VH domains identified above can be combined with a suitable constant domain to form a complete antibody.
In an especially preferred embodiment, the antibody contains a VL domain of SEQ ID No. 1 and a VH domain of SEQ ID No. 2, or a VL domain of SEQ ID No. 5 and a VH domain of SEQ ID No. 6, or a VL domain of SEQ ID No. 9 and a VH domain of SEQ ID No. 10, or a VL domain of SEQ ID No. 13 and a VH domain of SEQ ID No. 14 or 15, or a VL domain of SEQ ID No. 19 and a VH domain of SEQ ID No. 20, or a VL domain of SEQ ID No. 21 and a VH domain of SEQ ID No. 22, or a VL domain of SEQ ID No. 23 and a VH domain of SEQ ID No. 24. Herein, the sequence identities as defined above for the complete sequence as well as for the CDR apply. Most preferably, the antibody contains a VL domain of SEQ ID No. 1 and a VH domain of SEQ ID No. 2.
Alternatively, the antibody is defined by its light and heavy chains, which together form the antibody. Thus, in a preferred embodiment, the antibody contains a light chain selected from the group consisting of SEQ ID No. 3, 7, 11 and 16, and a heavy chain selected from the group consisting of SEQ ID No. 4, 8, 12, 17 and 18, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a more preferred embodiment, the antibody contains a light chain of SEQ ID No. 3 or 7, and a heavy chain selected from the group consisting of SEQ ID No. 4 or 8, wherein the sequence identity is at least 70%, preferably at least 75% or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%. In a most preferred embodiment, the antibody contains a light chain of SEQ ID No. 3, and a heavy chain selected from the group consisting of SEQ ID No. 4, wherein the sequence identity is at least 70%, preferably at least 75 % or at least 80%, more preferably at least 85% or at least 90% or at least 95%, most preferably at least 99% or even 100%.
The aforementioned sequence identities refer to the complete sequence of the light and heavy chains. While the entire sequences of these chains allow for some variation in the sequence without jeopardizing the binding to Trop-2, it is preferred that the CDRs have a higher sequence identity, to ensure that the binding to Trop-2 is not significantly jeopardized. The location of the CDRs is given in the tables below. Thus, it is preferred that the antibody contains a light chain selected from the group consisting of SEQ ID No. 3, 7, 11 and 16, and a heavy chain selected from the group consisting of SEQ ID No. 4, 8, 12, 17 and 18, wherein the sequence identity of the CDR is at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100%.
In an especially preferred embodiment, the antibody contains a light chain of SEQ ID No. 3 and a heavy chain of SEQ ID No. 4, or a light chain of SEQ ID No. 7 and a heavy chain of SEQ ID No. 8, or a light chain of SEQ ID No. 11 and a heavy chain of SEQ ID No. 12, or a light chain of SEQ ID No. 16 and a heavy chain of SEQ ID No. 17 or 18. Herein, the sequence identities as defined above for the complete sequence as well as for the CDR apply. Most preferably, the antibody contains a light chain of SEQ ID No. 3 and a heavy chain of SEQ ID No. 4, wherein the sequence identities as defined above for the complete sequence as well as for the CDR apply. Especially preferred is this antibody with exatecan as payload D.
In case reactive group F is directly connected to the antibody, or even part of the antibody structure, linker L6 that connects AB to F (for antibodies of structure (3)) or AB to Z (for conjugates of structure (1) is absent and b=0. This is for example the case for cysteine conjugation and lysine conjugation. Alternatively, reactive group F may also be introduced onto the antibody using a linker L6 that connects AB to F (for antibodies of structure (3)) or AB to Z (for conjugates of structure (1), in which case L6 is present and b=1. In case L6 is present, reactive group F is typically introduced at the glycan of the antibody. This is for example the case for conjugation via an artificially introduced reactive group F, such as for example using transglutaminase or by enzymatic glycan modification (e.g. glycosyltransferase or a-1,3-mannosyl-glycoprotein-2-b-N-acetylglucosaminyl-transferase). For example, a modified sugar residue S(F)x may be introduced at the glycan, extending the glycan with one monosaccharide residue S, which introduces x reactive groups F on the glycan of an antibody. In a most preferred embodiment, conjugation occurs via the glycan of the antibody and b=1. The site of conjugation is preferably at the heavy chain of the antibody.
If present, L6 is a linker that links AB to F or Z, and is represented by -GlcNAc(Fuc)w-(G)j-S-(L7)w′-, wherein G is a monosaccharide, j is an integer in the range of 0-10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1, w′ is 0, 1 or 2 and L7 is —N(H)C(O)CH2—, —N(H)C(O)CF2— or —CH2—. Typically, L6 is at least partly formed by the glycan of the antibody. All recombinant antibodies, generated in mammalian host systems, contain the conserved N-glycosylation site at the asparagine residue at or close to position 297 of the heavy chain, which is modified by a glycan of the complex type. This naturally occurring glycosylation site of antibodies is preferably used, but other glycosylation sites, including artificially introduced ones, may also be used for the connection of linker L6. Thus, in a preferred embodiment, L6 is connected to an amino acid of the antibody which is located at a position in the range of 250-350 of the heavy chain, preferably in the range of 280-310 of the heavy chain, more preferably in the range of 295-300 of the heavy chain, most preferably at position 297 of the heavy chain.
The -GlcNAc(Fuc)w-(G)j- of L6 is the glycan of the antibody, or part thereof. The -GlcNAc(Fuc)w-(G)j- of the glycan thus typically originates from the original antibody, wherein GlcNAc is an N-acetylglucosamine moiety and Fuc is a fucose moiety. Fuc is typically bound to GlcNAc via an α-1,6-glycosidic bond. Normally, antibodies may (w=1) or may not be fucosylated (w=0). In the context of the present invention, the presence of a fucosyl moiety is irrelevant, and similar effects are obtained with fucosylated (w=1) and non-fucosylated (w=0) antibody conjugates. The GlcNAc residue may also be referred to as the core-GlcNAc residue and is the monosaccharide that is directly attached to the peptide part of the antibody.
S may be directly connected to the core-GlcNAc(Fuc)w moiety, i.e. j=0, meaning that the remainder of the glycan is removed from the core-GlcNAc(Fuc)w moiety before S is attached. Such trimming of glycans is well-known in the art and can be achieved by the action of an endoglycosidase. Alternatively, there are one or more monosaccharide residues present in between the core-GlcNAc(Fuc)w moiety and S, i.e. j is an integer in the range of 1-10, preferably j=2-5. In a preferred embodiment, (G)j is an oligosaccharide fraction comprising j monosaccharide residues G, wherein j is an integer in the range of 2-5. (G)j is connected to the GlcNAc moiety of GlcNAc(Fuc)w, typically via a β-1,4 bond. In a preferred embodiment, j is 3, 4 or 5. Although any monosaccharide that may be present in a glycan may be employed as G, each G is preferably individually selected from the group consisting of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose and N-acetylneuraminic acid. More preferred options for G are galactose, N-acetylglucosamine, mannose. The inventors found that antibody-conjugates having j below 4 show no or hardly any binding to the Fc-gamma receptor, while antibody conjugates having j in the range of 4-10 do bind to the Fc-gamma receptor. Thus, by selecting a certain value for j, the desired extent of binding to the Fc-gamma receptor can be obtained. It is thus preferred that j=0, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably j=0, 3, 4 or 5, most preferably the antibody is trimmed and j=0.
S is a sugar or sugar derivative. The term “sugar derivative” is herein used to indicate a derivative of a monosaccharide sugar, i.e. a monosaccharide sugar comprising substituents and/or functional groups. Suitable examples for S include glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), amino sugars and sugar acids, e.g. glucosamine (GlcNH2), galactosamine (GalNH2) N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referred to as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA). Preferably, S is selected from Glc, Gal, GlcNAc and GalNAc. In an especially preferred embodiment, S is GalNAc.
x is an integer that denotes the number of connecting groups Z (for conjugate (I)) or reactive groups F (for antibody (3)) that are attached to sugar (derivative) S. Thus, the antibody according to the invention contains a moiety S comprising x reactive moieties F. Each of these reactive moieties F are reacted with a reactive moiety Q of the compound according to general structure (2), such that x connecting groups Z are formed and x compounds according to general structure (2) are attached to a single occurrence of S. x is 1 or 2, preferably x=1.
Connecting group Z (for conjugate (I)) or reactive group F (for antibody (3)) may be attached directly to S, or there may be a linker L7 present in between S and Z or F. L7 is a linker that links S with Z. L7 may be present (w′=1 or 2) or absent (w′=0). Typically, each moiety Z may be connected to S via a linker L7, thus in one embodiment w′=0 of x. Preferably, L7 is absent and each connecting moiety Z is directly attached to S. If present, L7 may be selected from —N(H)C(O)CH2—, —N(H)C(O)CF2— or —CH2—. In a preferred embodiment, x=1 and w′=0 or 1, most preferably x=1 and w′=0.
y is an integer that denotes the number of sugar(s) (derivative(s)) S, each having x reactive groups F or connected to x connecting groups Z, that are connected to the antibody. y is 1, 2, 3 or 4, preferably y=2 or 4, most preferably y=2. Thus, the antibody contains y moieties S, each of which comprises x reactive moieties F. Each of these reactive moieties F are reacted with reactive moiety Q of the compound according to general structure (2), such that x+y connecting groups Z are formed and x+y compounds according to general structure (2) are attached to a single antibody. Each compound according to general structure (2) may contain multiple payloads, e.g. by virtue of branching nitrogen atom N* in L. It is preferred that each compound according to general structure (2) contains 1 or 2 occurrences of D, most preferably 2 occurrences of D. In an especially preferred embodiment, linker L1 contains a branching nitrogen atom N* to which a second occurrence of D is connected.
The amount of payload (D) molecules attached to a single antibody is known in the art as the DAR (drug-antibody ratio). In the context of the present invention, it is preferred that DAR is an integer in the range 1-8, more preferably 2 or 4, most preferably DAR=4. Alternatively worded, the DAR is preferably an integer in the range (x+y) to [(x+y)×2], most preferably DAR=[(x+y)×2]. With preferred values for x of 1 and y of 2, the DAR is preferably 4. It will be appreciated that these are theoretical DAR values, and in practice the DAR may slightly deviate from this value, by virtue of incomplete conjugation. Typically, the conjugates are obtained as a stochastic mixture of antibody-drug conjugates, with DAR values varying between individual conjugates, and depending on the conjugation technique used the DAR may have a broad distribution (e.g. DAR=0-10) or a narrow distribution (e.g. DAR=3-4). In case of such mixture, DAR often refers to the average DAR of the mixture. This is well-known in the art of bioconjugation. However, in case the conjugation occurs via the glycan (i.e. b=1 and L6 is present), the antibody-conjugates according to the invention have a close-to-theoretical DAR. For example, when the theoretical DAR is 4, DAR values above 3.6 or even above 3.8 are readily obtained, indicating that most antibodies in the reaction mixture have reacted completely and have a DAR of 4.
Z is a connecting group, which covalently connects both parts of the conjugate according to the invention. The term “connecting group” herein refers to the structural element, resulting from the reaction between Q and F, connecting one part of the conjugate with another part of the same conjugate. As will be understood by the person skilled in the art, the nature of a connecting group depends on the type of reaction with which the connection between the parts of said compound is obtained. As an example, when the carboxyl group of R—C(O)—OH is reacted with the amino group of H2N—R′ to form R—C(O)—N(H)—R′, R is connected to R′ via connecting group Z, and Z may be represented by the group —C(O)—N(H)—. Since connecting group Z originates from the reaction between Q and F, it can take any form.
Since more than one reactive moiety F can be present or introduced in an antibody, the antibody-conjugate according to the present invention may contain per biomolecule more than one payload D, such as 1-8 payloads D, preferably 1, 2, 3 or 4 payloads D, more preferably 2 or 4 payloads D. The number of payloads is typically an even integer, in view of the symmetric nature of antibodies. In other words, when one side of the antibody is functionalized with F, the symmetrical counterpart will also be functionalized. Alternatively, in case naturally occurring thiol groups of the cysteine residues of a protein are used as F, the value of m can be anything and may vary between individual conjugates.
In a compound according to structure (1), connecting group Z connects D via linker L to AB, optionally via L6. Numerous reactions are known in the art for the attachment of a reactive group Q to a reactive group F. Consequently, a wide variety of connecting groups Z may be present in the conjugate according to the invention. In one embodiment, the reactive group Q is selected from the options described above, preferably as depicted in
For example, when F comprises or is a thiol group, complementary groups Q include N-maleimidyl groups and alkenyl groups, and the corresponding connecting groups Z are as shown in
For example, when F comprises or is an amino group, complementary groups Q include ketone groups and activated ester groups, and the corresponding connecting groups Z are as shown in
For example, when F comprises or is a ketone group, complementary groups Q include (O-alkyl)hydroxylamino groups and hydrazine groups, and the corresponding connecting groups Z are as shown in
For example, when F comprises or is an alkynyl group, complementary groups Q include azido groups, and the corresponding connecting group Z is as shown in
For example, when F comprises or is an azido group, complementary groups Q include alkynyl groups, and the corresponding connecting group Z is as shown in
For example, when F comprises or is a cyclopropenyl group, a trans-cyclooctene group or a cyclooctyne group, complementary groups Q include tetrazinyl groups, and the corresponding connecting group Z is as shown in
Additional suitable combinations of F and Q, and the nature of resulting connecting group Z3 are known to a person skilled in the art, and are e.g. described in G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd Ed. 2013 (ISBN:978-0-12-382239-0), in particular in Chapter 3, pages 229-258, incorporated by reference. A list of complementary reactive groups suitable for bioconjugation processes is disclosed in Table 3.1, pages 230-232 of Chapter 3 of G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd Ed. 2013 (ISBN:978-0-12-382239-0), and the content of this Table is expressly incorporated by reference herein.
In a preferred embodiment, connecting group Z is obtained by a cycloaddition or a nucleophilic reaction, preferably wherein the cycloaddition is a [4+2]cycloaddition or a 1,3-dipolar cycloaddition or the nucleophilic reaction is a Michael addition or a nucleophilic substitution. Such a cycloaddition or nucleophilic reaction occurs via a reactive group F, connected to S, and reactive group Q, connected to D via L. Conjugation reactions via cycloadditions or nucleophilic reactions are known to the skilled person, and the skilled person is capable of selecting appropriate reaction partners F and Q, and will understand the nature of the resulting connecting group Z.
In a first preferred embodiment, Z is formed by a cycloaddition. Preferred cycloadditions are a (4+2)-cycloaddition (e.g. a Diels-Alder reaction) or a (3+2)-cycloaddition (e.g. a 1,3-dipolar cycloaddition). Preferably, the conjugation is the Diels-Alder reaction or the 1,3-dipolar cycloaddition. The preferred Diels-Alder reaction is the inverse-electron demand Diels-Alder cycloaddition. In another preferred embodiment, the 1,3-dipolar cycloaddition is used, more preferably the alkyne-azide cycloaddition, and most preferably wherein Q is or comprises an alkyne group and F is an azido group. Cycloadditions, such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art, and the skilled person knowns how to perform them.
Preferably, Z contains a moiety selected from the group consisting of a triazole, a cyclohexene, a cyclohexadiene, a [2.2.2]-bicyclooctadiene, a [2.2.2]-bicyclooctene, an isoxazoline, an isoxazolidine, a pyrazoline, a piperazine, a thioether, an amide or an imide group. Triazole moieties are especially preferred to be present in Z. In one embodiment, Z comprises a (hetero)cycloalkene moiety, i.e. formed from Q comprising a (hetero)cycloalkyne moiety. In an alternative embodiment, Z comprises a (hetero)cycloalkane moiety, i.e. formed from Q comprising a (hetero)cycloalkene moiety. In a preferred embodiment, Z has the structure (Z1):
Herein, the bond depicted as is a single bond or a double bond. Furthermore:
In case the bond depicted as is a double bond, it is preferred that u+u′=4, 5, 6, 7 or 8. Preferably, the wavy bond labelled with * is connected to S and the wavy bond labelled with ** is connected to L.
It is especially preferred that Z comprises a (hetero)cycloalkene moiety, i.e. the bond depicted as is a double bond. In a preferred embodiment, Z is selected from the structures (Z2)-(Z20), depicted here below:
Herein, the connection to L is depicted with the wavy bond. B(−) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is formed by the cycloaddition reaction, and preferably is a triazole, a cyclohexene, a cyclohexadiene, a [2.2.2]-bicyclooctadiene, a [2.2.2]-bicyclooctene, an isoxazoline, an isoxazolidine, a pyrazoline or a piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have the structure selected from (Za)-(Zm) depicted below, wherein the carbon atoms labelled with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring of (Z2)-(Z20) to which ring Z is fused. Preferred rings Z are selected from (Za)-(Zj), more preferably from (Za), (Zd) and (Zh), most preferably ring Z has structure (Za). Since the connecting group Z is formed by reaction with a (hetero)cycloalkyne in the context of the present embodiment, the bound depicted above as is a double bond.
In a further preferred embodiment, Z is selected from the structures (Z21)-(Z38) and (Z8a), depicted here below:
Herein, the connection to L is depicted with the wavy bond. In structure (Z38), B(−) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is selected from structures (Za)-(Zm), preferably from structures (Za)-(Zj), as defined above.
In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety or a (hetero)cycloheptene moiety, preferably according to structure (Z8), (Z26), (Z27), (Z28) or (Z37) or (Z38a), more preferably according to structure (Z8), (Z26), (Z27), (Z28) or (Z37), which are optionally substituted. Each of these preferred options for Z are further defined here below.
Thus, in a preferred embodiment, Z comprises a heterocycloheptene moiety according to structure (Z37), which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Z37) is not substituted.
In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety according to structure (Z8), more preferably according to (Z29), which is optionally substituted. Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is not substituted. In the context of the present embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z39) as shown below, wherein V is (CH2)I and I is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, I is 0, 1, 2, 3 or 4, more preferably I is 0, 1 or 2 and most preferably I is 0 or 1. In the context of group (Z39), I is most preferably 1. Most preferably, Z is according to structure (Z42), defined further below.
In an alternative preferred embodiment, Z comprises a (hetero)cyclooctene moiety according to structure (Z26), (Z27) or (Z28), which are optionally substituted. In the context of the present embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z40) or (Z41) as shown below, wherein Y1 is O or NR11, wherein R11 is independently selected from the group consisting of hydrogen, a linear or branched C1-C12 alkyl group or a C4-C12 (hetero)aryl group. The aromatic rings in (Z40) are optionally O-sulfonylated at one or more positions, whereas the rings of (Z41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctene moiety according to structure (Z40) or (Z41) is not further substituted. Most preferably, Z is according to structure (Z43), defined further below.
In an alternative preferred embodiment, Z comprises a heterocycloheptenyl group and is according to structure (Z37).
In an especially preferred embodiment, Z comprises a cyclooctenyl group and is according to structure (Z42):
In a preferred embodiment of the group according to structure (Z42), R15 is independently selected from the group consisting of hydrogen, halogen, —OR16, C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R16 is hydrogen or C1-C6 alkyl, more preferably R15 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R15 are H. In a preferred embodiment of the group according to structure (Z42), R18 is independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, most preferably both R18 are H. In a preferred embodiment of the group according to structure (Z42), R19 is H. In a preferred embodiment of the group according to structure (Z42), I is 0 or 1, more preferably I is 1.
In an especially preferred embodiment, Z comprises a (hetero)cyclooctynyl group and is according to structure (Z43):
In a preferred embodiment of the group according to structure (Z43), R15 is independently selected from the group consisting of hydrogen, halogen, —OR16, —S(O)3(−), C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R16 is hydrogen or C1-C6 alkyl, more preferably R15 is independently selected from the group consisting of hydrogen and —S(O)3(−). In a preferred embodiment of the group according to structure (Z43), Y is N or CH, more preferably Y═N.
In an especially preferred embodiment, Z comprises a heterocycloheptynyl group and is according to structure (Z37) or (Z38a), preferably according to structure (Z37), wherein ring Z is a triazole ring:
In an alternative preferred embodiment, Z comprises a (hetero)cycloalkane moiety, i.e. the bond depicted as is a single bond. The (hetero)cycloalkane group may also be referred to as a heterocycloalkanyl group or a cycloalkanyl group, preferably a cycloalkanyl group, wherein the (hetero)cycloalkanyl group is optionally substituted. Preferably, the (hetero)cycloalkanyl group is a (hetero)cyclopropanyl group, a (hetero)cyclobutanyl group, a norbornane group, a norbornene group, a (hetero)cycloheptanyl group, a (hetero)cyclooctanyl group, a (hetero)cyclononnyl group or a (hetero)cyclodecanyl group, which may all optionally be substituted. Especially preferred are (hetero)cyclopropanyl groups, (hetero)cycloheptanyl group or (hetero)cyclooctanyl groups, wherein the (hetero)cyclopropanyl group, the trans-(hetero)cycloheptanyl group or the (hetero)cyclooctanyl group is optionally substituted. Preferably, Z comprises a cyclopropanyl moiety according to structure (Z44), a hetereocyclobutane moiety according to structure (Z45), a norbornane or norbornene group according to structure (Z46), a (hetero)cycloheptanyl moiety according to structure (Z47) or a (hetero)cyclooctanyl moiety according to structure (Z48). Herein, Y3 is selected from C(R23)2, NR23 or O, wherein each R23 is individually hydrogen, C1-C6 alkyl or is connected to L, optionally via a spacer, and the bond labelled
is a single or double bond. In a further preferred embodiment, the cyclopropanyl group is according to structure (Z49). In another preferred embodiment, the (hetero)cycloheptane group is according to structure (Z50) or (Z51). In another preferred embodiment, the (hetero)cyclooctane group is according to structure (Z52), (Z53), (Z54), (Z55) or (Z56).
Herein, the R group(s) on Si in (Z50) and (Z51) are typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is typically selected from structures (Zn)-(Zu), wherein the carbon atoms labelled with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring of (Z44)-(Z56) to which ring Z is fused, and the carbon a carbon labelled with * is directly connected to the peptide chain of the antibody. Preferred rings Z are selected from (Zo)-(Zr). Since the connecting group Z is formed by reaction with a (hetero)cycloalkene in the context of the present embodiment, the bound depicted above as is a single bond.
In a second preferred embodiment, Z is formed by a nucleophilic reaction, preferably by a nucleophilic substitution or a Michael addition, preferably by a Michael addition. A preferred Michael reaction is the thiol-maleimide ligation, most preferably wherein Q is maleimide and F is a thiol group. Preferably, the thiol is present in the sidechain of a cysteine residue. In a preferred embodiment, connection group Z comprises a succinimidyl ring or its ring-opened succinic acid amide derivative. Preferred options for connection group Z comprise a moiety selected from (Z57)-(Z71) depicted here below.
Herein, the wavy bond(s) labelled with an * is connected to the antibody Ab, optionally via a linker, and the wavy bond without label to the payload, optionally via a linker. In addition, R29 is C1-12 alkyl, preferably C1-4 alkyl, most preferably ethyl, and X1 is O or S, preferably X1═O. The nitrogen atom labelled with ** in (Z67)-(Z71) corresponds to the nitrogen atom of the side chain of a lysine residue of the antibody. The carbon atoms of the phenyl group of (Z69) and (Z70) are optionally substituted, preferably optionally fluorinated.
In a preferred embodiment, connection group Z comprise a moiety selected from (Z1)-(Z71).
Linker L connects payload D with connecting group Z (in the conjugate according to structure (1)) or connects payload D with reactive group Q (in the compound according to structure (2)). Linkers are known in the art and may be cleavable or non-cleavable. Linker L preferably contains a self-immolative group or cleavable linker, comprising a peptide spacer and a para-aminobenzyloxycarbonyl (PABC) moiety or derivative thereof.
In a preferred embodiment, linker L as the structure -(L1)n-(L2)o-(L3)p-(L4)q-, wherein (L4)q is connect to payload D and (L1)n is connected to Z or Q. Herein L1, L2, L3 and L4 are linkers or linking units and each of n, o, p and q are individually 0 or 1, wherein n+o+p+q is at least 1. In a preferred embodiment, at least linkers L1 and L2 are present (i.e. n=1; o=1; p=0 or 1; q=0 or 1), more preferably linkers L1, L2 and L3 are present and L4 is either present or not (i.e. n=1; o=1; p=1; q=0 or 1). In one embodiment, linkers L1, L2, L3 and L4 are present (i.e. n=1; o=1; p=1; q=1). In one embodiment, linkers L1, L2 and L3 are present and L4 is not (i.e. n=1; o=1; p=1; q=0).
A linker, especially linker L1, may contain one or more branch-points for attachment of multiple payloads to a single connecting group. In a preferred embodiment, the linker of the conjugate according to the invention contains a branching moiety. A “branching moiety” in the context of the present invention refers to a moiety that is embedded in a linker connecting three moieties. In other words, the branching moiety comprises at least three bonds to other moieties, typically one bond connecting to Z or Q, one bond to the payload D and one bond to a second payload D. The branching moiety, if present, is preferably embedded in linker L1, more preferably part of Sp2 or as the nitrogen atom of NR13. Any moiety that contains at least three bonds to other moieties is suitable as branching moiety in the context of the present invention. In a preferred embodiment, the branching moiety is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cycle or a polycyclic moiety. Most preferably, the branching moiety is a nitrogen atom.
Linker L1 is either absent (n=0) or present (n=1). Preferably, linker L1 is present and n=1. L1 may for example be selected from the group consisting of linear or branched C1-C200 alkylene groups, C2-C200 alkenylene groups, C2-C200 alkynylene groups, C3-C200 cycloalkylene groups, C5-C200 cycloalkenylene groups, C8-C200 cycloalkynylene groups, C7-C200 alkylarylene groups, C7-C200 arylalkylene groups, C8-C200 arylalkenylene groups, C9-C200 arylalkynylene groups. Optionally the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups may be substituted, and optionally said groups may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, said heteroatoms preferably being selected from the group consisting of O, S(O)y′ and NR21, wherein y′ is 0, 1 or 2, preferably y′=2, and R21 is independently selected from the group consisting of hydrogen, halogen, C1-C24 alkyl groups, C6-C24 (hetero)aryl groups, C7-C24 alkyl(hetero)aryl groups and C7-C24 (hetero)arylalkyl groups.
In a preferred embodiment, linker L1 contains a polar group. Such a polar group may be selected from (poly)ethylene glycoldiamines (e.g. 1,8-diamino-3,6-dioxaoctane or equivalents comprising longer ethylene glycol chains), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains and 1,z′-diaminoalkanes (wherein z′ is the number of carbon atoms in the alkane, preferably z′=1-10), —(O)a—C(O)—NH—S(O)2—NR13— (as further defined below, see structure (23)), —C(S(O)3(−))—, —C(C(O)2(−))—, —S(O)2—, —P(O)2(−)—, —O(CH2CH2O)t—, —NR30(CH2CH2NR30)t—, and the following two structures:
The polar group may also contain an amino acid, preferably selected from Arg, Glu, Asp, Ser and Thr. Herein, a and R13 are further defined below for structure (23). t is an integer in the range of integer in the range of 0-15, preferably 1-10, more preferably 2-5, most preferably t=2 or 4. Each R30 is individually H, C1-12 alkyl, C1-12 aryl, C1-12 alkaryl or C1-12 aralkyl. Linker L1 may contain more than one such polar group, such as at least two polar groups. The polar group may also be present in a branch of linker L1, which branches off a branching moiety as defined elsewhere. Preferable, a nitrogen or carbon atom is used as branching moiety. It is especially preferred to have a —O(CH2CH2O)t— polar group present in a branch.
In a preferred embodiment, Linker L1 is or comprises a sulfamide group, preferably a sulfamide group according to structure (23):
The wavy lines represent the connection to the remainder of the compound, typically to Q and L2, L3, L4 or D, preferably to Q and L2. Preferably, the (O)aC(O) moiety is connected to Q and the NR13 moiety to L2, L3, L4 or D, preferably to L2.
In structure (23), a=0 or 1, preferably a=1, and R13 is selected from the group consisting of hydrogen, C1-C24 alkyl groups, C3-C24 cycloalkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups, the C1-C24 alkyl groups, C3-C24 cycloalkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR14 wherein R14 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R13 is D connected to N via a spacer moiety, preferably Sp2 as defined below, in one embodiment D is connected to N via —(B)e-(A)f-(B)g—C(O)—.
In a preferred embodiment, R13 is hydrogen or a C1-C20 alkyl group, more preferably R13 is hydrogen or a C1-C16 alkyl group, even more preferably R13 is hydrogen or a C1-C10 alkyl group, wherein the alkyl group is optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR14, preferably O, wherein R14 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. In a preferred embodiment, R13 is hydrogen. In another preferred embodiment, R13 is a C1-C20 alkyl group, more preferably a C1-C16 alkyl group, even more preferably a C1-C10 alkyl group, wherein the alkyl group is optionally interrupted by one or more O-atoms, and wherein the alkyl group is optionally substituted with an —OH group, preferably a terminal —OH group. In this embodiment it is further preferred that R13 is a (poly)ethylene glycol chain comprising a terminal —OH group. In another preferred embodiment, R13 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, and even more preferably from the group consisting of hydrogen, methyl and ethyl. Yet even more preferably, R13 is hydrogen or methyl, and most preferably R13 is hydrogen.
In a preferred embodiment, L1 is according to structure (24):
Herein, a and R13 are as defined above, Sp1 and Sp2 are independently spacer moieties and b and c are independently 0 or 1. Preferably, b=0 or 1 and c=1, more preferably b=0 and c=1. In one embodiment, spacers Sp1 and Sp2 are independently selected from the group consisting of linear or branched C1-C200 alkylene groups, C2-C200 alkenylene groups, C2-C200 alkynylene groups, C3-C200 cycloalkylene groups, C5-C200 cycloalkenylene groups, C8-C200 cycloalkynylene groups, C7-C200 alkylarylene groups, C7-C200 arylalkylene groups, C8-C200 arylalkenylene groups and C9-C200 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR16, wherein R16 is independently selected from the group consisting of hydrogen, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups and C3-C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted. When the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups 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.
More preferably, spacer moieties Sp1 and Sp2, if present, are independently selected from the group consisting of linear or branched C1-C100 alkylene groups, C2-C100 alkenylene groups, C2-C100 alkynylene groups, C3-C100 cycloalkylene groups, C5-C100 cycloalkenylene groups, C8-C100 cycloalkynylene groups, C7-C100 alkylarylene groups, C7-C100 arylalkylene groups, C8-C100 arylalkenylene groups and C9-C100 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR16, wherein R16 is independently selected from the group consisting of hydrogen, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups and C3-C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted.
Even more preferably, spacer moieties Sp1 and Sp2, if present, are independently selected from the group consisting of linear or branched C1-C50 alkylene groups, C2-C50 alkenylene groups, C2-C50 alkynylene groups, C3-C50 cycloalkylene groups, C5-C50 cycloalkenylene groups, C8-C50 cycloalkynylene groups, C7-C50 alkylarylene groups, C7-C50 arylalkylene groups, C8-C50 arylalkenylene groups and C9-C50 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR16, wherein R16 is independently selected from the group consisting of hydrogen, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups and C3-C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted.
Yet even more preferably, spacer moieties Sp1 and Sp2, if present, are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, C2-C20 alkenylene groups, C2-C20 alkynylene groups, C3-C20 cycloalkylene groups, C5-C20 cycloalkenylene groups, C8-C20 cycloalkynylene groups, C7-C20 alkylarylene groups, C7-C20 arylalkylene groups, C8-C20 arylalkenylene groups and C9-C20 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR16, wherein R16 is independently selected from the group consisting of hydrogen, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups and C3-C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted.
In these preferred embodiments it is further preferred that the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups are unsubstituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR16, preferably O, wherein R16 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.
Most preferably, spacer moieties Sp1 and Sp2, if present, are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, the alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR16, wherein R16 is independently selected from the group consisting of hydrogen, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups and C3-C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted. In this embodiment, it is further preferred that the alkylene groups are unsubstituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR16, preferably O and/or S—S, wherein R16 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.
Preferred spacer moieties Sp1 and Sp2 thus include —(CH2)r—, —(CH2CH2)r—, —(CH2CH2O)r—, —(OCH2CH2)r—, —(CH2CH2O)rCH2CH2—, —CH2CH2(OCH2CH2)r—, —(CH2CH2CH2O)r—, —(OCH2CH2CH2)r—, —(CH2CH2CH2O)rCH2CH2CH2— 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 yet even more preferably in the range of 1 to 15. More preferably n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably 1, 2, 3, 4, 5 or 6, yet even more preferably 1, 2, 3 or 4.
Alternatively, preferred linkers L1 may be represented by —(W)k-(A)d-(B)e-(A)f-(C(O))g—, wherein:
In the context of the present embodiment, the wavy lines in structure (23) represent the connection to the adjacent groups such as (W)k, (B)e and (C(O))g. It is preferred that A is according to structure (23), wherein a=1 and R13═H or a C1-C20 alkyl group, more preferably R13═H or methyl, most preferably R13═H.
Preferred linkers L1 have structure —(W)k-(A)d-(B)e-(A)f-(C(O))g—, wherein:
In a preferred embodiment, linker L1 comprises a branching nitrogen atom, which is located in the backbone between Q or Z and (L2)o and which contains a further moiety D as substituent, which is preferably linked to the branching nitrogen atom via a linker. An example of a branching nitrogen atom is the nitrogen atom NR13 in structure (23), wherein R13 is connected to a second occurrence of D via a spacer moiety. Alternatively, a branching nitrogen atoms may be located within L1 according to structure —(W)k-(A)d-(B)e-(A)f-(C(O))g—. In one embodiment, L1 is represented by —(W)k-(A)d-(B)e-(A)f-(C(O))g—N*[-(A)d-(B)e-(A)f-(C(O))g′-]2, wherein A, B, W, d, e, f, g and k are as defined above and individually selected for each occurrence, and N* is the branching nitrogen atoms, to which two instances of -(A)d-(B)e-(A)f-(C(O))g′— are connected. Herein, both (C(O))g′ moieties are connected to -(L2)o-(L3)p-(L4)q-D, wherein L2, L3, L4, o, p, q and D are as defined above and are each selected individually. In a preferred embodiment, each of L2, L3, L4, o, p, q and D are the same for both moieties connected to (C(O))g′.
Preferred linkers L1 comprising a branching nitrogen atom have structure —(W)k-(A)d-(B)e-(A)f-(C(O))g—N*[-(A′)d′-(B′)e′-(A′)f′-(C(O))g′—]2 wherein:
Linker L2 is either absent (o=0) or present (o=1). Preferably, linker L2 is present and o=1. Linker L2 is a peptide spacer The peptide spacer is preferably defined by (NH—CR17—CO)n, wherein R17 represents an amino acid side chain as known in the art. Herein, the amino acid may be a natural or a synthetic amino acid. Preferably, the amino acid(s) are all in their L-configuration. n is an integer in the range of 1-5, preferably in the range of 2-5. Thus, the peptide spacer preferably contains 1-5 amino acids. Preferably, the peptide is a dipeptide (n=2), tripeptide (n=3) or tetrapeptide (n=4), most preferably the peptide spacer is a dipeptide. Although any peptide spacer may be used, preferably the peptide spacer is selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Asp-Val-Cit, iGlu-Val-Cit, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, Gly-Gly-Phe-Gly and Lys, more preferably Val-Cit, Val-Ala, Glu-Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably Val-Cit, Val-Ala, Ala-Ala-Asn, most preferably Val-Cit or Val-Ala. Herein, AcLys is acetyllysine and iGlu is isoglutamate. In one embodiment, L2=Val-Cit. In one embodiment, L2=Val-Ala.
R17 represents the amino acid side chain, preferably selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine and citrulline. Preferred amino acid side chains are those of Val, Cit, Ala, Lys, Arg, AcLys, Phe, Leu, lie, Trp, Glu, Asp and Asn, more preferably from the side chains of Val, Cit, Ala, Glu and Lys. Alternatively worded, R17 is preferably selected from CH3 (Ala), CH2CH(CH3)2 (Leu), CH2CH2CH2NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), CH2CH2CH2NHC(O)CH3 (AcLys), CH2CH2CH2NHC(═NH)NH2 (Arg), CH2Ph (Phe), CH(CH3)2 (Val), CH(CH3)CH2CH3 (Ile), CH2C(O)NH2 (Asn), CH2CH2C(O)OH (Glu), CH2C(O)OH (Asp) and CH2(1H-indol-3-yl) (Trp). Especially preferred embodiments of R17 are CH3 (Ala), CH2CH2CH2NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), CH2CH2C(O)OH (Glu) and CH(CH3)2 (Val). Most preferably, R17 is CH3 (Ala), CH2CH2CH2NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), or CH(CH3)2 (Val).
In an especially preferred embodiment, the peptide spacer may be represented by general structure (L3):
Herein, R17 is as defined above, preferably R17 is CH3 (Val) or CH2CH2CH2NHC(O)NH2 (Cit). The wavy lines indicate the connection to (L1)n and (L3)p, preferably L2 according to structure (L3) is connected to (L1)n via NH and to (L3)p via C(O).
Linker L3 is either absent (p=0) or present (p=1). Preferably, linker L3 is present and p=1. Linker L3 is a self-cleavable spacer, also referred to as self-immolative spacer. Preferably, L3 is para-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (L4):
Herein, the wavy lines indicate the connection to Q or Z, L1 or L2, and to L4 or D. Typically, the PABC derivative is connected via NH to Q, Z, L1 or L2, preferably to L2, and via O to L4 or D.
A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Suitable 5-membered rings are oxazole, thiazole and furan. Suitable 6-membered rings are phenyl and pyridyl. In a preferred embodiment, A is 1,4-phenyl, 2,5-pyridyl or 3,6-pyridyl. Most preferably, A is 1,4-phenyl.
R21 is selected from H, R26, C(O)OH and C(O)R26, wherein R26 is C1-C24 (hetero)alkyl groups, C3-C10 (hetero)cycloalkyl groups, C2-C10 (hetero)aryl groups, C3-C10 alkyl(hetero)aryl groups and C3-C10 (hetero)arylalkyl groups, which are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR28 wherein R28 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Preferably, R26 is C3-C10 (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably a polyethylene glycol or a polypropylene glycol, more preferably —(CH2CH2O)sH or —(CH2CH2CH2O)sH. The polyalkylene glycol is most preferably a polyethylene glycol, preferably —(CH2CH2O)sH, wherein s is an integer in the range 1-10, preferably 1-5, most preferably s=1, 2, 3 or 4. More preferably, R21 is H or C(O)R26, wherein R26=4-methyl-piperazine or morpholine. Most preferably, R21 is H.
Linker L4 is either absent (q=0) or present (q=1). Preferably, linker L4 is present and q=1. Linker L4 is selected from:
Linker L4 may be an aminoalkanoic acid spacer, i.e. —NR22—(Cz-alkylene)-C(O)—, wherein z is an integer in the range 1 to 20, preferably 1-10, most preferably 1-6. Herein, the aminoalkanoic acid spacer is typically connected to L3 via the nitrogen atom and to D via the carbonyl moiety. Preferred linkers L4 are selected from 6-aminohexanoic acid (Ahx, z=5), β-alanine (z=2) and glycine (Gly, z=1), even more preferably 6-aminohexanoic acid or glycine. In one embodiment, L4=6-aminohexanoic acid. In one embodiment, L4=glycine. Herein, R22 is H or C1-C4alkyl, preferably R22 is H or methyl, most preferably R22 is H.
Alternatively, linker L4 may be an ethyleneglycol spacer according to the structure —NR22—(CH2—CH2—O)e6—(CH2)e7—(C(O)—, wherein e6 is an integer in the range 1-10, preferably e6 is in the range 2-6, and e7 is an integer in the range 1-3, preferably e7 is 2. Herein, R22 is H or C1-C4 alkyl, preferably R22 is H or methyl, most preferably R22 is H.
Alternatively, linker L4 may be a diamine spacer according to the structure —NR22—(Cz-alkylene)-NR22—(C(O))h—, wherein h is 0 or 1, z is an integer in the range 1-20, preferably an integer in the range 2-6, even more preferably z=2 or 5, most preferably z=2. R22 is H or C1-C4 alkyl. Herein, R22 is H or C1-C4 alkyl, preferably R22 is H or methyl, most preferably R22 is methyl. Herein, h is preferably 1, in which case linker L4 is especially suited for conjugation via a phenolic hydroxyl group present on payload D.
D represents the target molecule D that is or is to be connected to the antibody, which is also referred to in the art as the payload. D is exatecan.
The compound according to general structure (2) may comprise more than one moiety D. When more than one cytotoxin D is present the cytotoxins D may be the same or different, typically they are the same. In a preferred embodiment, the compound according to general structure (2) contains 1 or 2 occurrences of D, most preferably 2 occurrence of D. Typically, the second occurrence of D is present within linker L, which may contain a branching moiety, typically a branching nitrogen atom, that is connected to the second occurrence of D. Preferably, both occurrences of D are connected to the branching moiety via the same linker. Likewise, the antibody-conjugate according to structure (1) may contain more than one moiety D per connecting group Z.
Preferred antibody-conjugates according to the first aspect are selected from the group consisting of compounds (I)-(III), more preferably (II) or (III), most preferably (II). More preferred antibody-conjugates are selected from (X)-(XIII). In one especially preferred embodiment, the antibody-conjugates is selected from (Xa), (XIb), (XIIg), (XIIh) and (XIIIe). In an even more preferred embodiment, the antibody-conjugates is selected from (XI) and (XIII), more preferably (XIb) or (XIIIe), more preferably the antibody-conjugates is according to (XIII), most preferably according to (XIIIe). The structures of these antibody-conjugates are defined here below.
Antibody-conjugate (I) has the following structure:
AB-[(L6)-{Z-(L1)-(L2)-(L3)-(L4)q-D}x]y (I)
wherein:
In the context of antibody-conjugate (I), it is preferred that for L1, d=1 (A according to structure (23), it is preferred that a=1 and R13═H), e=2, f=0 and g=1. In the context of antibody-conjugate (I), it is preferred that L2=Val-Cit. In the context of antibody-conjugate (I), it is preferred that for L3, R21═H. In the context of antibody-conjugate (I), it is preferred that in case q=1, then z=1 or 5.
In a particularly preferred embodiment, the antibody-conjugate according to structure (1) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably with exatecan as payload D.
Antibody-conjugate (II) has the following structure:
AB-[(L6)-{Z-(L1)-(L2)-(L3)-D}x]y (II)
wherein:
In the context of antibody-conjugate (II), it is preferred that for L1, e=2, A according to structure (23), it is preferred that a=1 and R13═H. In the context of antibody-conjugate (II), it is preferred that L2=Val-Cit.
In a particularly preferred embodiment, the antibody-conjugate according to structure (II) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably with exatecan as payload D.
Antibody-conjugate (III) has the following structure:
AB-[(L6)-{Z-(L1)-(L2)-(L3)-(L4)-D}x]y (III)
wherein:
In the context of antibody-conjugate (III), it is preferred that for L1, e=2, and with a=1 and R13═H. In the context of antibody-conjugate (III), it is preferred that L2=Val-Cit. In the context of antibody-conjugate (III), it is preferred that z=2.
In a particularly preferred embodiment, the antibody-conjugate according to structure (III) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably with exatecan as payload D.
Antibody-conjugate (X) has a linker-payload moiety according to the following structure:
wherein:
L2 may be present of absent, preferably L2 is present and o=1. For preferred antibody-conjugate (Xa), L2 is according to structure (L3) and R17 is CH3. For preferred antibody-conjugate (Xb), L2 is according to structure (L3) and R17 is CH2CH2CH2NHC(O)NH2. The antibody-conjugate (X) preferably has structure (Xa).
In a particularly preferred embodiment, the antibody-conjugate according to structure (X) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably with exatecan as payload D.
Antibody-conjugate (XI) has a linker-payload moiety according to the following structure:
wherein:
L2 may be present of absent, preferably L2 is present and o=1. For preferred antibody-conjugate (XIa), L2 is according to structure (L3) and R17 is CH3. For preferred antibody-conjugate (XIb), L2 is according to structure (L3) and R17 is CH2CH2CH2NHC(O)NH2. The antibody-conjugate (XI) preferably has structure (XIb).
In a particularly preferred embodiment, the antibody-conjugate according to structure (XI) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably with calicheamicin as payload D.
Antibody-conjugate (XII) has a linker-payload moiety according to the following structure:
wherein:
L2 may be present of absent, preferably L2 is present and o=1. For preferred antibody-conjugate (XIIa), L2 is according to structure (L3) and R17 is CH3. For preferred antibody-conjugate (XIIb), L2 is according to structure (L3) and R17 is CH2CH2CH2NHC(O)NH2. Preferably, R17═CH2CH2CH2NHC(O)NH2.
L4 may be present of absent. For preferred antibody-conjugate (XIIc), q=0 and L4 is absent. For preferred antibody-conjugate (XIId), q=1 and L4 is a diamine spacer according to the structure —NR22—(Cz-alkylene)-NR22—, wherein z is an integer in the range 1-20, and R22 is H or C1-C4 alkyl.
For preferred antibody-conjugate (XIIe), L2 is according to structure (L3), R17 is CH3, q=0 and L4 is absent. For preferred antibody-conjugate (XIIf), L2 is according to structure (L3), R17 is CH3, q=1 and L4 is a diamine spacer according to the structure —NR22—(Cz-alkylene)-NR22—, wherein z is an integer in the range 1-10, preferably wherein z=2, and R22 is H or C1-C4 alkyl.
For preferred antibody-conjugate (XIIg), L2 is according to structure (L3), R17 is CH2CH2CH2NHC(O)NH2, q=0 and L4 is absent. For preferred antibody-conjugate (XIIh), L2 is according to structure (L3), R17 is CH2CH2CH2NHC(O)NH2, q=1 and L4 is a diamine spacer according to the structure —NR22—(Cz-alkylene)-NR22—, wherein z is an integer in the range 1-20, and R22 is H or C1-C4 alkyl.
In the context of antibody-conjugate (XII), it is preferred that z is an integer in the range of 1-10, more preferably z=2-6, most preferably z=2. In the context of antibody-conjugate (XII), it is further preferred that R22 is H or CH3.
In the context of antibody-conjugate (XII), structures (XIIg) and (XIIh) are most preferred.
In a particularly preferred embodiment, the antibody-conjugate according to structure (XII) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably with exatecan as payload D.
Antibody-conjugate (XIII) has a linker-payload moiety according to the following structure:
wherein:
L2 may be present of absent, preferably L2 is present and o=1. For preferred antibody-conjugate (XIIIa), L2 is according to structure (L3) and R17 is CH3. For preferred antibody-conjugate (XIIIb), L2 is according to structure (L3) and R17 is CH2CH2CH2NHC(O)NH2. Preferably, R17═CH3.
L4 may be present of absent. For preferred antibody-conjugate (XIIIc), q=0 and L4 is absent. For preferred antibody-conjugate (XIIId), q=1 and L4 is a diamine spacer according to the structure —NR22—(Cz-alkylene)-NR22—, wherein z is an integer in the range 1-20, and R22 is H or C1-C4 alkyl.
For preferred antibody-conjugate (XIIIe), L2 is according to structure (L3), R17 is CH3, q=0 and L4 is absent. For preferred antibody-conjugate (XIIIf), L2 is according to structure (L3), R17 is CH3, q=1 and L4 is a diamine spacer according to the structure —NR22—(Cz-alkylene)-NR22—, wherein z is an integer in the range 1-10, preferably wherein z=2, and R22 is H or C1-C4 alkyl.
For preferred antibody-conjugate (XIIIg), L2 is according to structure (L3), R17 is CH2CH2CH2NHC(O)NH2, q=0 and L4 is absent. For preferred antibody-conjugate (XIIIh), L2 is according to structure (L3), R17 is CH2CH2CH2NHC(O)NH2, q=1 and L4 is a diamine spacer according to the structure —NR22—(Cz-alkylene)-NR22—, wherein z is an integer in the range 1-20, and R22 is H or C1-C4 alkyl.
In the context of antibody-conjugate (XIII), it is preferred that z is an integer in the range of 1-10, more preferably z=2-6, most preferably z=2. In the context of antibody-conjugate (XIII), it is further preferred that R22 is H or CH3.
In the context of antibody-conjugate (XIII), structure (XIIIe) is most preferred.
In a particularly preferred embodiment, the antibody-conjugate according to structure (XIII) contains a payload D selected from the group consisting of calicheamicin, MMAE, PF-06380101, exatecan and DXd, more preferably a payload D selected from the group consisting of calicheamicin, MMAE, and exatecan, most preferably with exatecan as payload D.
In one particularly preferred embodiment, the antibody-conjugate according to the invention is according to structure (XIIIe) as defined above, and the antibody is hRS7 as defined above and the payload is exatecan.
It is further preferred that these preferred antibody-conjugates are conjugated through the glycan, i.e. b=1, more preferably a trimmed glycan, i.e. j=0. Herein, it is further preferred that S=GalNAc and w′=0. Herein, it is further preferred that connecting group Z is formed by an azide-alkyne cycloaddition, preferably connecting group Z=(Z39), wherein ring Z=(Za) and V=CH2. Herein, it is further preferred that x=1. Herein, it is further preferred that y=2, more preferably that x=1 and y=2.
In a most preferred embodiment, the antibody-conjugate according to the invention is according to structure (XIIIe) as defined above, and the antibody is hRS7 as defined above and the payload is exatecan, wherein b=1, e=0, S=GalNAc, w′=0, connecting group Z=(Z39), wherein ring Z=(Za) and V=CH2, x=1 and y=2.
The compound has general structure (2):
Q-L-D (2)
wherein:
The compound of general structure (2) may also be referred to as a “linker-drug construct”, for containing linker L and payload D of the final conjugate. Compounds according to general formula (2) can be prepared by the skilled person using standard organic synthesis techniques, and as exemplified in the examples. Linker L and payload D are defined above in the context of the conjugate according to structure (1).
The compound according to general structure (2) comprises a reactive moiety Q. In the context of the present invention, the term “reactive moiety” may refer to a chemical moiety that comprises a reactive group, but also to a reactive group itself. For example, a cyclooctynyl group is a reactive group comprising a reactive group, namely a C—C triple bond. Similarly, an N-maleimidyl group is a reactive group, comprising a C—C double bond as a reactive group. However, a reactive group, for example an azido reactive group, a thiol reactive group or an alkynyl reactive group, may herein also be referred to as a reactive moiety.
Q serves as chemical handle for the connection to S(F)x. In other words, Q is reactive towards and complementary to F. Herein, a reactive group is denoted as “complementary” to a reactive group when said reactive group reacts with said reactive group selectively, optionally in the presence of other functional groups. Complementary reactive and functional groups are known to a person skilled in the art, and are described in more detail below. As such, the compound according to general structure (2) is conveniently used in a conjugation reaction, wherein a chemical reaction between Q and F takes place, thereby forming an antibody-conjugate comprising a covalent connection between payload D and the antibody.
The exact nature of Q, and F, depends on the type of conjugation reaction that is employed. The skilled person will be able to select the appropriate combination of Q and F. Preferably, Q, and thus also F, is reactive in a cycloaddition or a nucleophilic reaction. Thus, Q preferably comprises a click probe, a thiol, a thiol-reactive moiety, an amine or an amine-reactive moiety, more preferably Q is a click probe, a thiol-reactive moiety or an amine-reactive moiety, most preferably Q is a click probe. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an ortho-quinone, a dioxothiophene, a sydnone, an alkene moiety and an alkyne moiety. Preferably, the click probe comprises or is an alkene moiety or an alkyne moiety, more preferably wherein the alkene is a (hetero)cycloalkene and/or the alkyne is a terminal alkyne or a (hetero)cycloalkyne. Typical thiol-reactive moieties are selected from maleimide moiety, a haloacetamide moiety, an allenamide moiety, a phosphonamidite moiety, a cyanoethynyl moiety, a vinylsulfone, a vinylpyridine moiety or a methylsulfonylphenyloxadiazole moiety. Most preferably, the thiol-reactive moiety comprises or is a maleimide moiety. Typical amine-reactive moieties are selected from N-hydroxysuccinimidyl esters and other activated esters, p-nitrophenyl carbonates and other activated carbonates, isocyanates, isothiocyanates, haloacetamides and benzyl halides. In a preferred embodiment, Q is selected from an alkene moiety, an alkyne moiety, a thiol-reactive moiety or an amine-reactive moiety, more preferably an alkene moiety or an alkyne moiety, even more preferably an alkyne moiety. Herein, the alkene is preferably a (hetero)cycloalkene and the alkyne is preferably a terminal alkyne or a (hetero)cycloalkyne. Most preferably, Q is a cyclic (hetero)alkyne moiety. Each of these moieties are further defined here below.
Thus, in an especially preferred embodiment, Q comprises a cyclic (hetero)alkyne moiety. The alkynyl group may also be referred to as a (hetero)cycloalkynyl group, i.e. a heterocycloalkynyl group or a cycloalkynyl group, wherein the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptynyl group, a (hetero)cyclooctynyl group, a (hetero)cyclononynyl group or a (hetero)cyclodecynyl group. Herein, the (hetero)cycloalkynes may optionally be substituted. Preferably, the (hetero)cycloalkynyl group is an optionally substituted (hetero)cycloheptynyl group or an optionally substituted (hetero)cyclooctynyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, wherein the (hetero)cyclooctynyl group is optionally substituted.
In an especially preferred embodiment, Q comprises a (hetero)cycloalkynyl or (hetero)cycloalkenyl group and is according to structure (Q1):
Typically, v=(u+u′)×2 (when the connection to L, depicted by the wavy bond, is via Y2) or [(u+u′)×2]−1 (when the connection to L, depicted by the wavy bond, is via one of the carbon atoms).
In a preferred embodiment of structure (Q1), reactive group Q comprises a (hetero)cycloalkynyl group and is according to structure (Q1a):
In a preferred embodiment, u+u′=4, 5 or 6, more preferably u+u′=5. In a preferred embodiment, v=8, 9 or 10, more preferably v=9 or 10, most preferably v=10.
In a preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2)-(Q20) and (Q20a) depicted here below.
Herein, the connection to L, depicted with the wavy bond, may be to any available carbon or nitrogen atom of Q. The nitrogen atom of (Q10), (Q13), (Q14) and (Q15) may bear the connection to L, or may contain a hydrogen atom or be optionally functionalized. B(−) is an anion, which is preferably selected from (−)OTf, Cl(−), Br(−) or I(−), most preferably B(−) is (−)OTf. In the conjugation reaction, B) does not need to be a pharmaceutically acceptable anion, since B(−) will exchange with the anions present in the reaction mixture anyway. In case (Q19) is used for Q, the negatively charged counter-ion is preferably pharmaceutically acceptable upon isolation of the conjugate according to the invention, such that the conjugate is readily useable as medicament.
In a further preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21)-(Q38) and (Q38a) depicted here below.
In structure (Q38), B(−) is an anion, which is preferably selected from (−)OTf, Cl(−), Br(−) or I(−), most preferably B(−) is (−)OTf.
In a preferred embodiment, Q comprises a (hetero)cyclooctyne moiety or a (hetero)cycloheptyne moiety, preferably according to structure (Q8), (Q26), (Q27), (Q28), (Q37) or (Q38a), more preferably according to structure (Q8), (Q26), (Q27), (Q28) or (Q37), which are optionally substituted. Each of these preferred options for Q are further defined here below.
Thus, in a preferred embodiment, Q comprises a heterocycloheptyne moiety according to structure (Q37), also referred to as a TMTHSI, which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Q37) is not substituted.
In an alternative preferred embodiment, Q comprises a cyclooctyne moiety according to structure (Q8), more preferably according to (Q29), also referred to as a bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctyne moiety according to structure (Q8) or (Q29) is not substituted. In the context of the present embodiment, Q preferably is a (hetero)cyclooctyne moiety according to structure (Q39) as shown below, wherein V is (CH2)I and I is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, I is 0, 1, 2, 3 or 4, more preferably I is 0, 1 or 2 and most preferably I is 0 or 1. In the context of group (Q39), I is most preferably 1. Most preferably, Q is according to structure (Q42), defined further below.
In an alternative preferred embodiment, Q comprises a (hetero)cyclooctyne moiety according to structure (Q26), (Q27) or (Q28), also referred to as a DIBO, DIBAC, DBCO or ADIBO group, which are optionally substituted. In the context of the present embodiment, Q preferably is a (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) as shown below, wherein Y1 is O or NR11, wherein R11 is independently selected from the group consisting of hydrogen, a linear or branched C1-C12 alkyl group or a C4-C12 (hetero)aryl group. The aromatic rings in (Q40) are optionally O-sulfonylated at one or more positions, whereas the rings of (Q41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) is not further substituted. Most preferably, Q is according to structure (Q43), defined further below.
In an alternative preferred embodiment, Q comprises a heterocycloheptynyl group and is according to structure (Q37).
In an especially preferred embodiment, Q comprises a cyclooctynyl group and is according to structure (Q42):
In a preferred embodiment of the reactive group according to structure (Q42), R15 is independently selected from the group consisting of hydrogen, halogen, —OR16, C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R16 is hydrogen or C1-C6 alkyl, more preferably R15 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R15 are H. In a preferred embodiment of the reactive group according to structure (Q42), R18 is independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, most preferably both R18 are H. In a preferred embodiment of the reactive group according to structure (Q42), R19 is H. In a preferred embodiment of the reactive group according to structure (Q42), I is 0 or 1, more preferably I is 1.
In an especially preferred embodiment, Q comprises a (hetero)cyclooctynyl group and is according to structure (Q43):
In a preferred embodiment of the reactive group according to structure (Q43), R15 is independently selected from the group consisting of hydrogen, halogen, —OR16, —S(O)3(−), C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R16 is hydrogen or C1-C6 alkyl, more preferably R15 is independently selected from the group consisting of hydrogen and —S(O)3(−). In a preferred embodiment of the reactive group according to structure (Q43), Y is N or CH, more preferably Y═N.
In an especially preferred embodiment, Q comprises a heterocycloheptynyl group and is according to structure (Q37) or (Q38a), preferably according to structure (Q37)
In an alternative preferred embodiment, Q comprises a cyclic alkene moiety. The alkenyl group Q may also be referred to as a (hetero)cycloalkenyl group, i.e. a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, wherein the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a norbornene group, a norbornadiene group, a trans-(hetero)cycloheptenyl group, a trans-(hetero)cyclooctenyl group, a trans-(hetero)cyclononenyl group or a trans-(hetero)cyclodecenyl group, which may all optionally be substituted. Especially preferred are (hetero)cyclopropenyl groups, trans-(hetero)cycloheptenyl group or trans-(hetero)cyclooctenyl groups, wherein the (hetero)cyclopropenyl group, the trans-(hetero)cycloheptenyl group or the trans-(hetero)cyclooctenyl group is optionally substituted. Preferably, Q comprises a cyclopropenyl moiety according to structure (Q44), a hetereocyclobutene moiety according to structure (Q45), a norbornene or norbornadiene group according to structure (Q46), a trans-(hetero)cycloheptenyl moiety according to structure (Q47) or a trans-(hetero)cyclooctenyl moiety according to structure (Q48). Herein, Y3 is selected from C(R23)2, NR23 or O, wherein each R23 is individually hydrogen, C1-C6 alkyl or is connected to L, optionally via a spacer, and the bond labelled is a single or double bond. In a further preferred embodiment, the cyclopropenyl group is according to structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group is according to structure (Q50) or (Q51). In another preferred embodiment, the trans-(hetero)cyclooctene group is according to structure (Q52), (Q53), (Q54), (Q55) or (Q56).
Herein, the R group(s) on Si in (Q50) and (Q51) are typically alkyl or aryl, preferably C1-C6 alkyl.
In an alternative preferred embodiment, Q is a thiol-reactive probe. In this embodiment, Q is a reactive group compatible with cysteine conjugation. Such probes are known in the art and may be selected from the group consisting of a maleimide moiety, a haloacetamide moiety, an allenamide moiety, a phosphonamidite moiety, a cyanoethynyl moiety, a vinylsulfone, a vinylpyridine moiety or a methylsulfonylphenyloxadiazole moiety. Most preferably, Q comprises or is a maleimide moiety. Reagents may be monoalkylation type or may be a cross-linker for reaction with two cysteine side-chains.
In a further preferred embodiment, probe Q is selected from the group consisting of (Q57)-(Q71) depicted here below.
wherein:
In a preferred embodiment of thiol-reactive probe (Q57), the probe Q is selected from the group consisting of (Q72)-(Q74) depicted here below.
wherein:
In an alternative preferred embodiment, Q is an amine-reactive probe. In this embodiment, Q is a reactive group compatible with lysine conjugation. Such probes are known in the art and may be selected from the group consisting of N-hydroxysuccinimidyl groups, isocyanate groups, isothiocyanate groups and benzoyl halide groups. Most preferably, Q comprises or is an N-hydroxysuccinimidyl esters or a p-nitrophenyl carbonate moiety.
In a further preferred embodiment, probe Q is selected from the group consisting of (Q75)-(Q79) depicted here below.
Herein, X2 is halogen, preferably F.
In a preferred embodiment, Q is selected from the group consisting of (Q1)-(Q79).
The antibody has general structure (3):
AB-[(L6)b-{F}x]y (3)
wherein:
The antibody of general structure (3) may also be referred to as a “(modified) antibody”, for being an antibody containing reactive groups F, wherein the reactive groups F are naturally present or the antibody is modified to incorporate the reactive groups F. The (modified) antibody according to general formula (2) can be prepared by the skilled person using standard organic and/or enzymatic synthesis techniques, and as exemplified in the examples. Antibody AB, linker L6, b, x and y are defined above in the context of the conjugate according to structure (1).
F is reactive towards Q in the conjugation reaction defined below, preferably wherein the conjugation reaction is a cycloaddition or a nucleophilic reaction. As the skilled person will understand, the options for F are the same as those for Q, provided that F and Q are reactive towards each other. Thus, F preferably comprises a click probe, a thiol, a thiol-reactive moiety, an amine or an amine-reactive moiety, more preferably F is a click probe, a thiol or an amine, most preferably F is a click probe. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an ortho-quinone, a dioxothiophene, a sydnone, an alkene moiety and an alkyne moiety. Preferably, the click probe comprises or is an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an ortho-quinone, a dioxothiophene or a sydnone, most preferably an azide. Typical thiol-reactive moieties are selected from maleimide moiety, a haloacetamide moiety, an allenamide moiety, a phosphonamidite moiety, a cyanoethynyl moiety, an ortho-quinone moiety, a vinylsulfone, a vinylpyridine moiety or a methylsulfonylphenyloxadiazole moiety. Most preferably, the thiol-reactive moiety comprises or is a maleimide moiety. Typical amine-reactive moieties are selected from N-hydroxysuccinimidyl esters, isocyanates, isothiocyanates and benzyl halides. In a preferred embodiment, F is a click probe or a thiol, more preferably F is an azide or a thiol, most preferably F is an azide.
More than one reactive group F may be present in the antibody. The reactive group F in the antibody may be naturally present or may be placed in the antibody by a specific technique, for example a (bio)chemical or a genetic technique. The reactive group that is placed in the antibody is prepared by chemical synthesis, for example an azide or a terminal alkyne. Methods of preparing modified antibodies are known in the art, e.g. from WO 2014/065661, WO 2016/170186 and WO 2016/053107, which are incorporated herein by reference. From the same documents, the conjugation reaction between the modified antibody and a linker-drug construct is known to the skilled person.
Preferably, F is a click probe reactive towards a (hetero)cycloalkene and/or a (hetero)cycloalkyne, and is typically selected from the group consisting of azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, ortho-quinone, dioxothiophene and sydnone. Preferred structures for the reactive group are structures (F1)-(F10) depicted here below.
Herein, the wavy bond represents the connection to the payload. For (F3), (F4), (F8) and (F9), the payload can be connected to any one of the wavy bonds. The other wavy bond may then be connected to an R group selected from hydrogen, C1-C24 alkyl groups, C2-C24 acyl groups, C3-C24 cycloalkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups, C3-C24 (hetero)arylalkyl groups and C1-C24 sulfonyl groups, each of which (except hydrogen) may optionally be substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR32 wherein R32 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. The skilled person understands which R groups may be applied for each of the groups F. For example, the R group connected to the nitrogen atom of (F3) may be selected from alkyl and aryl, and the R group connected to the carbon atom of (F3) may be selected from hydrogen, alkyl, aryl, acyl and sulfonyl. Preferably, the reactive moiety F is selected from azides or tetrazines. Most preferably, the reactive moiety F is an azide.
In a second preferred embodiment, F is a thiol or precursor thereof. Thiol or precursor thereof F is used in the conjugation reaction to connect the linker-drug construct to the (modified) antibody. F is reactive towards thiol-reactive probe Q in a thiol ligation. The thiol preferably the thiol of the side chain of a cysteine amino acid, which are naturally present within the antibody AB, in which case linker L6 is not present (b=0), although it may also be synthetically introduced, optionally via a linker L6. Thiol precursors in the context of bioconjugation are known in the art, and include disulfides, which may be naturally occurring disulfide bridges present in the antibody or synthetically introduced disulfides, which are reduced as known in the art. Preferably, F is a thiol group of a cysteine side chain.
In a third preferred embodiment, F is an amine or precursor thereof, preferably an amine. Amine or precursor thereof F is used in the conjugation reaction to connect the linker-drug construct to the (modified) antibody. F is reactive towards amine-reactive probe Q in nucleophilic substitution. The amine is typically a primary amine, preferably the amine of the side chain of a lysine amino acid, which are naturally present within the antibody AB, in which case linker L6 is not present (b=0), although it may also be synthetically introduced, optionally via a linker L6. Preferably, F is a primary amine group of a lysine side chain.
In a further aspect, the present invention relates to a process for the preparation of the antibody-conjugate according to the invention, the process comprising the step of reacting Q of the compound according to the invention with a reactive group F of an antibody. The compound according to general structure (2), and preferred embodiments thereof, are described in more detail above. The present process occurs under conditions such that Q is reacted with F to covalently link the antibody AB (3) to the payload D. In the process according to the invention, Q reacts with F, forming a covalent connection between the antibody and the compound according to the invention. Complementary reactive groups Q and reactive groups F are known to the skilled person and are described in more detail below.
Any conjugation technique known in the art can be employed to prepare the multifunctional antibody constructs according to the invention. Suitable conjugation techniques include thiol ligation, lysine ligation, cycloadditions (e.g. copper-catalysed click reaction, strain-promoted azide-alkyne cycloaddition, strain-promoted quinone-alkyne cycloaddition). Preferred conjugation techniques used in the context of the present invention include nucleophilic reactions and cycloadditions, preferably wherein the cycloaddition is a [4+2]cycloaddition or a [3+2]cycloaddition and the nucleophilic reaction is a Michael addition or a nucleophilic substitution. Suitable conjugation techniques are for example disclosed in G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd Ed. 2013 (ISBN:978-0-12-382239-0), WO 2014/065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, PCT/EP2021/050594, PCT/EP2021/050598 and NL 2026947.
Thus, in a preferred embodiment of the conjugation process according to the invention, conjugation is accomplished via a nucleophilic reaction, such as a nucleophilic substitution or a Michael reaction. A preferred nucleophilic reaction is the acylation of a primary amino group with an activated ester. A preferred Michael reaction is the maleimide-thiol reaction, which is widely employed in bioconjugation.
Thus, in a preferred embodiment of the conjugation process according to the invention, conjugation is accomplished via a cycloaddition. Preferred cycloadditions are a (4+2)-cycloaddition (e.g. a Diels-Alder reaction) or a (3+2)-cycloaddition (e.g. a 1,3-dipolar cycloaddition). Preferably, the conjugation reaction is the Diels-Alder reaction or the 1,3-dipolar cycloaddition. The preferred Diels-Alder reaction is the inverse-electron demand Diels-Alder cycloaddition. In another preferred embodiment, the 1,3-dipolar cycloaddition is used, more preferably the alkyne-azide cycloaddition, and most preferably wherein Q is or comprises an alkyne group and F is an azido group. Cycloadditions, such as Diels-Alder reactions and 1,3-dipolar cycloadditions are known in the art, and the skilled person knowns how to perform them.
The process according to the present aspect preferably concerns a click reaction, more preferably a 1,3-dipolar cycloaddition, most preferably an alkyne/azide cycloaddition. Most preferably, Q is or comprises an alkyne group and F is an azido group. Click reactions, such as 1,3-dipolar cycloadditions, are known in the art, and the skilled person knows how to perform them.
Thus, the process for preparing the antibody-conjugate according to the invention according to this aspect comprises reacting the modified antibody of structure (3) with a compound according to structure (2), to obtain the antibody-conjugate of structure (1).
In a preferred embodiment, the process for preparing the antibody-conjugate according to the invention comprises:
AB-[GlcNAc(Fuc)w-S{F}x]y (26)
Q-L-D (2)
In step (i), an antibody comprising 1, 2, 3 or 4 core N-acetylglucosamine moieties is contacted with a compound of the formula S(F)x—P in the presence of a catalyst, wherein S(F)x is a sugar derivative comprising x reactive groups F capable of reacting with a reactive group Q, x is 1 or 2 and P is a nucleoside mono- or diphosphate, and wherein the catalyst is capable of transferring the S(F)x moiety to the core-GlcNAc moiety. Herein, the antibody is typically an antibody that has been trimmed to a core-GlcNAc residue as described further below. Step (i) affords a modified antibody according to Formula (26).
The starting material, i.e. the antibody comprising a core-GlcNAc substituent, is known in the art and can be prepared by methods known by the skilled person. In one embodiment, the process according to the invention further comprises the deglycosylation of an antibody glycan having a core N-acetylglucosamine, in the presence of an endoglycosidase, in order to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein said core N-acetylglucosamine and said core N-acetylglucosamine substituent are optionally fucosylated. Depending on the nature of the glycan, a suitable endoglycosidase may be selected. The endoglycosidase is preferably selected from the group consisting of EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT and EndoSH and/or a combination thereof, the selection of which depends on the nature of the glycan. EndoSH is described in PCT/EP2017/052792, see Examples 1-3, and SEQ. ID No: 1, which is incorporated by reference herein.
Structural features S and x are defined above for the antibody-conjugate according to the invention, which equally applies to the present aspect. Compounds of the formula S(F)x—P, wherein a nucleoside monophosphate or a nucleoside diphosphate P is linked to a sugar derivative S(F)x, are known in the art. For example Wang et al., Chem. Eur. J. 2010, 16, 13343-13345, Piller et al., ACS Chem. Biol. 2012, 7, 753, Piller et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459-5462 and WO 2009/102820, all incorporated by reference herein, disclose a number of compounds S(F)x—P and their syntheses. In a preferred embodiment nucleoside mono- or diphosphate P in S(F)x—P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP), more preferably P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP) and cytidine diphosphate (CDP), most preferably P=UDP. Preferably, S(F)x—P is selected from the group consisting of GalNAz-UDP, F2-GalNAz-UDP (N-(azidodifluoro)acetylgalactosamine), 6-AzGal-UDP, 6-AzGalNAc-UDP (6-azido-6-deoxy-N-acetylgalactosamine-UDP), 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, GlcNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP and 2-(but-3-yonic acid amido)-2-deoxy-galactose-UDP. Most preferably, S(F)x—P is GalNAz-UDP or 6-AzGalNAc-UDP.
Suitable catalyst that are capable of transferring the S(F)x moiety to the core-GlcNAc moiety are known in the art. A suitable catalyst is a catalyst wherefore the specific sugar derivative nucleotide S(F)x—P in that specific process is a substrate. More specifically, the catalyst catalyses the formation of a β(1,4)-glycosidic bond. Preferably, the catalyst is selected from the group of galactosyltransferases and N-acetylgalactosaminyltransferases, more preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases (GalNAcT) and β(1,4)-galactosyltransferases (GalT), most preferably from the group of 3(1,4)-N-acetylgalactosaminyltransferases having a mutant catalytic domain. Suitable catalysts and mutants thereof are disclosed in WO 2014/065661, WO 2016/022027 and WO 2016/170186, all incorporated herein by reference. In one embodiment, the catalyst is a wild-type galactosyltransferase or N-acetylgalactosaminyltransferase, preferably an N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or N-acetylgalactosaminyltransferases, preferably a mutant N-acetylgalactosaminyltransferase. Mutant enzymes described in WO 2016/022027 and WO 2016/170186 are especially preferred. These galactosyltransferase (mutant) enzyme catalysts are able to recognize internal sugars and sugar derivatives as an acceptor. Thus, sugar derivative S(F)x is linked to the core-GlcNAc substituent in step (i), irrespective of whether said GlcNAc is fucosylated or not.
Step (i) is preferably performed in a suitable buffer solution, such as for example phosphate, buffered saline (e.g. phosphate-buffered saline, tris-buffered saline), citrate, HEPES, tris and glycine. Suitable buffers are known in the art. Preferably, the buffer solution is phosphate-buffered saline (PBS) or tris buffer. Step (i) is preferably performed at a temperature in the range of about 4 to about 50° C., more preferably in the range of about 10 to about 45° C., even more preferably in the range of about 20 to about 40° C., and most preferably in the range of about 30 to about 37° C. Step (i) is preferably performed a pH in the range of about 5 to about 9, preferably in the range of about 5.5 to about 8.5, more preferably in the range of about 6 to about 8. Most preferably, step (i) is performed at a pH in the range of about 7 to about 8.
In step (ii), the modified antibody is reacted with a compound according to general structure (2), comprising a reactive group Q capable of reacting with reactive group F and a payload D, to obtain the antibody-conjugate according to structure (1), containing connecting group Z resulting from the reaction between Q and F. Such reaction occurs under condition such that reactive group Q is reacted with the reactive group F of the biomolecule to covalently link the antibody to the compound according to general structure (2). Step (ii) may also be referred to as the conjugation reaction.
In a preferred embodiment, in step (ii) an azide on an azide-modified antibody reacts with an alkynyl group, preferably a terminal alkynyl group, or a (hetero)cycloalkynyl group of the compound according to general structure (2), via a cycloaddition reaction. This cycloaddition reaction of a molecule comprising an azide with a molecule comprising a terminal alkynyl group or a (hetero)cycloalkynyl group is one of the reactions that is known in the art as “click chemistry”. In the case of a linker-conjugate comprising a terminal alkynyl group, said cycloaddition reaction needs to be performed in the presence of a suitable catalyst, preferably a Cu(I) catalyst. However, in a preferred embodiment, the linker-conjugate comprises a (hetero)cycloalkynyl group, more preferably a strained (hetero)cycloalkynyl group. When the (hetero)cycloalkynyl is a strained (hetero)cycloalkynyl group, the presence of a catalyst is not required, and said reaction may even occur spontaneously by a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions known in the art as “metal-free click chemistry”.
The invention further concerns a method for the treatment of a subject in need thereof, comprising the administration of the antibody-conjugate according to the invention as defined above. The subject in need thereof is typically a cancer patient. The use of antibody-conjugates, such as antibody-drug conjugates, is well-known in the field of cancer treatment, and the antibody-conjugates according to the invention are especially suited in this respect. The method as described is typically suited for the treatment of cancer. In the method according to this aspect, the antibody-conjugate is typically administered in a therapeutically effective dose. The present aspect of the invention can also be worded as an antibody-conjugate according to the invention for use in the treatment of a subject in need thereof, preferably for the treatment of cancer. In other words, this aspect concerns the use of an antibody-conjugate according to the invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of a subject in need thereof, preferably for use in the treatment of cancer. In the present context, treatment of cancer is envisioned to encompass treating, imaging, diagnosing, preventing the proliferation of, containing and reducing tumours.
This aspect of the present invention may also be worded as a method for targeting Trop-2-expressing cells, in particular Trop-2-expressing tumour cells, comprising contacting the antibody-conjugate according to the invention with cells that may possibly be Trop-2-expressing. The method according to this aspect is thus suitable to determine whether the cells are Trop-2-expressing. These Trop-2-expressing cells may be present in a subject, in which case the method comprises administering to a subject in need thereof the antibody-conjugate according to the invention. In a preferred embodiment, the cells that may possibly be Trop-2-expressing are Trop-2-expressing cells. The targeting of Trop-2-expressing cells preferably includes one or more of treating, imaging, diagnosing, preventing the proliferation of, containing and reducing Trop-2-expressing cells, in particular Trop-2-expressing tumour cells. The method according to this embodiment may be medical or non-medical. Non-medical methods according to the present aspect may be directed to in vitro or ex vivo targeting Trop-2-expressing cells, wherein the cells that may possibly be Trop-2-expressing are present in a sample, e.g. taken from a patient. Such a non-medical method is typically used for the diagnosis of cancer, in particular Trop-2-positive cancer.
In the context of the present invention, the subject may suffer from a disorder selected from oral cancer, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, breast cancer and lung cancer. Thus, the treatment of a subject in need thereof preferably refers to the treatment of oral cancer, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, breast cancer and lung cancer.
The inventors have surprisingly found that the antibody-conjugates according to the invention are superior to conventional Trop-2-targeting antibody-conjugates in terms of safety and/or efficacy, such that the therapeutic index of the antibody-conjugate according to the invention is increased with respect to conventional Trop-2-targeting antibody-conjugates.
In the context of the present invention, the “mode of conjugation” refers to the process that is used to conjugate a payload D to an antibody AB, as well as to the structural features of the resulting antibody-conjugate, in particular of the linker that connects the payload to the antibody, that are a direct consequence of the process of conjugation. Thus, in one embodiment, the mode of conjugation refers to a process for conjugation a payload to an antibody. In an alternative embodiment, the mode of conjugation refers to structural features of the linker and/or to the attachment point of the linker to the antibody that are a direct consequence of the process for conjugation a payload to an antibody.
In a further aspect, the invention concerns the use of a mode of conjugation for increasing the therapeutic index of an antibody-conjugate in the treatment of Trop-2-expressing tumours, wherein the mode of conjugation is being used to connect antibody AB with payload D via a linker L. The mode of conjugation mode of conjugation comprises:
AB-[GlcNAc(Fuc)w-S{F}x]y (26)
Q-L-D (2)
Preferably, increasing the therapeutic index of an antibody-conjugate is selected from:
Increase in therapeutic efficacy of the antibody-conjugates according to the invention may take the form of a reduction in tumour size and/or a prolonged period of regression, when compared to conventional Trop-2-targeting ADC. Increase in tolerability of the antibody-conjugates according to the invention may take the form of a reduction in signs of toxicity, compared to administration of a Trop-2-targeting ADC made with a conventional technology. The reduction in sings may also be referred to as a reduction in symptoms or side-effects of cancer treatment, and may involve one or more clinical signs such as reduced reduction in body weight, reduced reduction in mobility, reduced reduction in food intake and/or one or more toxicity parameters, such as improved blood chemistry, hematology, and/or histopathology.
General procedure for transient expression and purification of monoclonal antibodies: Various IgGs (hRS7 and hTINA) were transiently expressed in CHO K1 cells by Evitria (Zurich, Switzerland) at 300 mL scale. The supernatant was purified using a HiTrap MabSelect sure column. The supernatant was loaded onto the column followed by washing with at least 10 column volumes of 25 mM Tris pH 7.5, 150 mM NaCl (TBS). Retained protein was eluted with 0.1 M AcOH pH 2.7. The eluted product was immediately neutralized with 2.5 M Tris-HCl pH 8.8 and dialyzed against 20 mM histidine, 150 mM NaCl, pH 7.5. Next the IgG was concentrated (>20 mg/mL) using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). The sequences of the IgGs are given here below:
General procedure for analytical RP-UPLC (DTT treated samples): Prior to RP-UPLC analysis, IgG (10 μL, 1 mg/mL in PBS pH 7.4) was added to 12.5 mM DTT, 100 mM TrisHCl pH 8.0 (40 μL) and incubated for 15 minutes at 37° C. The reaction was quenched by adding 49% acetonitrile, 49% water, 2% formic acid (50 μL). RP-UPLC analysis was performed on a Waters Acquity UPLC-SQD. The sample (5 μL) was injected with 0.4 mL/min onto Bioresolve RP mAb 2.1*150 mm 2.7 μm (Waters) with a column temperature of 70° C. A linear gradient was applied in 9 minutes from 30 to 54% acetonitrile in 0.1% TFA and water. Absorbance of eluted peaks was measured at 215 nm followed by automated integration (MassLynx, Waters) to determine reaction conversion.
General procedure for mass spectral analysis of (modified) monoclonal antibodies: Prior to mass spectral analysis, IgG was treated with IdeS, which allows analysis of the Fc/2 fragment. For analysis of both light and heavy chain, a solution of 20 μg (modified) IgG was incubated for 5 minutes at 37° C. with 100 mM DTT in a total volume of 4 μL. If present, azide-functionalities are reduced to amines under these conditions. For analysis of the Fc/2 fragment, a solution of 20 μg (modified) IgG was incubated for 1 hour at 37° C. with IdeS/Fabricator™ (1.25 U/μL) in phosphate-buffered saline (PBS) pH 6.6 in a total volume of 10 μL. Samples were diluted to 80 μL followed by analysis electrospray ionization time-of-flight (ESI-TOF) on a JEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.
General procedure for enzymatic remodeling of IgG to mAb-(6-N3-GalNAc)2: IgG (15 mg/mL) was incubated with 1% w/w EndoSH (as described in PCT/EP2017/052792, see Examples 1-3, and SEQ. ID No: 1, which is incorporated by reference herein), 3% w/w His-TnGalNAcT (as described in PCT/EP2016/059194, see Examples 3 and 4, and SEQ. ID No: 49, which is incorporated by reference herein), 0.01% AP (Roche) and UDP 6-N3-GalNAc (compound 2d in
According to the general procedure for enzymatic remodeling, hRS7 was converted to hRS7-(6-N3-GalNAc)2 Mass spectral analysis of a sample after IdeS treatment showed one major Fc/2 product (observed mass 24360 Da, approximately 80% of total Fc/2), corresponding to the expected product.
Compound 10 (163 mg, 240 μmol) was added to a mixture of exatecan mesylate (125 mg, 235 μmol) and DIPEA (61 mg, 82 μL, 0.47 mmol) in dry DMF (0.9 mL). After 20 h, the reaction mixture was diluted to 9 mL DCM and purified by gradient column chromatography (0→40% MeOH/DCM) to afford 11 (155 mg, 159 μmol, 68%). LCMS (ESI+) calculated for C55H54FN6O10+ (M+H)+977.39, found 977.72. In addition to 11, free base of exatecan (82.4 mg, 189 μmol, 20%) was recovered. LCMS (ESI+) calculated for C24H23FN3O4+ (M+H)+436.46, found 436.54.
The synthesis of BCN-HS-(va-PABC-Ex)2 (3) is also described in PCT/EP2021/075401 (example 4), incorporated herein. To a solution of compound 11 (155 mg, 159 μmol) in DMF (1.6 mL) were added Et3N (73 mg, 101 μL, 0.72 mmol) and a solution of compound 12 (65 mg, 72 μmol) in DMF (1.4 mL). The reaction mixture was stirred for 18 h, diluted with DCM (20 mL) and purified by gradient column chromatography (0→40% MeOH/DCM) to afford 3 as a pale-yellow solid (94 mg, 44 μmol, 28%). LCMS (ESI+) calculated for C102H118F2N16O29S22++(M/2+H)+1066.88, found 1067.12.
A solution of BCN-HS-PEG2-b-(Glu(OFm)—OH)2 (8, 12.1 mg, 10 μmol, 1.0 eq) dissolved in anhydrous DMF (180 μL) was added to a solution of NH2-Val-Ala-PABC-exatecan (5b, Fmoc-deprotected 5, 19 mg, 25 μmol, 2.5 eq) in anhydrous DCM (180 μL), DIPEA (11 μL, 63 μmol, 6.2 eq) and HATU (8.9 mg, 23 μmol, 2.3 eq). After stirring for 2 h at room temperature, the reaction mixture was further diluted with DCM (800 μL) and purified by flash column chromatography over silicagel (0%→20% MeOH in DCM) to give the product as a clear oil (difficult to determine yield due to presence of DMF). LCMS (ESI+) calculated for C140H150F2N17O33S+ (M/2+H+) 1334.01, found 1334.79.
This compound was dissolved in DMF (300 μL) and triethylamine (21 μL, 150 μmol, 15 eq) was added. After 17 h at room temperature, the reaction mixture was diluted with DCM (700 μL) and purified by flash column chromatography over silica gel (0%→45% MeOH in DCM) to give compound 9 in 44% yield as a yellow solid (10.2 mg, 4.4 μL). LCMS (ESI+) calculated for C112H130F2N17O33S+ (M/2+H+) 1156.2, found 1156.74.
To a solution of hRS7(6-N3-GalNAc)2 (764.5 μL, 15.0 mg, 19.62 mg/ml in TBS pH 7.5) was added sodium deoxycholate (150 μL, 110 mM) and BCN-HS-PEG2-HS-(va-PAB-Ex)23 (50 μL, 10 mM solution in DMF) and propylene glycol (400 μL). The reaction was incubated overnight at rt. To remove the excess of free payload, 15 mg of active charcoal (Carbon RHC, Filtrox AG) was added and rotated for 2 hours. The charcoal was removed by centrifugation and subsequently filtered over a PES syringe filter (pore 0.20 μm, Corning). Subsequently the solution was buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1M NaOH and equilibrated with PBS. Since the free payload levels were still too high, another 10 mg of active charcoal was added and incubated overnight. The charcoal was removed by centrifugation and subsequently filtered over a PES syringe filter (pore 0.20 μm, Corning). Subsequently the solution was buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1 M NaOH and equilibrated with 20 mM histidine, 6% sucrose buffer pH 6.0, and 0.04% Tween-20 was added before filter sterilization. Mass spectral analysis of the sample after IdeS treatment showed one major Fc/2 product (observed mass 26497 Da, approximately 90% of total Fc/2), corresponding to the conjugate hRS7-3. RP-UPLC analysis of the sample after IdeS treatment showed an average DAR of 3.90.
To a solution of hTINA (1780 μL, 10 mg, 5.63 mg/mL in PBS pH 7.4) was added PBS (27 μL), EDTA (40 μL, 500 mM) and TCEP (153 μL, 1 mM, 2.3 equiv.). The mixture was incubated for 90 minutes at room temperature. Subsequently, the conjugation was performed by adding deruxtecan (200 μL, 1.8 mM, Achemblock) and incubating it for 90 minutes at room temperature. Subsequently the solution was buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1M NaOH and equilibrated with PBS pH 7.4. Subsequently the solution was buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1M NaOH and equilibrated with 20 mM histidine, 6% sucrose at pH 6.0, and 0.04% Tween-20 was added before filter sterilization. RP-UPLC analysis of the reduced conjugate showed an average DAR of 3.97.
To a solution of hRS7 (6-N3-GalNAc)2 (22.8 mL, 448 mg, 19.64 mg/ml in TBS pH 7.5) was added sodium deoxycholate (4.48 mL, 110 mM) and BCN-HS-PEG2-(eva-PAB-Ex)29 (895 μL, 10 mM solution in DMF) and propylene glycol (8.1 mL). The reaction was incubated overnight at rt. Next the conjugate was purified using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2M NaOH and equilibrated with PBS pH 7.4 on an AKTA Pure (Cytiva). To remove the excess of free payload, 100 mg of active charcoal (Carbon RHC, Filtrox AG) was added and rotated for 2 h. The charcoal was removed by centrifugation and subsequently filtered over a PES syringe filter (pore 0.20 μm, Corning). Subsequently the solution was dialyzed to 20 mM histidine, 6% sucrose buffer pH 6.0. The material was concentrated and 0.04% Tween-20 was added before filter sterilization. Mass spectral analysis of the sample after IdeS treatment showed one major Fc/2 product (observed mass 26676 Da, approximately 90% of total Fc/2), corresponding to the conjugate hRS7-9. RP-UPLC analysis of the sample treated with fabricator (IdeS) showed an average DAR of 3.86.
NCI-N87, human gastric carcinoma model cell line was maintained in vitro and the cells in an exponential growth phase were harvested and counted for tumor inoculation.
CB.17 SCID mice (female) of 8-12 weeks old at the start date, received a subcutaneous injection in the flank region with 1×107 tumor cells in 0.1 m1 of PBS mixed with Matrigel (1:1) for tumor development. When tumors reached an average size of 150-200 mm3, randomization was performed into 3 groups of 8 mice and treatment began.
The test article administration was performed via intravenous injection, and the dosing volume was 10 mL/kg. Treatment was initiated on the same day of randomization. Dosing was conducted in a Laminar Flow Cabinet.
After tumor cells inoculation, the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals are checked for any adverse effects of tumor growth and treatments on normal behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effects. Tumor volumes were measured every 3-4 days in two dimensions using an caliper, and the volume data are expressed in mm3 using the formula: V=(L×W×W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements will be conducted in a Laminar Flow Cabinet. The endpoint of the experiment is a tumor volume of 800 mm3, body weight loss over 20% or 45 days, whichever comes first.
Data for efficacy study with the test items above is depicted in
Colo-205, colon cancer xenograft model cell line was maintained in vitro using RPMI-1640 medium supplemented with 10% FBS in humidified cell culture incubator at 37° C. with standard 5% CO2 specs. The cells in an exponential growth phase were harvested and counted for tumor inoculation.
BALB/c nude mice (female) of 7-9 weeks old received a subcutaneous injection in the right front flank region with 5×106 tumor cells in 0.1 m1 of PBS mixed with Matrigel (1:1) for tumor development. When tumors reached an average size of 100-200 mm3, randomization was performed into 5 groups of 8 mice and treatment began. Randomization will be performed based on “Matched distribution” method (Study Director™ Software, version 3.1.399.19). The date of randomization will be denoted as day 0.
Electronic caliper measurement were performed 2 times a week. Daily observations for clinical signs, food and water consumption, behavioral changes, animals were weighed 2 times per week. The endpoint of the experiment is a tumor volume of 3,000 mm3, body weight loss over 20% or 77 days, whichever comes first.
Randomization: The randomization was performed when the mean tumor size reached approximately 143 mm3. Totally 40 mice were enrolled in NCI-H446 model study and randomly allocated to 5 groups, with 8 mice per group. Randomization will be performed based on “Matched distribution” method (Study Director™ Software, version 3.1.399.19). The date of randomization will be denoted as day 0. Due to the cachectic nature of the tumor model, all animals received supplemental gel from the day of randomization.
Test article administration: The test article administration was performed via intravenous injection through tail vein, and the dosing volume was 10 mL/kg. Treatment was initiated on the same day of randomization. Dosing was conducted in a Laminar Flow Cabinet.
Observation and data collection: After tumor cells inoculation, the animals are checked daily for morbidity and mortality. At the time of routine monitoring, the animals are checked for any adverse effects of tumor growth and treatments on normal behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effects. Tumor volumes are measured every 3-4 days in two dimensions using an caliper, and the volume data are expressed in mm3 using the formula: V=(L×W×W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements will be conducted in a Laminar Flow Cabinet.
Tumor volume over time and mice body weight in the efficacy study and the corresponding Kaplan-Meier plot with the test items above are depicted in
Stability of ADCs in human plasma was tested. Prior to the assay, the plasma was depleted from all IgG using CaptivA® Protein A agarose (1 mL agarose/mL serum). ADCs were added to the depleted human serum to a final concentration of 0.1 mg/mL followed by incubation at 37° C. At each time point, 0.5 mL was snap frozen and stored at −80° C. until further analysis. To isolate the ADCs after incubation, 20 μl CaptivA® Protein A agarose resin was added to the samples and incubated for 1 hour at room temperature. The resin was washed three times with PBS and subsequently 0.1 M Glycine-HCl pH 2.7 (0.4 mL) was added to elute the ADCs. After elution, the samples were immediately neutralized with 1.0 M Tris pH 8.0 (0.1 mL). The samples were spin-filtered against PBS for three times using Amicon Ultra spin-filter 0.5 mL MWCO 10 kDa (Merck Millipore) and the volume was reduced to 40 μL, yielding a final ADC concentration of approximately 1 mg/mL. Samples were analyzed on RP-UPLC (DTT reduced) at specific days to determine the DAR and the results are shown in the table below. At t=7 days, the difference with t=0 days is given in percentage.
The stability of ADCs was tested at elevated temperatures either at physiological conditions (PBS, pH 7.4, 37° C.) or enhanced stress conditions (citrate buffered saline, CBS, pH 5.0, 40° C.). The ADCs were buffer exchanged using a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.1M NaOH and equilibrated with either PBS or CBS on an AKTA Pure (Cytiva). The solution was concentrated using a Vivaspin Turbo 4 10 kDa MWCO ultrafiltration unit (Sartorius) to a concentration>1 mg/mL. The concentration of the ADCs was measured, they were diluted to 1 mg/mL and the first measurement, t=0, was taken for SE-HPLC analysis as described above. The samples were placed at either 37° C. or 40° C. and samples were taken at several timepoints (days) and aggregation levels were determined, see tables 4-6 below.
Samples were diluted to 1 mg/mL in PBS. HIC analysis is performed on an Agilent 1200 series using a TSKgel® Butyl-NPR HPLC Column (3.5 cm×4.6 mm, 2.5 μm). 10 μL sample is injected at a flow rate of 0.5 mL/min using a gradient starting from 100% buffer A (2 M ammonium sulfate in 50 mM potassium phosphate pH 6.0) to 100% buffer B (50 mM potassium phosphate pH 6.0+20% isopropanol) in 20 minutes. The native mAb (hRS7 or hTINA) is measured and its retention time is set as 1. The other measured samples are relative retention times compared to the native mAb. The results are depicted in
| Number | Date | Country | Kind |
|---|---|---|---|
| 22163943.8 | Mar 2022 | EP | regional |
This application is a continuation of International Patent Application No. PCT/EP2023/057561, filed Mar. 23, 2023, which claims priority to European Patent Application Serial No. 22163943.8, filed Mar. 23, 2022, the entire disclosures of which are hereby incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2023/057561 | Mar 2023 | WO |
| Child | 18892270 | US |
