Bis(2-haloacetamide) compounds for use as linking agents and resulting products comprising antibodies, antibody fragments, and antibody fragments

Abstract

Described are bis(2-haloacetamide) compounds for use as linkers for chemically crosslinking multiple thiol groups, particularly, but not limited to, the thiol groups of cysteine ​​amino acids in peptide chains, as well as their use as linking agents and the resulting products (e.g., antibody-protein conjugates and antibody-drug conjugates) including antibodies, half antibodies, and antibody fragments having thiol groups attached to said linkers, and methods for making said conjugates and products (Formula I). TIFF2022538253000049.tif7863

Description

Technical Field

[0001] The present invention relates to the use of bis(2-haloacetamide) derivatives as compounds for chemically crosslinking a plurality of thiol groups, particularly, but not limited to, the thiol groups of cysteine amino acids in a peptide chain.

[0002] Accordingly, the present invention provides arylbis(2-haloacetamide) compounds for use as linking compounds, antibodies, half-antibodies, and antibody fragment conjugates having thiol groups attached to the linker, products comprising antibodies, half-antibodies, and antibody fragments having thiol groups attached to the linker (e.g., antibody-protein conjugates and antibody-drug conjugates), and methods for making the conjugates and products.

Background Art

[0003] Antibody-drug conjugates (ADCs) are one of the state-of-the-art antibody-based therapeutic approaches. Therefore, they have received a great deal of attention over the past two decades. An ADC comprises a monoclonal antibody (mAb) employed as a vehicle for delivering a cytotoxic drug to specific malignant cells or tissues. Specific linker properties are required for the construction of an ADC to achieve plasma-stable antibody-drug conjugates.

[0004] For the construction of first-generation ADCs, the intrinsic nucleophilic functionality of amino acid residues within the antibody, mainly cysteine and lysine amino acids, was utilized. A pre-reduction step of the interchain disulfide bonds is required to generate cysteine-based conjugates. The IgG1 subclass of immunoglobulin G antibodies (IgG) is the most abundant subclass in human serum and the most common type employed in mAb-based therapies. IgG1 has a total of four interchain disulfide bonds, two of which are between the heavy chain / light chain (HC / LC), and two of which are between the heavy chain / heavy chain (HC / HC) in the hinge region.

[0005] Numerous studies have shown the importance of the four inter-chain disulfide bonds not only in maintaining the structure of mAbs and optimal antigen binding, but also in retaining the effector functions of mAbs. When mutant IgGs lacking inter-heavy chain disulfide bonds were tested, a reduction in complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in chimeric mouse / human IgGs was observed.

[0006] While the utility of half-antibodies is increasingly recognized, it has been found that even partial reduction of the four inter-chain disulfide bonds of IgG results in a dramatic reduction in its complement binding function and CDC.

[0007] If disulfide bonds are essential for maintaining the proper folding, structure, and more importantly, the function of mAbs, it is essential to maintain the structure of the reduced recombinant antibody prior to the construction of mAb conjugates. In an attempt to avoid complex and expensive gene reconstruction of mAbs to maintain the structure of the reduced mAb and the defined conjugation protocol, Abzena has developed a novel method (ThioBridge® technology) for the generation of homogeneous and stable ADCs based on the re-bridging of reduced inter-chain disulfide bonds.

[0008] ThioBridge® technology is a bis-sulfone reagent that re-anneals the reduced inter-chain disulfide bonds of mAbs that form a 3-carbon bridge, thereby maintaining the disulfide structure of the antibody after reduction. The available reduced disulfide bonds are first initiated by Michael addition to the thiolate group of this reduced disulfide bond, followed by elimination of p-toluenesulfinic acid to generate a second Michael acceptor (α-β unsaturated carbonyl), and are cross-linked via a sequential addition-elimination mechanism. Next, the adjacent thiolate group reacts via a second Michael addition to form a 3-carbon bond between the two thiols of cysteine.

[0009] Since the main purpose of using the bis-sulfone linker is to achieve better plasma stability of the ADC, enhanced plasma stability was evaluated by comparing fluorescein-labeled trastuzumab using a maleimide bond with fluorescein-labeled bis-alkylated trastuzumab. The latter showed significant serum stability over 5 days (compared to the maleimide-bonded antibody) in the presence of a high concentration of albumin.

[0010] Furthermore, in the construction of the ADC of trastuzumab, it has been confirmed that using a bis-sulfone reagent with monomethyl auristatin E (MMAE) completely retains the binding activity of trastuzumab to the HER2 receptor and shows strong growth inhibitory activity in HER2-positive cell lines.

[0011] The bis-reactive maleimide linker was developed as a disulfide crosslinking agent via a 2-carbon bridge to obtain bis-thiosuccinamide bonds. The fast reaction rate of the bis-reactive maleimide derivative provides higher structural integrity of the protein and a wider reaction pH range (6 - 8) compared to the bis-sulfone derivative.

[0012] Bis-sulfone reagents and dibromomaleimide derivatives are considered the fastest and most established crosslinking reagents for reducing disulfides. Nevertheless, each has related drawbacks. These include the poor water solubility of the reagent and the lack of selectivity between reducing disulfide bonds and unpaired cysteine residues in the reduced state. Crosslinking is possible in both heavy-chain to light-chain and heavy-chain to heavy-chain, resulting in a mixture of products.

[0013] Therefore, there is interest in discovering additional linkers to replace sulfide crosslinking in antibodies. Examples of other skeletons include pyridazinedione-based compounds such as dibromopyridazinedione that crosslinks disulfide bonds via a 2-carbon bridge. Di-TCEP substituted dithiophenol pyridazinedione has also been used and can function simultaneously as both a reducing agent and a stable crosslinking agent. Other approaches have introduced clickable chemical properties into pyridazinedione-based linkers to allow for the acceptance of additional functionality.

[0014] EP3335734 relates to the use of a drug moiety linked to a linker that can be used to form an anti-drug conjugate via replacement of a leaving group to form a thioether. For example, auristatin E (MMAE) and auristatin F were linked to the linker via amide coupling. Herceptin-linker-drug units are disclosed.

[0015] The re-crosslinking strategy is considered an elegant approach that allows for the introduction of reactive functional groups to reduced mAbs while maintaining the well-defined structure of the mAb after reduction. Despite recent advances and significant research efforts in this area, the aforementioned re-crosslinking approaches via the formation of either 2- and 3-carbon bridges still have practical limitations that require further addressing. This includes low yields and the products being mixtures.

[0016] The present invention was devised in consideration of the above-mentioned considerations.

Summary of the Invention

Means for Solving the Problems

[0017] The present invention provides arylbis(2-haloacetamide) derivatives as next-generation re-crosslinking agents. These bis-haloacetamide derivatives, as described above, have utility in conventional re-crosslinking, but may be particularly useful in the hetero-re-crosslinking of full mAbs (binding of two or more different re-crosslinking linkers) and the generation of half-antibody conjugates.

[0018] To obtain a fully cross-linked antibody (150 KDa), the aforementioned re-crosslinking method (see Background Art), which focuses as much as possible on fully cross-linking the inter-chain disulfide bonds of the heavy chain-light chain and heavy chain-heavy chain, has overlooked the potential advantages in the construction of half-antibodies (75 KDa) as the intended product. For example, half-antibodies may have utility in endosome recycling and tumor penetration. In addition, as far as the inventors know, no particular selectivity has been observed between the heavy chain-heavy chain and heavy chain-light chain disulfide bonds in conventional re-crosslinking techniques. Therefore, site-selective protein labeling to obtain hetero-bifunctional mAbs has been an unresolved issue in the past.

[0019] It is an object of the present invention to address these problems. Linker compound In a first aspect, the present invention provides a linker compound having a bis-(2-haloacetamide) motif. The linker is suitable for re-crosslinking (ligating) the sulfide crosslinks in the antibody structure and / or for ligating within the heavy chain rather than between the heavy chains to form half-antibodies. These ligated antibody structures are referred to as "conjugates".

[0020] Accordingly, in a first aspect, the present invention provides a compound of formula (I):

[0021]

Chemical formula

[0022] (wherein, each X is independently F, Cl, Br, or I, R 1 is H, COOR A , CONH2, CONHR A , CONR A 2, CONHL, or CONR A L, L, when present, is a chain terminating with the reactive group R 3 , each RA is, when present, C 1~4 independently selected from alkyl, n is 0, 1, 2, or 3, each R 2 is, when present, independently selected from F, Cl, Me, CF3, OMe, and OCF3, or R 2 is a group as defined for R 1 (), or a salt thereof. A linker compound is provided.

[0023] When L is present, a polyether or polythioether is suitable for the chain. Preferably, X is Cl, Br, or I, more preferably Br or I. This is because bromo and iodo groups are better leaving groups than chloro groups.

[0024] each R 2 is, when present, independently selected from F, Cl, Me, CF3, OMe, and OCF3, or R 2 is a group as defined for R 1 . R 2 is a group as defined for R 1 when it is the group as defined for R 1 , preferably, n is 1. In other words, the linker compound may have two groups according to the definition for R

[0025] Preferably, each R 2 is, when present, independently selected from F, Cl, Me, CF3, OMe, and OCF3. each R A is, when present, C 1~4 independently selected from alkyl, preferably methyl or ethyl, most preferably methyl.

[0026] The haloacetamide groups are suitably arranged ortho or meta to each other. For example, the linker compound is of formula (Ia) or (Ib):

[0027] [Chemical formula]

[0028] It may also be a compound of The linker compound of formula (Ia) may be called ortho or 1,2-, while the linker compound of formula (Ib) may be called meta or 1,3-.

[0029] Preferably, n is 0. That is, R 2 does not exist. Preferably, in the compound of formula (Ib), R 1 group and two haloacetamide groups are placed in a 1,3,5 configuration.

[0030] Therefore, the linker compound is of formula (IIa), (IIb), or (IIc):

[0031] [Chemical formula]

[0032] It may also be a compound of R 1 When is H, it will be understood that the compounds of formulas (IIa) and (IIc) are equivalent.

[0033] The linker compound of formula IIa is referred to as 3,4-substituted. The linker compound of formula (IIb) is referred to as 3,5-substituted. The linker compound of formula (IIc) is referred to as 2,3-substituted. These names are usually used when R 1 is not H.

[0034] In the linker compound, R 1 is H, COOR A , CONH2, CONHR A , CONR A 2, CONHL, or CONR A L, and L, when present, is the reactive group R 3 (-Sp -R 3 a spacer S having -R as the terminus p and each R A , when present, is independently selected from C 1~4 alkyl, preferably methyl or ethyl, and most preferably methyl.

[0035] In a preferred compound of the present invention, R 1 is H, COOR A , CONH2, or CONHL, more preferably H, COOMe, CONH2, or CONHL. L group L is a chain having a reactive group at the terminus. Thus, additional functionality can be introduced into the conjugate via a reaction at the reactive group. The reactive group may be referred to as R 3 .

[0036] In other words, L may be -S p -R 3 (wherein S p is a spacer and R 3 is a reactive group). Spacer S p may be, for example, a polyether or thioether up to 32 atoms in length. For example, spacer S p may be a polyether or thioether having 10 to 32 atoms in length, for example, 10 to 23 atoms in length.

[0037] For example, spacer S p may be polyethylene glycol. Thus, in some embodiments, spacer S p is of the formula:

[0038]

Chemical formula

[0039] (wherein ---- is a bonding point) polyether. In other words, the spacer may be a PEG chain. m may be selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some cases, m is selected from 3, 4, 5, 6, 7, 8, 9, and 10. In some cases, m is selected from 3, 4, 5, 6, and 7. Spacers where m is 3 and m is 7 are exemplified herein and may be preferred.

[0040] R 3 is a reactive group. R 3 may be a head group of formula (H), or any group that can subsequently be replaced or modified for the purpose of attaching another molecule. The term head group, as used herein, refers to formula (H)

[0041]

Chemical formula

[0042] (wherein X, R 2 , and n are as defined above, and ---- represents a point of attachment) refers to the arylbis(2-haloacetamide) moiety described above, or the corresponding moiety in any compound of formulas (I)-(VII).

[0043] The options and preferences for formula (I) above apply to formula (H). Thus, R 3 may be (Hi) or (Hii):

[0044]

Chemical formula

[0045] may be the moiety described in Preferably, X is Br or I. Preferably, n is 0. Preferably, the configuration in (Hi) is 1,3,4 (further any R 2 groups).

[0046] The linker compound is spacer Sp When including two arylen bis-haloacetamide head groups linked by p , these arylen bis-haloacetamide head groups may be the same or different.

[0047] In other words, for the purpose of binding to formula (H), or another molecule, it may be any group that can subsequently be replaced or modified. For example, R 3 is -N3 (azide), alkynyl (e.g., -C≡C-H or other groups containing a C≡C bond, e.g., dibenzocyclooctyne (DBCO) substituent), a protected amine (e.g., an NHP group such as NHBoc, NHFmoc, NHCbz), a leaving group, or a moiety of formula (H), preferably, R 3 may be N3 (azide), alkynyl (e.g., -C≡C-H), or a moiety of formula (H).

[0048] Suitable leaving groups will be apparent to those skilled in the art and include, for example, -I, -Br, -Cl, -OH, -OP, -O-aryl (e.g., a phenoxide leaving group), -OTs, -OMs, -SP, -S-C 1~4 alkyl, -OSO2-C 1~4 alkyl may be included.

[0049] P is a protecting group. Suitable protecting groups are known in the art and are described, for example, in Greene’s Protective Groups in Organic Synthesis (4th Edition) (Peter G.M.Wuts, Theodora W.Greene), the content of which is incorporated by reference.

[0050] In some embodiments, R 3 is -N3 or a C≡C bond, e.g., a group containing -C≡C-H or DBCO. In these cases, the compound is suitable for reactions by click chemistry using methods known in the art. -N3 and DBCO may be preferred because click chemistry can be achieved without using a copper catalyst.

[0051] In some embodiments, R 3 is - N3. The azide group may be used in click chemistry reactions and may be reduced to unmask an amine. Accordingly, the linker compound may be a compound of formula (III):

[0052]

Chemical formula

[0053] (when n is not 0, the nR 2 groups may be present as in formula (I), and preferably, n is 0). For example, the linker compound may be a compound of formula (IIIa) or (IIIb):

[0054]

Chemical formula

[0055] (when n is not 0, the nR 2 groups may be present as in formula (I), and preferably, n is 0). Preferably, the regiochemical configuration in formula (IIIa) is 1,3,4.

[0056] In some embodiments, R 3 is a moiety of formula (H). All of the above preferences and options regarding formulas (I) through (VII) apply equally. Accordingly, the linker compound may be a compound of formula (IVa), (IVb), or (IVc):

[0057]

Chemical formula

[0058] (when n is not 0, the nR 2The base may be present as in formula (I), preferably, n may be 0) and may be a compound. Preferably, the regiochemical configuration in formulas (IVa) and (IVc) (the left head groups) is 1,3,4. The X groups may be the same or different. In some embodiments, each X is Br or I. In some embodiments, for two X, one arylene is Br and for two X or the other arylene is I.

[0059] The compounds of formula (IV) may be useful in the preparation of thio-bridged fAb conjugates, which may then be conjugated to an additional moiety, for example, an antibody as shown in Example 10.

[0060] In some embodiments, L is absent. In other words, R 1 is selected from H, COOR A , CONH2, CONHR A , and CONR A 2, and preferably, R 1 is selected from H, COOR A , and CONH2, and most preferably, R 1 is selected from H, COOMe, and CONH2.

[0061] In some embodiments, R 1 is H. Thus, the linker compound may be of formula (Va) or (Vb):

[0062]

Chemical formula

[0063] and may be a compound of. In some embodiments, R 1 is COOR A . Thus, the linker compound may be of formula (VIa), (VIb), or (VIc):

[0064]

Chemical formula

[0065] (When n is not 0, nR 2 groups may be present as in formula (I), and preferably, n is 0) may be a compound. Preferably, R A is Me.

[0066] In some embodiments, R 1 is CONH2, CONHR A , or CONR A 2 (all represented by CONR2). Thus, the linker compound is of formula (VIIa), (VIIb), or (VIIc):

[0067]

Chemical formula

[0068] (When n is not 0, nR 2 groups may be present as in formula (I), and preferably, n is 0) may be a compound. In some embodiments, each X in the compounds of any of the formulas described herein is chloro.

[0069] In some embodiments, each X in the compounds of any of the formulas described herein is bromo. In some embodiments, each X in the compounds of any of the formulas described herein is iodo. Exemplary linker compounds of the present invention Exemplary linker compounds 1-18 are shown below. Compounds 1, 2, 3, 4, 5, 6, and 7 are compounds of formulas (VIa), (VIb), and (VIc) substituted with methyl esters. Compounds 8, 9, 10, 11, 12, and 13 are compounds where R 1 is H. These are compounds of formulas (Va) and (Vb).

[0070] Compounds 14, 15, 16, 17, and 18 contain polyethylene glycol (PEG) chain pendants from the aryl ring. This corresponds to spacer S p This spacer, when used, may terminate with a second aryl bis(2-haloacetamide) moiety (e.g., Compound 18), which is a compound of formula (IVa). Alternatively, this spacer may terminate with a different group (referred to herein as R 3 ), which is less reactive towards thiol substitution than the halogen of the haloacetamide group. In this latter type, the compound may be considered to be orthogonally activated. This aryl bis(2-haloacetamide) group may form a bridge between two thiol groups, and then its spacer end group may react with or de-mask and further react with additional thiol groups of an antibody, antibody fragment, drug, and / or other protein, or different functional groups of an antibody, antibody fragment, drug, and / or other protein. In Compounds 14, 15, 16, and 17, their end groups are azides, and these compounds are compounds of formula (IIIa) and (IIIb).

[0071] In some embodiments, the linker compounds of the invention are:

[0072]

Chemical formula

[0073] selected from. Conjugate In a further aspect, the invention may provide an antibody, half-antibody, or antibody fragment having a linker as described herein. These may be referred to as antibody conjugates, half-antibody conjugates, or antibody fragment conjugates.

[0074] The linker usually crosslinks two thiols generated by the reduction of disulfide bridges in the antibody structure. This crosslinking may be a heavy chain-light chain crosslink, a heavy chain-heavy chain crosslink, or the linker may form a crosslink within the same heavy chain.

[0075] Accordingly, the present invention provides a conjugate comprising a motif of formula (VIII):

[0076]

Chemical formula

[0077] (wherein all substituents are as described herein and the linker may optionally be as depicted in any of formulas (I)-(VII)). The preferences for each of these structures apply equally.

[0078] The motif is considered to be the residue of the linker compound described herein, i.e., the structure of the linker compound remaining after the reaction. In other words, the "linker" or "residue" is the portion remaining after replacement of the halide leaving group. In some embodiments, the linker of any conjugate of the present invention is the residue of any one of exemplary compounds 1-18.

[0079] Figure 1 schematically shows certain conjugate structures of the present invention. Figure 1(1) shows a half-antibody having a linker that re-bridges the heavy and light chains and a second (identical) linker that links the intra-heavy chain disulfides rather than between the heavy chains. Figure 1(3) shows a half-antibody conjugate in which those linkers are different. This may refer to a hetero-bifunctionalized half-antibody. Figure 1(2) shows a conjugate that is a full-length antibody in which a single linker bridges the heavy-light chain. When that linker has a chain terminated with a reactive group (defined as "L" in the claims), that linker can be orthogonally activated for further cross-coupling, for example, to generate a trispecific antibody or an antibody-protein conjugate. Refer to Figures 1(4) and 1(8). It will be understood that the head group portion of the linker may be the same or different. Figure 1(5) shows a physical state in which the half-antibody is observed, which is chemically identical to the structure shown in Figure 1(1). Figure 1(6) shows a physical state in which the half-antibody is observed, which is chemically identical to the structure shown in Figure 1(3). Figure 1(7) shows a conjugate that is a full-length antibody in which two linkers bridge the heavy-light chain.

[0080] Thus, in some embodiments, the present invention may provide a product comprising an antibody having a linker of formula (VIII) or any linker described herein. Thus, in some embodiments, the present invention may provide a product comprising a half-antibody having a linker of formula (VIII) or any linker described herein. In other words, the present invention may provide a half-antibody having a linker of formula (VIII) or any linker described herein that bridges within two sulfide moieties on the same heavy chain.

[0081] In some embodiments, the invention provides a half antibody having a linker as defined above that crosslinks within two sulfide moieties on the heavy chain, and an additional linker as described in formula (VIII) that crosslinks the heavy chain and the light chain. These linkers of formula (VIII) can be the same (Figure 1(1)) or different (Figure 1(3)).

[0082] Accordingly, in some embodiments, the invention may provide a product comprising an antibody fragment having a linker as described in formula (VIII) or any linker described herein.

[0083] In some embodiments, the invention relates to a trispecific antibody. Thus, specific Fab-Mab conjugates can be generated (see (4) in Figure 1). Accordingly, in some embodiments, the invention provides a thio-bridged Fab. In other words, it is a Fab fragment re-bridged by a linker compound as described in formula (VIII) or any linker described herein. Preferably, the linker compound contains L. For example, it may be a linker compound of formula (IIIa), (IIIb), (IVa), (IVb), or (IVc).

[0084] In other words, the invention provides a thio-bridged Fab, wherein the bridging is by formula (IXa), (IXb), or (IXc)

[0085]

Chemical formula

[0086] (wherein ---- represents the binding point to the protein chain, and m is selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10), and can provide a thio-bridged Fab. In some cases, m is selected from 3, 4, 5, 6, 7, 8, 9, and 10. In some cases, m is selected from 3, 4, 5, 6, and 7. It may be preferred that m is 3 or m is 7.

[0087] In some embodiments, the present invention may provide an antibody conjugate, a half - antibody conjugate, or an antibody fragment conjugate (e.g., a thiol - bridged Fab) that binds to a drug and / or a protein or other compound (e.g., a fluorescent label or a radioactive label). Such compounds may sometimes be referred to herein as products. Further considerations regarding methods of making the products of the present invention are found below. Thus, in some embodiments, the present invention is a product, wherein its linker compound comprises L, and R 3 is replaced by an antibody, a half - antibody, an antibody fragment, a protein, a polypeptide, a drug, or a fluorescent label or a radioactive label, and provides a product.

[0088] In some embodiments, the present invention provides a product comprising a linker motif as shown in formula (IXa) above (wherein the X group is replaced by an antibody, a half - antibody, an antibody fragment, a protein, a polypeptide, a drug, or a fluorescent label or a radioactive label). For example, each X may be replaced by an S----- resulting from the replacement of the X leaving group by a thiol group of an antibody, a half - antibody, an antibody fragment, a protein, or a polypeptide. Method In a further aspect, the present invention may provide a method of making a conjugate and a product comprising the conjugate described herein.

[0089] Thus, in a further aspect, the present invention relates to a method comprising the steps of forming a conjugate, treating an antibody, a half - antibody, or an antibody fragment with a reducing agent such as TCEP that effects reduction or partial reduction, and then treating the reduced or partially reduced antibody, half - antibody, or antibody fragment with a linker compound of any of the formulas described herein to obtain the corresponding conjugate.

[0090] Preferably, after the reduction step, the reaction is quenched prior to the addition of the linker compound. This prevents any reaction between any residual TCEP and the linker compound. A suitable quenching agent is the penta-PEG azide described by Kantner et al.

[2017] . Antibody conjugate In some embodiments, the present invention relates to a method for generating a monofunctionalized antibody as shown in FIG. 1(2). That is, a conjugate having a single linker that crosslinks the heavy and light chains. This is: (i) partial reduction of the antibody with a reducing agent such as TCEP, followed by treatment with a linker compound described in any one of formulas (I)-(VII) described herein, or (ii) utilizing the selectivity of a particular linker compound of the present invention for preferential re-crosslinking of the heavy chain-light chain can be obtained by.

[0091] Accordingly, the present invention may provide a method for generating a monofunctionalized antibody having the motif shown in formula (VIII) or any linker described herein, the method comprising treating a partially reduced antibody with a linker compound described in any one of formulas (I)-(VII) described herein. In some embodiments, the method further comprises the step of preparing a partially reduced antibody by reducing the antibody using about 1 to 1.1 equivalents, for example, about 1.1 equivalents of a reducing agent such as TCEP. Such a product is shown in FIG. 1(1).

[0092] In some embodiments, the method of the invention is a method of generating a bifunctional antibody having two motifs each represented by formula (VIII) or any linker described herein, the method comprising treating a partially reduced antibody with a linker compound according to any one of formulas (I)-(VII) described herein. At least 2 equivalents of the linker compound will be used. Usually, this linker compound will be provided in excess of 2 equivalents. In some embodiments, the method further comprises preparing a partially reduced antibody by reducing the antibody using about 2-2.2 equivalents, such as about 2.2 equivalents, of a reducing agent such as TCEP.

[0093] In some embodiments, the linker compound is a compound in which R 1 is COOR A or H. As described above, the inventors have recognized that an unsubstituted linker may be preferred for light chain-heavy chain crosslinking. Thus, in some methods, the compound is of formula (Va) or (Vb), preferably a compound of (Va). That is, R 1 is H and n is 0. Preferably, X is bromo. Thus, the linker may be selected from compounds 9 and 12, preferably compound 12.

[0094] In other embodiments, the linker compound is a compound in which R 1 is COOR A , preferably COOMe. Thus, in some methods, the compound is of formula (VIa), (VIb), or (VIc), preferably a compound of (VIa). That is, R 1 is COOMe and n is 0. Preferably, X is bromo. Thus, the linker may be selected from compounds 2 and 5, preferably compound 5.

[0095] In some embodiments, R 1 contains L. For example, R 1is de-masked and may be conjugated to a drug moiety, additional antibody, half-antibody, or antibody fragment, protein, polypeptide, drug, or other compound due to orthogonal functionalization, linker compounds 14, 15, 16, 17, and 18, preferably CONHL as in linker compounds 14, 15, 16, and 17. Thus, the method may further comprise the step of attaching an additional moiety, such as an antibody, half-antibody, antibody fragment, protein, polypeptide, drug, or other compound (e.g., a fluorescent or radioactive label). In such methods, the structures shown in FIGS. 1(4) and 1(8) may be produced, where FIG. 1(4) illustrates the product obtained when the two head groups are the same and FIG. 1(8) illustrates the product obtained when the two head groups are different.

[0096] In some embodiments, methods for generating the monofunctionalized antibodies shown in FIG. 1(2) are initiated using selectively partially reduced antibodies. These methods utilize the preference of the specific linker compounds of the invention, in particular the linker compounds of formula (Ia) for re-crosslinking heavy chain-light chain. X may be selected from Br and I, preferably X is Br.

[0097] Accordingly, in some embodiments, the invention provides a method for generating a monofunctionalized antibody having the motif shown in formula (VIII) or any linker described herein, the method comprising treating a fully reduced antibody with about 1 to 1.1 equivalents of a linker compound of formula Ia. In some embodiments, X is Br.

[0098] Preferably, the linker compound is a compound of formula (IIa). In some embodiments, the linker compound is a compound in which R 1 is COOR A or H. As noted above, the inventors have recognized that an unsubstituted linker may be preferred for light chain-heavy chain crosslinking. Thus, in some methods, the compound is a compound of formula (Va). That is, R1 is H and n is 0. Thus, the first linker may be Compound 12.

[0099] In other embodiments, the linker compound is R 1 is COOR A , preferably a compound that is COOMe. Thus, in some ways, the compound is a compound of formula (VIa). That is, R 1 is COOMe and n is 0. Thus, the first linker may be Compound 5.

[0100] In some embodiments, R 1 contains L. For example, R 1 is deprotected and may be conjugated to a drug moiety, an additional antibody, a half-antibody, or an antibody fragment, or a protein, and may be CONHL as in linker compounds 16, 17, and 18, preferably linker compound 16 or 17, due to orthogonal functionalization. Thus, the method may further comprise the step of binding an additional moiety, such as an antibody, a half-antibody, an antibody fragment, a protein, a polypeptide, a drug, or another compound (e.g., a fluorescent label or a radioactive label). Half-antibody conjugate In some embodiments, the method produces a half-antibody having a linker motif that crosslinks within a sulfide moiety in the same heavy chain (examples are shown in FIGS. 1(1) and (3)).

[0101] Thus, in some embodiments, the present invention provides a method for producing a half-antibody (so-called half-antibody conjugate) having a motif of formula (VIII) that crosslinks the -HC-HC intramolecular cysteine residues in the hinge region or any linker described herein, the method comprising treating a fully reduced antibody with a linker compound described in any one of formulas (I)-(VII) described herein. Such half-antibodies are the labeled HC-LC in the figure, see, for example, FIG. 5.

[0102] The inventors have recognized that the main product using both the ortho and meta compounds described herein in excess (4 equivalents or more) is a half-antibody conjugate having two motifs described in formula (VIII) or any linker described herein. Thus, the half-antibody product contains two identical linkers as shown in Figure 1(1), the first crosslinking within the sulfide moiety in the heavy chain and the second crosslinking the heavy and light chains.

[0103] When using the ortho compound, there are few by-products observed. Thus, the linker compound may preferably be a compound of formula (Ia), for example, a compound of formula (IIa) or (IIc). Preferably, n is 0. The compound may be a compound of formula (IIIa), (IVa), (Va), (VIa), (VIc), (VIIa), or (VIIc), preferably (IIIa), (IVa), (Va), (VIa), or (VIIa).

[0104] Preferably, X is Br or I, preferably Br. In some embodiments, the linker compound is selected from Compounds 2, 5, 9, 12, 16, and 17, preferably Compounds 5 and 12.

[0105] The linker compound is provided in at least 4 equivalents, for example, at least 5 equivalents of stoichiometry, and may be provided, for example, in about 8 equivalents. When the linker compound is provided in less than 4 equivalents, for example, about 1 - 1.1 or 2 - 2.2 equivalents of stoichiometry, the inventors have recognized the regioselectivity in the compounds of the present invention. For example, the inventors have recognized a clear preference for crosslinking within -HC in the meta compound, particularly when X is I.

[0106] Accordingly, in some embodiments, the present invention provides a method for generating a half-antibody having a motif of formula (VIII) that crosslinks -HC-HC intramolecular cysteine residues in the hinge region or any linker described herein, the method comprising treating a fully reduced antibody with a linker compound of formula (Ib) described herein.

[0107] Preferably, n is 0. That is, the linker compound is a compound of formula (IIb). For example, the linker compound may be a compound of formula (IIIb), (IVb), (IVc), (Vb), or (VIb), preferably (IIIb), (IVb), (IVc), (Vb), or (VIb).

[0108] Preferably, X is I or Br, most preferably I. Preferably, R 1 is H, COOMe, CONH2, or CONHL. In some embodiments, the linker compound is selected from compounds 2, 3, 9, 10, 14, and 15, preferably compounds 3, 10, and 15.

[0109] If at least one of the linker compounds contains L, the method may include the step of attaching an additional moiety, such as an antibody, half-antibody, antibody fragment, protein, polypeptide, drug, or other compound (e.g., a fluorescent or radioactive label). Compound 15 is a preferred example.

[0110] In some embodiments, the amount of the linker compound is about 2 to 2.2 equivalents. Next, the method may further include the step of re-crosslinking the HC-LC disulfide. This re-crosslinking may, where possible, be by oxidation or, more preferably, by treating the half-antibody with an additional linker compound as described in the present invention to generate a disulfide bridge. In the latter case, the linker compound may be any of the linker compounds described herein and may be used at about 1 to 1.1 equivalents or more per half-antibody conjugate, i.e., about 2 to 2.2 equivalents or more per fully reduced antibody, to create the conjugate shown in Figure 1(3).

[0111] In some embodiments, the method produces a half-antibody having a linker that crosslinks within the sulfide moiety in the heavy chain (as shown in Figure 1(3)) and a different linker that crosslinks the heavy chain and the light chain.

[0112] Accordingly, in some embodiments, the present invention provides a method for producing a hetero-bifunctionalized half-antibody having a first linker that is a motif described in formula (VIII) that crosslinks the heavy chain and the light chain or any linker described herein, and a second linker that is a motif described in formula (VIII) that crosslinks the -HC-HC intradomain cysteine residues in the hinge region or any linker described herein, the method comprising: (i) treating a partially reduced antibody with a first linker compound as described in any one of formulas (I)-(VII) herein to produce a first conjugate; and then (ii) further reducing the first conjugate to produce a reduced conjugate; and then (iii) treating the reduced conjugate with a second linker compound as described in any one of formulas (I)-(VII) herein to produce the hetero-bifunctionalized half-antibody conjugate, wherein the first and second linker compounds are different.

[0113] In some embodiments, one of the first and second linker compounds comprises an L group. In other words, at least one of the linker compounds may be a compound of formula (III) (e.g., IIIa or IIIb, preferably, IIa), (IVa), (IVb), or (IVc) (preferably, IVb). Preferably, the second linker compound comprises L as in formula (III), (IIIa), (IIIb), (IVa), (IVb), and (IVc), more preferably, (IIb) and (IIIb). For example, the second linker compound may be selected from compounds 14, 15, 16, and 17, preferably, may be compound 15.

[0114] In some embodiments, the first linker compound does not comprise L. In some embodiments, the first linker compound is a compound where R 1 is COOR A or H. The inventors have recognized that unsubstituted linker compounds may be preferred for light chain - heavy chain crosslinking. Thus, in some methods, the linker compound is a compound of formula (Va) or (Vb), preferably, (Va). That is, R 1 is H and n is 0. Preferably, X is bromo. Thus, the first linker compound may be selected from compounds 9 and 12, preferably, may be compound 12.

[0115] When at least one of the linker compounds comprises L, the method may include the step of binding an additional moiety, such as an antibody, half - antibody, antibody fragment, protein, polypeptide, drug, or other compound (e.g., a fluorescent or radioactive label).

[0116] In some embodiments, the first linker compound is a compound where R 1 is COOR A , preferably, COOMe. Thus, in some methods, the linker compound is a compound of formula (VIa), (VIb), or (VIc), preferably, (VIa). That is, R 1is COOMe and n is 0. Preferably, X is bromo. Thus, the first linker compound may be selected from Compounds 2 and 5, and preferably may be Compound 5.

[0117] In some embodiments, the method further includes preparing a partially reduced antibody by partially reducing the antibody using a reducing agent such as about 2 - 2.2 equivalents, e.g., about 2.2 equivalents of TCEP.

[0118] In some embodiments, the step of further reducing the conjugate uses a reducing agent such as about 2 - 2.2 equivalents, e.g., about 2.2 equivalents of TCEP. Suitably, after the reduction step, the reaction is quenched, e.g., using penta - PEG azide, prior to the addition of the linker compound.

[0119] In some embodiments, the present invention provides a method for generating a hetero - bifunctionalized half - antibody conjugate, comprising treating a fully reduced antibody with a first linker compound of formula (Ia) wherein X is Br and a second linker compound of formula (Ib) wherein X is I. The first and second linker compounds may be added simultaneously. The first linker compound re - crosslinks the heavy and light chains, and the second linker compound crosslinks the - HC - HC internal cysteine residues in the hinge region. The product is shown in Figure 1(3), where L1 represents the first linker and L2 represents the second linker. In some embodiments, the first linker compound is a compound described by formula (IIa) and the second linker compound is a compound described by formula (IIb). Fab conjugate In some embodiments, the present invention relates to a method for generating a stable thio - bridged fAb conjugate. That is, a product comprising a light chain and a digested (cleaved) heavy chain linked by a linker of formula (VIII) described herein.

[0120] The fAb starting material can be obtained commercially or by methods described in the art, for example, by digestion of an antibody using papain. See Andrew & Titus

[2000] .

[0121] Methods for generating thio-bridged fAb conjugates may (i) reducing the fAb fragment to produce a reduced fAb precursor, and then (ii) treating the reduced fAb precursor with a linker compound described in any one of formulas (I)-(VII) herein to produce the thio-bridged fAb conjugate and may include.

[0122] Preferably, the linker compound contains an L group for additional functionalization / conjugation. That is, R 1 contains L. For example, R 1 may be CONHL as in linker compounds 14, 15, 16, 17, and 18, preferably compound 18.

[0123] In other words, the linker compound may be a compound of formula (III) (preferably, IIIa), (IVa), (IVb), or (Vc) (preferably, VIa). Most preferably, the linker compound is a compound of formula (IVa), (IVb), or (IVc), preferably (IVa). X may be Br, and preferably, X is Br.

[0124] Thus, the method may further include treating a conjugated product, such as an fAb-mAb or fAb-protein product, with the thio-bridged fAb conjugate using a reduced or partially reduced antibody, half-antibody, antibody fragment, protein, polypeptide, drug, or other compound.

[0125] The inventors have found that in the methods described herein, the selectivity is improved when the pH of the reaction is about 7.5. Thus, in some of the methods described herein, the pH is less than about 8, preferably between about 6 and about 8, and most preferably about 7.5. The preferred pH values have been found to result in higher yields. The inventors note that the reaction proceeds further towards completion, presumably as a result of reagent hydrolysis at high pH and lower reactivity at low pH (due to thiol protonation).

[0126] In some embodiments, the antibody in any of the methods described herein is selected from trastuzumab and rituximab. The invention includes the described aspects and combinations of preferred features, except where such combinations are clearly not permitted or are clearly avoided.

[0127] Aspects and experiments illustrating the principles of the invention will be discussed further below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0128]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12-1

Figure 12-2

Figure 13-1

Figure 13-2

Figure 13-3

Figure 13-4

Figure 14

Mode for Carrying Out the Invention

[0129] Aspects and embodiments of the present invention will be discussed further below with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference. Stability of Compounds The linker compounds of the present invention have desirable stability in water and are not readily hydrolyzed.

[0130] Significant differences were observed in the aqueous solution stability of methyl bis(2-haloacetamido)benzoates stored in phosphate buffer at pH = 7. Generally, the 3,5-compounds (meta-configuration) were much more stable than the 2,3- and 3,4-compounds (ortho-configuration). Interestingly, the stability did not directly correlate with the halide leaving groups within each group of the compounds. The stability in 3,4-substituted benzoic acids was in the order of I < Br < Cl (most stable). The stability in 3,5-substituted benzoic acids was in the order of Br < Cl < I (most stable).

[0131] Interestingly, when the aryl has an amide group (i.e., R 1 is amide), contrary to the normal trend in halide stability, iodine is more stable than the corresponding bromo compound.

[0132] R 1 In compounds having an amide group, the stability of the ortho and corresponding meta compounds was more similar. Exchanging the ester functional group for an amide functional group reversed the trend in halide stability (iodo- is more stable than bromo-), and the 2,3-modification had a stability similar to that of the 1,3-modification. The presence of the L group improves solubility.

[0133] Unsubstituted compounds (R 1 is H) showed the same stability trend as the ester derivatives, but generally had lower stability. Without being bound by any particular theory, the inventors believe that the inductive electron-donating group / mesomerically electron-withdrawing group R 1The presence of 1 is not H.

[0134] All are sufficiently stable for use. Reactivity towards thiols The relative reactivity of the compounds towards small thiols, including glutathione which is a peptide, was evaluated (pH = 7, room temperature). Bis-(2-chloroacetamide) shows a significantly slower reaction rate than the bromo- or iodo-derivatives. The 3,4-bis-Cl compound (4) was found to react significantly faster than the 3,5-bis-Cl compound (1). Without being bound by any particular theory, the inventors speculate that the differences in reactivity and stability between the ortho and meta compounds may be due to differences in the 3D structure of each compound. Methyl 3,5-bis(2-chloroacetamide)benzoate (1) is predicted to have a planar conformation. Thus, steric hindrance can slow down the backside attack of the thiolate at the carbon bonded to the halogen (via the S N 2 mechanism). This can explain the greater reactivity of ortho compounds such as methyl 3,4-bis(2-chloroacetamide)benzoate (4). Compound (4) is not planar and can provide a carbon that is more accessible in the backside attack.

[0135] Thus, in some of the compounds, conjugates, and methods described herein, the linker compound may be selected such that it is not a compound of formula (VIb).

[0136] The chloro-derivatives react with thiols and are viable linking compounds, but in the examples described herein, mainly bromo- and iodo-acetamides are used because the reaction rate is faster. In other words, preferably, X is Br or I. All have sufficient reactivity for use in the methods of the invention.

[0137] Selectivity of the compounds In ester and amide substituted compounds and unsubstituted arylene compounds, when reacted with trastuzumab, the pattern of thiol crosslinking in ortho- and meta-substituted linker compounds is different.

[0138] When the linker compound is provided in excess, this reaction produces a half-antibody product bound to two linkers that crosslink one intra-heavy chain (H-H) disulfide. The inventors have recognized that linker compounds having a meta (i.e., 1,3-bis-(2-haloacetamido)-) configuration produce more by-products by generating more between LC-LC and H-H. Therefore, when the antibody is fully reduced and the linker is provided in excess (more than 4 equivalents), it may be desirable to use the ortho linker compound of formula (Ia).

[0139] However, the inventors have recognized that the ortho results in fewer "by-products" (between LC-LC, -HC), while the meta compound has a preference for -HC internal linkages and the formation of half-antibodies. Therefore, when treating a fully reduced antibody with less than 4 equivalents of linker, it is desirable to use the meta compound (i.e., the linker compound of formula (Ib)) for the formation of the half-antibody conjugate.

[0140] The inventors have also noted that, regardless of the positional chemical structure, aryl bis(2-iodoacetamide) compounds (X = I) exhibit a higher preference for -HC internal linkages than the corresponding aryl bis(2-bromoacetamide) compounds (X = Br). Therefore, for the preferential formation of half-antibody conjugates ((1) and (3) in Figure 1), compounds where X is I are preferred.

[0141] In the generation of HC-LC conjugates (i.e., antibodies having a linker that crosslinks the heavy and light chains), the inventors have recognized that ortho compounds exhibit higher selectivity. Thus, when treating a fully reduced antibody with less than 4 equivalents of a linker compound, it is desirable to use an ortho compound (i.e., a linker compound of formula (Ia)) for the generation of a monofunctionalized HC-LC antibody conjugate (i.e., one linker crosslinks the heavy and light chains).

[0142] The inventors have also recognized that, regardless of the regiochemical structure, arylbis(2-bromoacetamide) compounds (X = Br) exhibit higher preference for HC-LC ligation than the corresponding arylbis(2-iodoacetamide) compounds (X = I). Thus, for the preferential generation of HC-LC antibody conjugates ((2) in FIG. 1), compounds where X is Br are preferred.

[0143] When stoichiometric and near-stoichiometric (slightly excess) amounts of a reducing agent are used, the position of the linker in the conjugate can be determined by the regioselectivity of disulfide bridge reduction.

[0144] The inventors have recognized that a compound undergoes selective reduction of a single HC-LC disulfide bridge to generate one HC-LC conjugate per mAb. In reactions using such partially reduced antibodies, unsubstituted arylene linkers have been shown to allow the reaction to proceed better. When less than 4 equivalents of a reducing agent are used, unsubstituted arylene linkers may be preferred in the generation of HC-LC conjugates.

[0145] Example 11 provides an exemplary illustration of this positional selectivity. The fully reduced Tmab (reduced four disulfides) was treated with only 2 equivalents of the linker compound. The 3,4-compound (X = I) showed a high preference for LC-HC and little preference for the -HC internal cross-link. The 3,5-compound (X = I) showed a high preference for within -HC, and almost all HCs have one linker, but an HC-LC conjugate using two linkers is also formed (this means that the first -HC internal product with one linker can then react with another linker to bind LC). No HC-LC product containing only one linker molecule was observed.

[0146] The inventors believe that these selectivities extend to other mAbs by the examples regarding rituximab described herein. Therefore, it will be recognized that by utilizing the selectivity of the linker compound, site-selective monofunctionalization of mAbs such as trastuzumab can be obtained by treating the fully reduced mAb with a limited amount (less than 4 equivalents) of any linker compound of the present invention. The present invention further shows the possibility of achieving hetero-difunctionalization of mAbs either through sequential reduction and treatment with linker compounds or by simultaneously hetero-difunctionalizing multiple mAbs using a mixture of linker compounds having different site selectivities.

[0147] Stability of the conjugate The positional chemistry of the diacetamide compound has a small difference in the stability of its conjugate (antibody, semi-antibody, and antibody fragment containing the linker of the present invention). This 3,4-linked conjugate is slightly more stable than the 2,3- and 3,5-linked conjugates. All diacetamide conjugation products were found to be significantly more stable than the conjugation products of maleimide using glutathione. When stored in the presence of dithiothreitol (DTT), all conjugates showed the same level of stability as the samples stored without DTT.

[0148] definition antibody The term antibody is well understood in the art and is synonymous with immunoglobulin (Ig). In humans, there are five types of antibodies: IgG, IgA, IgM, IgE, and IgD. While the term antibody is intended to encompass all types, it will be recognized that the antibody type herein will be exclusively IgG. The inventors have observed common characteristics among IgG isotypes; for example, IgG1 and IgG4 have been tested and found to have comparable selectivity for linker compounds and products. It will also be apparent to those skilled in the art that the term antibody is intended to encompass monoclonal antibodies. The examples use the monoclonal antibodies trastuzumab, ipilimumab, and rituximab. While these monoclonal antibodies may be preferred in some cases, it should be understood that the present invention is not intended to be limited thereto.

[0149] As used herein, unless the context dictates otherwise, the term antibody refers to a whole antibody, ie, both heavy and light chains in a monomeric form in a Y-configuration.

[0150] half antibody The term half antibody is art-recognized and is used herein to describe a heavy chain-light chain pair.

[0151] antibody fragment As used herein, unless otherwise specified, the term antibody fragment refers to an antigen-crosslinking fragment that can be generated from the variable region of an antibody. Thus, the term antibody fragment is intended to encompass F(ab')2, Fab, Fab', and Fv antibody fragments. The antibody fragment will be selected according to the purpose, and it will be understood that the selection and linking of appropriate antibody fragments is within the ability of one skilled in the art. A particularly preferred fragment is a Fab fragment.

[0152] In some embodiments, the present invention relates to trispecific antibodies. Thus, specific Fab-Mab conjugates can be generated (see (4) and (8) of FIG. 1). Antibody conjugate As used herein, the term "conjugate" refers to the presence of the linker described in the present invention in the product. This linker is attached to an antibody, a half-antibody, or an antibody fragment. The linker that can be attached may crosslink two chains or may link intra-chain disulfides rather than between heavy chains.

[0153] The linker may bind to additional moieties, such as an antibody, a half-antibody, an antibody fragment, a protein, a polypeptide, a drug, or another compound (e.g., a fluorescent label or a radioactive label). For example, a linker compound such as linker compound 18 contains two bis(haloacetamide) head groups that can crosslink thiol linkages and can thus be used, for example, for the formation of an mAb protein conjugate (see Example 10), while linker compounds 14, 15, 16, and 17 contain azides that can be used to attach additional moieties, for example, via click chemistry or amide coupling.

[0154] When the conjugates described herein are linked to additional moieties, the term "conjugate product" is used. Examples of conjugate products include fAb-mAb conjugates, antibody-drug conjugates, mAb-protein conjugates, and labeled conjugates (e.g., mAb-fluorophore conjugates).

[0155] Protein or polypeptide The linker of the present invention can be used, for example, to generate mAb-protein conjugates. The term protein includes antibodies, antibody fragments, or antibody derivatives, but as used herein, the term is intended to include non-antibody proteins. The term polypeptide refers to short chains of amino acids. This can be conjugated (via reduction and peptide coupling) via the azide moieties of linker compounds 14, 15, 16, and 17, or introduced directly via the use of compound 18. Many polypeptide drugs are known in the art and may be useful in the methods and products of the present invention.

[0156] Abbreviations In the art, it will be understood that the capitalization of antibody nomenclature may vary. The following definitions are not intended to be limited to the combinations of upper and lower case letters used in the following list.

[0157] Ab - Antibody Mab / mAb - Monoclonal antibody TCEP - Tris(2 - carboxyethyl)phosphine (often supplied as the HCl salt).

[0158] Tmab / TmAb - Trastuzumab IFab / IfAb - Fab fragment of ipilimumab Imab / ImAb - Ipilimumab Features expressed in terms of the foregoing description, the following claims, or the features disclosed in the accompanying drawings, their specific forms or the means for performing the disclosed functions, or the methods or processes for obtaining the disclosed results may, if necessary, be used to understand the present invention in its various forms, individually or in any combination.

[0159] Although the present invention has been described in the conjugation according to the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is provided. Accordingly, the above exemplary embodiments of the present invention are considered to be illustrative and not restrictive. Various changes to the described embodiments may be made without departing from the spirit and scope of the present invention.

[0160] To avoid any doubt, all theoretical explanations provided herein are provided for the purpose of enhancing the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0161] All headings of the terms used herein are for organizational purposes only and should not be considered as limiting the subject matter described. Throughout this specification, including the following claims, unless the context requires otherwise, the words "comprise", "include", and variations such as "comprises", "comprising", and "including" are to be understood to mean including the stated integer or step or group of integers or steps but not to mean excluding any other integer or step or group of integers or steps.

[0162] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as "about" one particular value and / or "about" another particular value. When such a range is expressed, another aspect includes from one particular value and / or to another particular value. Similarly, when values are expressed as approximations, it will be understood that the use of the preceding "about" causes a particular value to form another aspect. The term "about" associated with a numerical value is optional and, for example, means ±10%.

Example

[0163] Example 1 General synthesis of methyl 3,5-bis(2-haloacetamido)benzoate Methyl 3,5-bis(2-haloacetamido)benzoate, which links the compounds of the present invention, can be prepared according to the following general synthesis (wherein, if necessary, X is Br or Cl).

[0164]

Chemical formula

[0165] Compound 1 2-Chloroacetyl chloride (1.35 g, 12.04 mmol, 2 equivalents) was added dropwise to a cooled DCM solution of methyl 3,5-diaminobenzoate (1 g, 6.02 mmol). The mixture was warmed and stirred at room temperature for 2 hours. The resulting solution was washed with water, saturated ammonium chloride solution, dried over MgSO4, and the solvent was distilled off under reduced pressure to obtain acetamide (1) as a pale yellow solid (1.7 g, 89%). 1 H NMR (CDCl3, 400 MHz): δ = 8.31 (s, 2H, NH), 8.20 (s, 1H, Ar), 7.93 (s, 2H, Ar), 4.16 (s, 2H, 2x CH2), 3.88 (s, 3H, Me). 13 C NMR (CDCl3, 100 MHz): δ 165.82, 164.10, 137.58, 131.88, 117.45, 115.56, 52.45, 42.71.ESI-HRMS: Predicted value C 12 H 12 Cl2N2O4Na(M+Na + )=m / z341.0066. Measured value: m / z341.0077.

[0166] Compound 2 Methyl 3,5-diaminobenzoate (1.0 g, 6.02 mmol) was dissolved in DMF (10 mL) and bromoacetyl bromide (2.4 equiv, 1.26 mL, 14.45 mmol) was added dropwise with stirring over 10 minutes. The solution was stirred and left for 24 hours, then diluted to 150 mL with ethyl acetate, washed with water (4 × 30 mL) and brine (30 mL), and dried over MgSO4. The solvent was distilled off under reduced pressure to obtain pale orange crystals (2) (1.86 g, 76% yield). 1 H NMR, 400 MHz, CDCl3: δ 9.48 (s, 1H), 8.1 (s, 1H), 7.9 (s, 2H) 3.88 (s, 4H), 3.77 ppm (s, 3H); 13 C NMR, 500MHz, CDCl3: δ166.3, 165.0, 138.8, 131.2, 116.5, 115.1, 52.1, 26.8 ppm;HRMS-ESI: Calcd for C12H12N2O4Br2+: 406.9237; Found: 406.9261.

[0167] Compound 3 KI (1.56 g, 9.43 mmol, 2 equiv) was added to a solution of methyl 3,5-bis(2-chloroacetamido)benzoate (1) (1 g, 3.14 mmol) in anhydrous acetone (20 mL). The mixture was refluxed for 3 hours. The resulting mixture was filtered and the solvent was distilled off under reduced pressure to obtain acetamide (3) as a yellow solid (1 g, 63%). 1 H NMR (CD3COCD3, 500 MHz): δ 9.91 (s, 1H), 8.20 (s, 1H), 8.16 (s, 2H), 4 (s, 4H), 3.89 (s, 3H). 13 C NMR (CD3COCD3, 126 MHz): δ 166.82, 166.09, 140.01, 127.53, 131.52, 115.56, 114.00, 51.85, -0.00.HRMS: Predicted for C 12 H 13 I2N2O4(M+H + ) = m / z 502.8969, Found: m / z 502.8959. Synthesis of Methyl 3,4-Bis(2-haloacetamido)benzoate Methyl 3,4-bis(2-haloacetamido)benzoate, which links the compounds of the present invention, can be prepared according to the following general synthesis (wherein, if necessary, X is Br or Cl).

[0168]

Chemical formula

[0169] Compound 4 2-Chloroacetyl chloride (1.35 g, 12.04 mmol, 2 equivalents) was added dropwise to a cooled DCM solution of methyl 3,4-diaminobenzoate (1 g, 6.02 mmol). The mixture was warmed and stirred at room temperature for 2 hours. The resulting solution was washed with water and saturated ammonium chloride solution, dried over MgSO4, and the solvent was distilled off under reduced pressure to obtain acetamide (X) as a pale yellow solid (1.5 g, 79%). 1 H NMR (CDCl3, 400 MHz): δ 8.84 (s, 1H, NH), 8.54 (s, 1H, NH), 8.04 (d, J = 2 Hz, 1H, Ar), 7.94 (dd, J = 8.4, 2 Hz, 1H, Ar), 7.75 (d, J = 8.4 Hz, 1H, Ar), 4.19 (d, J = 12 Hz, 4H, 2 x CH2), 3.88 (s, 3H, Me). 13 C NMR (CDCl3, 100 MHz): δ 165.82, 164.10, 137.58, 131.88, 117.45, 115.56, 52.45, 42.71.ESI-HRMS: Predicted value C 12 H 12 Cl2N2O4Na(M+Na + )=m / z341.0066. Found: m / z341.0086.

[0170] Compound 5 2-Bromoacetyl bromide (2.91 g, 14.44 mmol, 2.4 equiv) was added dropwise to a solution of methyl 3,4-diaminobenzoate (1.0 g, 6.02 mmol, 1.0 equiv) in dimethylformamide (DMF) at room temperature. The reaction mixture was stirred continuously at room temperature for 24 h. Approximately 150 mL of ethyl acetate was added to make a solution, which was washed three times with distilled water and once with saturated ammonium chloride, dried over MgSO4, and the residual solvent was distilled off under reduced pressure to obtain methyl 3,4-bis(2-bromoacetamido)benzoate (5) as a pale yellow powder (2.40 g, 98%). Further, recrystallization was performed using DCM. 1 H NMR (500 MHz, chloroform-d) δ 8.75 (s, 1H, NH), 8.47 (s, 1H, NH), 8.05 (s, 1H, Ar), 7.98 (d, J = 8 Hz, 1H, Ar), 7.77 (d, J = 8 Hz, 1H, Ar), 4.07 (s, 2H, CH2), 4.04 (s, 2H, CH2), 3.91 (s, 3H, CH3). 13 C NMR (100 MHz, CDCl3) δ167.79, 167.15, 165.31, 134.72, 128.82, 127.21, 126.40, 124.76, 52.43, 29.11.Esi-HRMS: Predicted value C 12 H 12 N2O4Br2(M+H + )=m / z404.9091, found: m / z404.9130.

[0171] Compound 6 KI (1.56 g, 9.43 mmol, 2 equiv) was added to a solution of methyl 3,4-bis(2-chloroacetamido)benzoate (4) (1 g, 3.14 mmol) in anhydrous acetone (20 mL). The mixture was refluxed for 3 h. The resulting mixture was filtered, and the solvent was distilled off under reduced pressure to obtain acetamide (6) as a yellow solid (1 g, 63%). 11H NMR (CDCl3, 500 MHz): δ 9.73 (s, 1H), 9.61 (s, 1H), 8.02 (1H), 7.80 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 3.86 (s, 3H), 3.86 (s, 4H). 13 13C NMR (CDCl3, 126 MHz): δ 167.79, 167.15, 165.94, 127.53, 127.36, 126.65, 124.22, 52.11, -0.74, -1.0. HRMS: Calculated value for C 12 H 13 I2N2O4(M+H + ) = m / z 502.8969, Found: m / z 502.8959.

[0172] Compound 7 2,3 - Diaminobenzoic acid (1.0 g, 6.57 mmol, 1 equiv) was dissolved in 10 mL of anhydrous methanol (MeOH), and 4 drops of concentrated H2SO4 were added to acidify to pH = 1. The reaction solution was heated to 50 °C and refluxed with stirring for 72 h. Subsequently, the product was neutralized with sodium carbonate and concentrated under reduced pressure. Next, the residual solution was washed three times with distilled water and once with saturated ammonium chloride, dried over MgSO4, and the excess solvent was distilled off under reduced pressure to obtain methyl 2,3 - diaminobenzoate as a dark brown solid (0.70 g, 64%). Next, bromoacetyl bromide (0.58 g, 2.88 mmol, 2.4 equiv) was added dropwise to a solution of methyl 2,3 - diaminobenzoate (0.2 g, 1.20 mmol, 1.0 equiv) in anhydrous DCM at room temperature. The reaction flask was continuously stirred at room temperature for 24 h. Next, the resulting solution was washed three times with distilled water and once with concentrated ammonium chloride, dried over MgSO4, and the excess solvent was distilled off under reduced pressure to obtain methyl 2,3 - bis(2 - bromoacetamido)benzoate (7) as a pale yellow solid (0.38 g, 78%). 11H NMR (CDCl3, 500 MHz): δ 10.70 (s, 1H, NH), 9.07 (s, 1H, NH), 7.92 (d, J = 8 Hz, 2H, Ar), 7.38 (t, J = 8 Hz, 1H, Ar), 4.09 (s, 2H, CH2), 3.99 (s, 2H, CH2), 3.95 (s, 3H, CH2). 13 13C NMR (CDCl3, 100 MHz): δ167.33, 166.17, 164.80, 131.99, 128.79, 126.3, 122.61, 52.89, 29.28, 28.61. ESI-HRMS: Calculated for C 12 H 12 N2O4Br2(M+H + ) = m / z 406.9237, found: m / z 406.9251.

[0173] Compound 8 Chloroacetyl chloride (9.3 g, 84 mmol, 6 equiv) was added dropwise to a cooled aqueous solution of NaOH (0.55 M) containing benzene-1,3-diamine (1.5 g, 14 mmol). The mixture was heated and stirred overnight at room temperature. The resulting precipitate was filtered, washed 5-6 times with water, and dried completely under high vacuum to give acetamide (8) as a white solid (0.97 g, 27%). 1 1H NMR (DMSO-d6, 400 MHz): δ 10.34 (s, 2H, NH), 7.96 (t, J = 2.0 Hz, 1H, Ar), 7.37 - 7.25 (m, 3H, Ar), 4.25 (s, 4H, 2 x CH2). 13 13C NMR (DMSO-d6, 100 MHz): δ 164.61, 138.82, 129.14, 114.81, 110.36, 45.53. ESI-HRMS: Calculated for C 10 H 10 Cl2N2O2Na(M+Na + ) = m / z 283.0012. found: m / z 283.0049.

[0174] Compound 9 2-Bromoacetyl bromide (2.05 g, 10.2 mmol, 2.2 equiv) was added dropwise to a cooled DCM solution of benzene-1,3-diamine (0.5 g, 4.6 mmol) and TEA (1.35 g, 13.3 mmol, 2.2 equiv). The resulting precipitate was filtered, washed 5-6 times with water and then with ether. This solid compound was dried under high vacuum to give acetamide (9) as a pale yellow solid (2.48 g, 77%). 1 H NMR (DMSO-d6, 500 MHz): δ 10.42 (s, 2H, 2 X NH), 7.96 (t, J = 2.0 1H, Ar), 7.37-7.24 (m 3H, Ar), 4.04 (s, 4H, 2 X CH2). 13 C NMR (DMSO-d6, 126 MHz): δ 164.79, 138.95, 129.14, 114.72, 110.17, 30.36.ESI-HRMS: Calculated for C 10 H 10 Br2N2O2Na(M+Na + )=m / z370.9001. Found: m / z370.9039.

[0175] Compound 10 KI (1.3 g, 7.7 mmol, 4 equiv) was added to a solution of N,N’-(1,3-phenylene)bis(2-chloroacetamide) (8) (0.50 g, 1.9 mmol) in anhydrous acetone (20 mL). The mixture was refluxed for 3 h. The resulting mixture was filtered and the solvent was evaporated under reduced pressure to give acetamide (10) as a yellow solid (0.63 g, 74%). 1 H NMR (DMSO-d6, 500 MHz): δ 10.36 (s, 2H, NH), 7.92 (t, J = 2.0 Hz, 1H, Ar), 7.33-7.22 (m, 3H, Ar), 3.83 (s, 4H, 2 X CH2). 13 C NMR (DMSO-d6, 126 MHz): δ 166.56, 139.18, 129.10, 114.36, 109.86, 1.58.HRMS: Calculated for C 10 H 10 I2N2O2Na(M+Na+ ) = m / z 466.8724, measured value: m / z 466.8758. HPLC: Column: HiQ Sil HS (150×4.60 mm). Mobile phase: Isocratic: (0.75 mL / min), 35% MeCN: 65% water. Detection at 280 nm. Retention time, 8.89 minutes, purity, 93.4%.

[0176] Compound 11 2-Chloroacetyl chloride (9.3 g, 84 mmol, 6 eq) was added dropwise to a cooled aqueous solution of NaOH (0.55 M) of benzene-1,2-diamine (1.5 g, 14 mmol). The mixture was warmed and stirred overnight at room temperature. The resulting precipitate was filtered and washed 5 - 6 times with water, and the obtained solid compound was dried completely under high vacuum to obtain acetamide (11) as a white solid (1.8 g, 51%). 1 H NMR (DMSO-d6, 400 MHz): δ 9.69 (s, 2H, NH), 7.55 (dd, J = 7.4, 3.7 Hz, 2H, Ar), 7.23 (dd, J = 6.1, 3.5, 2 Hz, 2H, Ar), 4.34 (s, 4H, 2 x CH2). 13 C NMR (CDCl3, 100 MHz): δ 165.12, 130.17, 125.61, 125.08, 43.19. ESI-HRMS: Predicted value C 10 H 10 Cl2N2O2Na (M+Na + ) = m / z 283.0012. Measured value: m / z 283.0010.

[0177] Compound 12 2-Bromoacetyl bromide (2.05 g, 10.2 mmol, 2.2 eq) was added dropwise to a cooled DCM solution of benzene-1,2-diamine (0.50 g, 4.6 mmol) and TEA (1.30 g, 10.2 mmol, 2.2 eq). The resulting precipitate was filtered and washed 5 - 6 times with water and then with ether. This solid compound was dried under vacuum to obtain acetamide (12) as a yellow solid (1.87 g, 58%). 11H NMR (DMSO-d6, 500 MHz): δ 9.71 (s, 2H, NH), 7.53 (dd, J = 7.5, 3.7 Hz, 2H, Ar), 7.23 (dd, J = 6.0, 3.5 Hz, 2H, Ar), 4.13 (s, 4H, 2 X CH2). 13 13C NMR (DMSO-d6, 126 MHz): δ 165.14, 130.27, 125.56, 124.93, 30.14. ESI-HRMS: Calculated for C 10 H 10 Br2N2O2Na (M+Na + ) = m / z 370.9001. Found: m / z 370.9012.

[0178] Compound 13 KI (1.3 g, 7.7 mmol, 4 equiv) was added to a solution of N,N'-(1,3-phenylene)bis(2-chloroacetamide) (11) (0.50 g, 1.9 mmol) in anhydrous acetone (20 mL). The mixture was refluxed for 3 h. The resulting mixture was filtered and the solvent was evaporated under reduced pressure to give acetamide (13) as a yellow solid (0.63 g, 45%). 1 1H NMR (DMSO-d6, 500 MHz): δ 9.63 (s, 2H, NH), 7.49 (dd, J = 7.4, 3.7 Hz, 2H, Ar), 7.20 (dd, J = 6.1, 3.5 Hz, 2H, Ar), 3.90 (s, 4H, 2 X CH2). 13 13C NMR (DMSO-d6, 126 MHz): δ 166.92, 130.42, 125.35, 124.70, 1.49. HRMS: Calculated for C 10 H 10 I2N2O2Na (M+Na + ) = m / z 466.8724. Found: m / z 466.8770.

[0179] Compound 14 Synthesis of 3,5-bis(2-chloroacetamido)benzoic acid (19)

[0180]

Chem.

[0181] 2-Chloroacetyl chloride (1.62 g, 14.5 mmol, 2.2 equiv) was added dropwise to a cooled THF solution of 3,4-diaminobenzoic acid (1.0 g, 6.6 mmol). The mixture was warmed and stirred at room temperature for 2 h. The resulting precipitate was filtered, washed 5 - 6 times with water, and then dried completely under high vacuum to give acetamide (19) as a pale yellow solid (1.8 g, 90%). 1 H NMR (CD3COCD 3, 400 MHz): δ 9.64 (s, 2H, NH), 8.24 (d, J = 78.3 Hz, 3H, Ar), 4.30 (s, 4H, CH2). 13 C NMR (CD3COCD 3, 101 MHz) δ 166.91, 140.07, 132.58, 117.11, 115.48, 44.07.ESI-HRMS: Calculated for C 11 H9Cl2N2O4(M-H + ) = m / z 302.9939. Found: m / z 302.9956. Synthesis of 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-chloroacetamido)benzoate (20)

[0182]

Chemical Structure

[0183] A solution of EDC·HCl (0.63 g, 3.3 mmol, 1.1 equiv) in DMF (5 mL) was added to a stirred THF solution of acid (19) (1 g, 3 mmol) and N-hydroxysuccinimide (0.38 g, 3.3 mmol, 1.1 equiv) at room temperature. Next, the reaction mixture was stirred at room temperature for 2 h and then concentrated under reduced pressure. The resulting residue was dissolved in EtOAc, washed with water, dried over MgSO4, and the solvent was distilled off under reduced pressure to give a foamy solid. This crude product was further purified by precipitation (EtOAc / petroleum ether) to give acetamide (20) as a yellow solid (0.75 g, 57%). 1 H NMR (CD3COCD 3, 400 MHz,): δ 9.78 (s, 2H, NH), 8.34 (d, J = 64.1 Hz, 3H, Ar), 4.32 (s, 4H, 2 x ClC H 2CO), 2.99 (s, 4H, COC H 2C H 2CO). 13 C NMR (CD3COCD 3, 101 MHz): δ 170.86, 170.43, 170.37, 165.95, 162.44, 140.81, 127.32, 117.21, 117.15, 44.06, 26.40. ESI-HRMS: calculated for C 15 H 13 Cl2N3O6Na (M+Na + ) = m / z 424.0074. Found: m / z 424.0106. 2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethan-1-amine (22)

[0184]

Chemical Structure

[0185] A solution of Ph3P (0.54 g, 2.1 mmol, 1 equivalent) in ether (5 mL) was added dropwise to an aqueous solution of tri-PEG azide (21) (0.50 g, 2.1 mmol) in HCl (5%, 5 mL), and the mixture was stirred and left for 24 hours. Next, the ether was removed under reduced pressure, and the aqueous layer was extracted with DCM until no Ph3P oxide was detected in the aqueous layer. The pH of the aqueous layer was adjusted to pH = 12, and the azide-linked amine was extracted from the aqueous layer with DCM. The combined DCM solutions were evaporated under reduced pressure to obtain the azide-linked amine (22) as a pale yellow liquid (0.25 g, 56%). 1 H NMR (CDCl3, 400 MHz): δ 3.65 - 3.53 (m, 11H), 3.44 (td, J = 5.2, 1.3 Hz, 2H), 3.41 - 3.22 (m, 3H), 2.79 (td, J = 5.3, 1.4 Hz, 2H). 13 C NMR (CDCl 3, 101 MHz): δ 73.41, 70.65, 70.60, 70.58, 70.23, 69.95, 50.63, 41.75.ESI-HRMS: Predicted value for C8H 19 N4O3(M+H + )=m / z219.1452. Found: m / z219.1464. N,N’-(5((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-chloroacetamide) (23)

[0186]

Chemical Structure

[0187] 2-(2-Azidoethoxy)ethan-1-amine (22) (0.34 g, 1.6 mmol, 1.3 equiv) was added to a solution of activated ester (20) (0.50 g, 1.2 mmol) in anhydrous THF. Next, the reaction mixture was stirred at room temperature for 1 h and then concentrated under reduced pressure. The resulting residue was dissolved in DCM, washed with water, dried over MgSO4, and the solvent was distilled off under reduced pressure. This crude product was further purified by silica gel chromatography: (5% - 20% MeOH / DCM) to give azido-linked acetamide (23) as a white solid (0.39 g, 62%). 1 H NMR (CDCl3, 400 MHz,): δ 8.78 (s, 2H, N H CH), 8.02 (s, 1H, Ar), 7.68 (s, 2H, Ar), 7.20 (d, J = 8.8 Hz, 1H, CON H CH2), 4.13 (s, 4H, 2 x ClC H 2CO), 3.75 - 3.41 (m, 14H), 3.27 (t, J = 5.0 Hz, 2H, CH2 C H2N3). 13 C NMR (CDCl 3, 101 MHz): δ166.77, 164.69, 137.88, 135.96, 115.04, 114.31, 70.57, 70.51, 70.47, 70.24, 69.84, 69.55, 50.57, 42.96, 40.08.ESI-HRMS: Calculated for C 15 H 13 Cl2N3O6Na(M+Na + )=m / z424.0074. Found: m / z424.0106. HPLC: Column: HiQ Sil HS(150×4.60mm). Mobile phase: Isocratic: (0.75 mL / min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 6.54 min, Purity: 99.3%. N,N’-(5-((2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-bromoacetamide)(14)

[0188] [Chemical]

[0189] KBr (1.4 g, 12 mmol, 6 eq) was added to a solution of N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-chloroacetamide) (23) (1.0 g, 2.0 mmol) in anhydrous acetone (20 mL). The mixture was refluxed for 4 days. The resulting mixture was filtered, and the solvent was distilled off under reduced pressure. The crude product was further purified by silica gel chromatography: (50% - 70% acetone / chloroform) to obtain azido-linked acetamide 14 as a yellow solid (0.6 g, 51%). 1 H NMR (CDCl3, 500 MHz,): δ 8.79 (s, 2H, 2 x N H CH), 7.96 (s, 1H, Ar), 7.67 (s, 2H, Ar), 7.10 (s, 1H, N H CH), 3.95 (s, 4H, BrC H 2CO), 3.84 - 3.44 (m, 14H), 3.27 (t, J = 5.0 Hz, 2H, CH C H2N3). 13 C NMR (CDCl3,126 MHz): δ 167.04, 164.62, 138.22, 135.84, 114.87, 114.14, 70.67, 70.59, 70.55, 70.36, 69.94, 69.59, 50.64, 40.24, 29.42. ESI-HRMS: Predicted value for C 19 H 27 Br2N6O6(M+H + ) = m / z 593.0353. Found: m / z 593.0344. HPLC: Column: HiQ Sil HS (150×4.60 mm). Mobile phase: Isocratic: (0.75 mL / min), 40% MeCN: 60% water. Detection at 280 nm. Retention time: 6.52 minutes, purity: 99.4%.

[0190] Compound 15 N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-iodoacetamide)(15)

[0191]

Chem.

[0192] KI (1.0 g, 8.0 mmol, 4 equiv) was added to a solution of N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-chloroacetamide)(23) (1.0 g, 2.0 mmol) in anhydrous acetone (20 mL). The mixture was refluxed for 3 h. The resulting mixture was filtered, and the solvent was evaporated under reduced pressure. The crude product was further purified by silica gel chromatography: (50% - 70% acetone / chloroform) to give the azido-linked acetamide(15) as a yellow solid (1.19 g, 88%). 1 1H NMR (CD3COCD3, 500 MHz): δ 9.98 (s, 2H, 2 x N H CH), 7.93 (d, J = 3.7 Hz, 3H, Ar), 7.69 (d, J = 5.3 Hz, 1H, CON H CH2), 3.95 (s, 4H, IC H 2CO), 3.60 - 3.11 (m, 16H). 13 13C NMR (CD3COCD 3, 126 MHz): δ 170.49, 167.51, 140.48, 137.24, 114.46, 113.41, 71.14, 70.58, 51.42, 40.42, 1.34. ESI-HRMS: calculated for C 19 H 27 Cl2N6O6(M+H +) = m / z 505.1364. Measured value: m / z 505.141600. HPLC: Column: HiQ Sil HS (150×4.60 mm). Mobile phase: Isocratic: (0.75 mL / min), 35% MeCN: 65% water. Detected at 280 nm. Retention time: 10.31 minutes, purity: 97.8%.

[0193] Compound 16 3,4 - Bis(2 - chloroacetamido)benzoic acid (24)

[0194]

Chemical formula

[0195] 2 - Chloroacetyl chloride (1.62 g, 14.5 mmol, 2.2 equivalents) was added dropwise to a cooled THF solution of 3,4 - diaminobenzoic acid (1.0 g, 6.6 mmol). The mixture was warmed and stirred at room temperature for 2 hours. The resulting precipitate was filtered and washed 5 - 6 times with water, and the obtained solid compound was dried completely under high vacuum to obtain acetamide (24) as a white solid (1.8 g, 90%). 1 H NMR (DMF - d7, 500 MHz): δ 10.09 (d, J = 20.4 Hz, 2H, NH), 8.30 (d, J = 1.9 Hz, 1H, Ar), 8.00 - 7.83 (m, 2H, Ar), 4.45 (d, J = 7.7 Hz, 4H, CH2). 13 C NMR (126 MHz, DMF - d7): δ 166.86, 166.02, 165.84, 135.31, 129.94, 128.03, 127.15, 127.00, 124.12, 43.64, 43.56. ESI - HRMS: Predicted value C 11 H 11 Cl2N2O4Na (M + Na +) = m / z 305.0090. Measured value: m / z 305.0092. HPLC: Column: HiQ Sil HS (150×4.60 mm). Mobile phase: Isocratic: (0.75 mL / min), 35% MeCN: 65% water. Detected at 280 nm. Retention time: 4.38 minutes, purity: 93%. 3,4 - Bis(2 - chloroacetamido)benzoic acid 2,5 - dioxopyrrolidin - 1 - yl (25)

[0196] [Chemical formula]

[0197] A solution of EDC.HCl (0.63 g, 3.3 mmol, 1.1 eq) in DMF (5 mL) was added to a stirred THF solution of acid (24) (1 g, 3 mmol) and N - hydroxysuccinimide (0.38 g, 3.3 mmol, 1.1 eq) at room temperature. Next, the reaction mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The resulting residue was dissolved in EtOAc, washed with water, dried over MgSO4, and the solvent was distilled off under reduced pressure to obtain a foamy solid. This crude product was further purified by precipitation (EtOAc / petroleum ether) to obtain acetamide (25) as a yellow solid (0.6 g, 45%). 1 H NMR (CD3COCD 3, 400 MHz): δ 9.51 (d, J = 16.2 Hz, 2H, NH), 8.34 (s, 1H, Ar), 8.18 - 7.89 (m, 2H, Ar), 4.38 (s, 4H, 2 x ClC H 2CO), 2.98 (s, 4H, COC H 2C H 2CO). 13 C NMR (CD3COCD3, 100 MHz): δ 170.48, 166.85, 166.31, 161.95, 138.26, 130.66, 129.04, 128.65, 125.21, 122.83, 43.88, 26.39. ESI - HRMS: Predicted value C 15 H 13 Cl2N3O6Na (M + Na +) = m / z 424.0074. Measured value: m / z 424.0094. N,N’-(4-((2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-chloroacetamide) (26)

[0198]

Chemical formula

[0199] 2-(2-Azidoethoxy)ethan-1-amine (22) (0.34 g, 1.6 mmol, 1.3 equivalents) was added to a solution of activated ester (25) (0.50 g, 1.2 mmol) in anhydrous THF. Next, the reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The obtained residue was dissolved in DCM, washed with water, dried over MgSO4, and the solvent was distilled off under reduced pressure. This crude product was further purified by silica gel chromatography: (5% - 20% MeOH / DCM) to obtain azide-linked acetamide (26) as a white solid (0.25 g, 40%). 1 1H NMR (CDCl3, 500 MHz): δ 9.05 (s, 2H, 2 x N H CH), 7.66 (s, 1H, Ar), 7.58 - 7.40 (m, 2H, Ar), 7.14 (t, J = 5.4 Hz, 1H, CON H CH2), 4.15 (d, J = 10.6 Hz, 4H, 2 x ClC H 2CO), 3.69 - 3.46 (m, 14H), 3.26 (t, J = 5.0 Hz, 2H, CH2C H 2N3). 13 13C NMR (CDCl3, 126 MHz): δ166.29, 166.09, 165.49, 132.96, 132.74, 129.15, 125.57, 125.10, 124.86, 70.63, 70.60, 70.51, 70.24, 69.94, 69.65, 50.63, 42.91, 42.68, 40.01. ESI-HRMS: Predicted value C 19 H27 Cl2N6O6(M + H + ) = m / z 505.1364. Measured value: m / z 505.1365. HPLC: Column: HiQ Sil HS (150 × 4.60 mm). Mobile phase: Isocratic: (0.75 mL / min), 35% MeCN: 65% water. Detected at 280 nm. Retention time: 6.76 minutes, purity: 99.8%. N,N’-(4-((2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-bromoacetamide)(16)

[0200]

Chemical Structure

[0201] KBr (1.4 g, 12 mmol, 6 equivalents) was added to a solution of N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-chloroacetamide)(26) (1.0 g, 2.0 mmol) in anhydrous acetone (20 mL). The mixture was refluxed for 4 days. The resulting mixture was filtered, and the solvent was distilled off under reduced pressure. The crude product was further purified by silica gel chromatography: (50% - 70% acetone / chloroform) to obtain azido-linked acetamide 16 as a white solid (0.5 g, 43%). 1 1H NMR (CDCl3, 500 MHz,): δ 9.02 (s, 1H, N H CH), 8.96 (s, 1H, N H CH), 7.79 (d, J = 2.0 Hz, 1H, Ar), 7.70 - 7.60 (m, 2H, Ar), 7.15 (s, 1H, CON H CH2), 4.09 (d, J = 12.2 Hz, 4H, BrC H 2CO), 3.78 - 3.50 (m, 14H), 3.37 (t, J = 5.0 Hz, 2H, CHCH2N3). 1313C NMR (CDCl3, 126 MHz): δ 166.31, 165.79, 165.13, 133.28, 132.55, 132.28, 125.68, 125.14, 124.79, 70.64, 70.62, 70.52, 70.26, 69.96, 69.63, 50.65, 40.10, 29.71, 29.09, 28.71. ESI-HRMS: Calculated for C 19 H 27 Br2N6O6 (M + H + ) = m / z 593.0353. Found: m / z 593.0344. HPLC: Column: HiQ Sil HS (150 × 4.60 mm). Mobile phase: Isocratic: (0.75 mL / min), 35% MeCN: 65% water. Detected at 280 nm. Retention time: 7.62 min, Purity: 99.1%.

[0202] Compound 17 N,N’-(4-((2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-iodoacetamide) (17)

[0203]

Chem.

[0204] KI (1.0 g, 8.0 mmol, 4 equiv) was added to a solution of N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-chloroacetamide) (26) (1.0 g, 2.0 mmol) in anhydrous acetone (20 mL). The mixture was refluxed for 3 h. The resulting mixture was filtered and the solvent was evaporated under reduced pressure. The crude product was further purified by silica gel chromatography: (50% - 70% acetone / chloroform) to give the azido-linked acetamide (17) as a yellow solid (1 g, 73%). 1 1H NMR (CDCl3, 500 MHz): δ 9.30 (s, 1H, 2 x N HCH), 9.17 (s, 1H, NH), 7.61 (s, 1H, Ar), 7.41 (s, 1H, CON H CH2), 7.27 (s, 2H, Ar), 3.93 (d, J = 10.8 Hz, 4H, 2 x IC H 2CO), 3.75 - 3.48 (m, 14H), 3.36 (t, J = 5.0 Hz, 2H, CH2C H 2N3). 13 C NMR (CDCl 3, 126 MHz): δ170.97, 168.13, 166.49, 129.71, 125.31, 125.01, 70.48, 70.31, 70.24, 69.73, 50.60, 40.09, -0.33, -0.53. ESI-HRMS: Calculated value for C 19 H 27 I2N6O6(M+H + ) = m / z 689.0076. Measured value: m / z 689.0108. HPLC: Column: HiQ Sil HS(150×4.60 mm). Mobile phase: Isocratic: (0.75 mL / min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 9.10 minutes, Purity: 96.2%.

[0205] Compound 18 3,6,9,12,15,18,21-Heptaoxatricosane-1,23-ditosylate (27)

[0206]

Chemical Structure

[0207] 4-Toluenesulfonyl chloride (1.33 g, 7.02 mmol, 2.6 equiv) was added to a solution of anhydrous pyridine (0.51 g, 6.5 mmol, 2.4 equiv) and 3,6,9,12,15,18,21-heptaoxatricosane-1,23-diol (1.0 g, 2.7 mmol) in anhydrous DCM (10 mL). The mixture was stirred overnight under N2 at room temperature. Next, the solution was concentrated under reduced pressure and standard workup (EtOAc) was performed. Next, the resulting residue was purified by silica gel chromatography (40 - 80% EtOAc / petroleum ether) to give 3,6,9,12,15,18,21-heptaoxatricosane-1,23-ditosylate (27) as a colorless oil (1.1 g, 60%). 1 1H NMR (CDCl3, 400 MHz): δ 7.81 - 7.68 (m, 4H, Ar), 7.34 - 7.26 (m, 4H, Ar), 4.23 - 4.01 (m, 4H, 2x SO2OC H 2), 3.70 - 3.48 (m, 28H), 2.39 (d, J = 1.5 Hz, 6H, CHCC H 3). 13 13C NMR (CDCl3, 101 MHz): δ 144.74, 132.87, 129.77, 127.92, 70.67, 70.54, 70.49, 70.44, 69.20, 68.60, 21.60. ESI-HRMS: predicted C 30 H 47 O 13 S2(M+H + ) = m / z 679.2453. Found: m / z 679.2448. 1,23-Diazido-3,6,9,12,15,18,21-heptaoxatricosane (28)

[0208]

Chemical Structure

[0209] Sodium azide (2 g, 30 mmol, 10 equiv) was added to a DMF solution of ditosylate (27) (2.0 g, 3.0 mmol), and the mixture was stirred at 80 °C overnight. Next, the mixture was concentrated under reduced pressure to remove DMF, and the product was extracted with ethyl acetate (3 × 20 mL). The organic extract was then washed with saturated brine solution, dried over MgSO4, and concentrated to give the diazide (28) as a colorless oil (1 g, 80.6%). 1 H NMR (CDCl3, 400 MHz): δ 3.66 - 3.54 (m, 28H), 3.36 - 3.28 (m, 4H, 2x N3CH2). 13 C NMR (CDCl3, 101 MHz): δ 13 C NMR δ 70.62, 69.96, 50.60. ESI-HRMS: calculated for C 16 H 33 O7N6(M+H + ) = m / z 421.2405. Found: m / z 421.2466. 1,23-Diamino-3,6,9,12,15,18,21-heptaoxatricosane (29)

[0210]

Chem.

[0211] Triphenylphosphine (1.87 g, 7.14 mmol, 3 equiv) was added portionwise to a stirred anhydrous THF solution of diazide (28) (1.0 g, 2.4 mmol). The reaction mixture was stirred at room temperature overnight. Water (50 mL) was added, and the reaction mixture was stirred at room temperature overnight. THF was distilled off under reduced pressure, the reaction mixture was filtered, and the filtrate was then washed with DCM (3 × 100 mL) to remove phosphine oxide. The filtrate was distilled off under reduced pressure to give the diamino-PEG (29) as a yellow oil (0.50 g, 57%). 1 H NMR (CDCl3, 400 MHz,): δ 3.58 (dd, J = 2.4, 1.1 Hz, 24H), 3.44 (t, J = 5.2 Hz, 4H, 2x NH2CH2C H2O), 2.80 (t, J = 5.2 Hz, 4H, 2x NH2C H 2CH2O), 1.67 (s, 4H, 2x N H 2). 13 C NMR (CDCl3, 101 MHz,): δ 73.27, 70.48, 70.18, 41.68. ESI-HRMS: Calculated value for C 16 H 37 O7N2(M+H + ) = m / z 369.2600. Measured value: m / z 369.2850. N,N’,N’’,N’’’-((5,8,11,14,17,20,23-Heptaoxa-2,26-diazapentacosanedioyl)bis(benzene-4,1,2-triyl))tetrakis(2-chloroacetamide)(30)

[0212]

Chemical Structure

[0213] 1,23-Diamino-3,6,9,12,15,18,21-heptaoxatricosane(29) (150 mg, 0.407 mmol) was added to an anhydrous THF solution of the activated ester (25) (409 mg, 1.02 mmol, 2.5 equivalents). Next, the reaction mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. This crude product was purified by silica gel chromatography: (5% - 20% MeOH / DCM) to obtain the acetamide (30) as a white solid (340 mg, 89%). 1 H NMR (DMSO-d6, 500 MHz): δ 9.81 (d, J = 25.2 Hz, 4H), 8.54 (t, J = 5.6 Hz, 2H), 7.98 (s, 2H), 7.73 (d, J = 1.4 Hz, 4H), 4.35 (d, J = 9.3 Hz, 8H), 3.64 - 3.45 (m, 28H), 3.41 (q, J = 5.9 Hz, 4H). 1313C NMR (DMSO-d6, 126 MHz): δ 172.72, 165.38, 165.20, 165.17, 133.26, 131.12, 129.13, 124.98, 124.59, 123.79, 69.71, 69.56, 68.83, 43.28, 43.21. ESI-HRMS: Calculated for C 38 H 53 Cl4N6O 13 (M + H + ) = m / z 941.2419. Found: m / z 941.2451. HPLC: Column: HiQ Sil HS (150 × 4.60 mm). Mobile phase: Isocratic: (0.75 mL / min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 5.83 min, purity: 98%. N,N’,N’’,N’’’-((5,8,11,14,17,20,23-Heptaoxa-2,26-diazapentacosanedioyl)bis(benzene-4,1,2-triyl))tetrakis(2-iodoacetamide)(18)

[0214]

Chemical Structure

[0215] KI (176 mg, 1.06 mmol, 10 equiv) was added to a mixed solution of N,N’,N’’,N’’’-((5,8,11,14,17,20,23-heptaoxa- 2,26-diazapentacosanedioyl)bis(benzene-4,1,2-triyl))tetrakis(2-chloroacetamide)(30) (100 mg, 0.106 mmol) in anhydrous acetone (400 mL). The mixture was refluxed for 3 h. The resulting mixture was filtered and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel chromatography: (50% - 70% acetone / chloroform) to give acetamide (18) as a white solid (130 mg, 93.5%). 11H NMR (DMSO-d6, 500 MHz): δ 9.62 (d, J = 16.8 Hz, 4H), 8.31 (d, J = 4.9 Hz, 3H), 7.98 (s, 2H), 7.84 - 7.59 (m, 4H), 3.98 (d, J = 7.0 Hz, 8H), 3.75 - 3.30 (m, 32H). 13 13C NMR (DMSO-d6, 126 MHz): δ 167.18, 167.09, 165.31, 133.53, 130.78, 129.30, 124.57, 124.29, 123.34, 69.64, 68.87, 1.67, 1.59. ESI-HRMS: Calculated for C 38 H 52 I4N6O 13 Na1 (M+Na + ) = m / z 1330.9669. Found: m / z 1330.9765. HPLC: Column: HiQ Sil HS (150×4.60 mm). Mobile phase: Isocratic: (0.75 mL / min), 35% MeCN: 65% water. Detection at 280 nm. Retention time: 9.55 min, purity: 98%.

[0216] Example 2 Aqueous solution stability The stability of ester- and amide-functionalized linker compounds was measured and compared.

[0217] In the first experiment, the aqueous solution stability of linker compounds 1 - 6 was observed over 4 days. The stability in phosphate buffer (100 mM, pH 7.5) in the presence of 10% DMF-d7 was 1 measured using 1H NMR solvent suppression. This data shows that ortho-substituted (1,2-) di-haloacetamides have lower aqueous solution stability than meta-substituted (1,3-) ones. See Figure 2.

[0218] Next, a comparison of the aqueous solution stability of ester and amide-functionalized aryl bis-haloacetamide linkers was carried out by determining the percentage of bis-haloacetamide derivatives (2, 3, 5, 6 and 14 - 16) remaining over 4 days. Again, the stability in phosphate buffer (100 mM, pH 7.5) using 10% DMF-d7 as the final concentration was evaluated using the solvent suppression method. Refer to Figure 3.

[0219] Surprisingly, the inventors found that exchanging the ester functional group for an amide functional group reverses the trend in halide stability (iodide is more stable than bromide), and that 3,4-modifications have similar stability to 3,5-modifications.

[0220] Example 3 Reactivity with the thiol side chain of amino acids The reactivity of the linker compounds of the present invention was tested by reaction with glutathione. The following representative reactions are described. Similar reactions were carried out using amide-functionalized linker compounds. Reactivity of glutathione with methyl 3,5-bis(2-haloacetamido)benzoate

[0221]

Chemical formula

[0222] Glutathione (290 mg, 0.943 mmol, 3 eq) was dissolved in an aqueous sodium phosphate buffer (100 mM, pH 7.5, 8 mL). Methyl 3,5-bis(2-chloroacetamido)benzoate (1) (100 mg, 0.314 mmol) was dissolved in THF (2 mL) and slowly added to the glutathione aqueous solution and maintained at room temperature overnight. Subsequently, the reaction solution was concentrated under reduced pressure and purified by C-18 chromatography (100% H2O to 20% MeCN / H2O) to obtain product 4.17 as a sticky solid (0.20 mg, 74%). 11H NMR (D2O, 500 MHz): δ 7.73 (s, 1H, Ar), 7.56 (s, 2H, Ar), 4.57 - 4.54 (m, 2H, 2 x NHC H CO), 3.79 (s, 3H, OMe), 3.73 - 3.63 (m, 6H, 2 x NHC H 2COOH, CH2 C HNH2), 3.40 (s, 4H, 2 x SC H 2CO), 3.13 - 2.89 (m, 4H, 2 x SC H 2CH), 2.43 (t, J = 8 Hz, 4H, 2 x CH2C H 2CO), 2.06 - 2.01 (m, 4H, 2 x C H 2CH2CO). 13 13C NMR (126 MHz, D2O): δ 176.17, 174.81, 174.00, 171.69, 170.39, 167.65, 137.89, 130.43, 117.32, 54.11, 52.92, 52.89, 43.42, 36.24, 33.77, 31.37, 26.19. HRMS: Calculated for C 32 H 44 O 16 S2N8Na (M + Na + ) = m / z 883.2209. Found: m / z 883.2187. HPLC: Column: HiQ Sil HS (150 × 4.60 mm). Mobile phase: Isocratic: (1 mL / min), 10% MeCN: 90% water: 0.1% TFA. Detected at 214 nm. Retention time: 36.39 minutes, purity: 99%. Reactivity of glutathione with methyl 3,4 - bis(2 - haloacetamido)benzoate

[0223]

Chem.

[0224] Glutathione (290 mg, 0.943 mmol, 3 eq) was dissolved in an aqueous sodium phosphate buffer (100 mM, pH 7.5, 8 mL). Methyl 3,4-bis(2-chloroacetamido)benzoate (4) (100 mg, 0.314 mmol) was dissolved in THF (2 mL) and slowly added to the glutathione aqueous solution, and the mixture was maintained at room temperature overnight. Subsequently, the reaction solution was concentrated under reduced pressure and purified by C-18 chromatography (100% H2O~20% MeCN / H2O) to obtain the product (32) as a sticky solid (0.17 mg, 63%). 1 H NMR (500 MHz, D2O): δ 8.00 (s, 1H, Ar), 7.93 (dd, J = 8.5, 2 Hz, 1H, Ar), 7.63 (d, J = 8.5 Hz, 1H, Ar), 4.70-4.56 (m, 2H, 2 x NHC H CO), 3.86 (s, 3H, OMe), 3.76-3.65 (m, 6H, 2 x NHC H 2COOH, CH2 C HNH2), 3.52-3.45 (m, 4H, 2 x SC H 2CO), 3.15-2.90 (m, 4H, 2 x SC H 2CH), 2.47-2.43 (m, 4H, 2 x CH2C H 2CO), 2.07 -2.02 (m, 4H, 2 x C H 2CH2CO). 13 C NMR (126 MHz, D2O): δ 176.11, 174.88, 173.88, 171.63, 171.22, 167.95, 135.56, 128.79, 128.22, 128.04, 125.81, 54.08, 52.90, 52.81, 43.35, 35.72, 35.52, 33.85, 33.79, 31.36, 26.09.HRMS: Calculated for C 32 H 44 O 16 S2N8Na(M+Na +) = m / z 883.2209. Measured value: m / z 883.2187. HPLC: Column: HiQ Sil HS (150×4.60 mm). Mobile phase: Isocratic: (1 mL / min), 10% MeCN: 90% water: 0.1% TFA. Detection at 214 nm. Retention time: 14.25 minutes, purity: 99%. Reactivity of Glutathione with Methyl 2,3-Bis(2-bromoacetamido)benzoate

[0225] [Chemical formula]

[0226] Glutathione (0.11 g, 3.66 mmol, 3 equivalents) was dissolved in 6 mL of 0.1 M phosphate buffer pH = 7 and added to a solution of 4 mL of methyl 2,3-bis(2-bromoacetamido)benzoate (7) (0.05 g, 1.22 mmol, 1 equivalent) in THF. The mixture was continuously stirred at room temperature for 24 hours. The resulting product was purified using a reverse-phase column (C18), and the product was obtained from the fractions eluted with 10% acetonitrile / water (0.05 g, 48%). Comparison of Amide-Functionalized Compounds The inventors compared the reactivity of various linker compounds of the present invention by determining the percentage remaining of bis-haloacetamide derivatives 14 - 17 in an aqueous phosphate buffer (100 mM, pH 7.5) in the presence of glutathione (2.2 equivalents). Refer to Figure 4.

[0227] The inventors observed that, although there is no difference between 3,4- and 3,5-amide-modified derivatives in hydrolysis (reaction with water), there are significant differences between the thiol of glutathione and their reactions. The 3,4-modified derivatives react faster with thiol than the 3,5-modified compounds. The ester compound reacted very rapidly, so the reaction rate could not be measured.

[0228] Example 4 Reaction of Bromoacetamide Linker with Trastuzumab Linker compounds 2, 5, 9, and 12 were reacted with trastuzumab under various stoichiometries, and the reaction products were analyzed using SDS-PAGE.

[0229]

Chemical formula

[0230] This reaction with the bis-bromoacetamide linker used fully reduced Tmab (0.034 mM) in Tris.HCl buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 7.5). Tmab (0.033 mM) was reduced with 5 equivalents of TCEP at room temperature for 2 hours.

[0231] This analysis and the resulting products are shown in Figure 5. L: Protein ladder, Lane 1: Tmab incubated with 5 equivalents of methyl 3,4-bis(2-bromoacetamido)benzoate (5) Lane 2: Tmab incubated with 8 equivalents of methyl 3,4-bis(2-bromoacetamido)benzoate (5) Lane 3: Tmab incubated with 5 equivalents of methyl 3,5-bis(2-bromoacetamido)benzoate (2) Lane 4: Tmab incubated with 8 equivalents of methyl 3,5-bis(2-bromoacetamido)benzoate (2) Lane 5: Tmab incubated with 5 equivalents of N,N'-(1,2-phenylene)bis(2-bromoacetamide) (12) Lane 6: Tmab incubated with 8 equivalents of N,N'-(1,2-phenylene)bis(2-bromoacetamide) (12) Lane 7: Tmab incubated with 5 equivalents of N,N'-(1,3-phenylene)bis(2-bromoacetamide) (9) Lane 8: Tmab incubated with 8 equivalents of N,N'-(1,3-phenylene)bis(2-bromoacetamide) (9).

[0232] Legend: HC: heavy chain, LC: light chain, LC-LC: light chain homodimer, HC-HC: heavy chain homodimer. The protein sample was digested by reducing SDS-PAGE (4-12% gel). The inventors recognized that the pattern of thiol cross-linking differed between ortho-(1,2- and 3,4-) and meta-(1,3- and 3,5-) substituted linking compounds. When an excess (5 or 8 equivalents) of the linker was used, the meta-substituted linker was less selective because more LC-LC (47 kDa) and HC-HC (106 kDa) were produced. The main product in all reactions was the half-antibody conjugate, HC-LC (75 kDa) with two linkers attached.

[0233] The products in the reaction with linker compound 5 (labeled HC-LC) were analyzed by mass spectrometry. Tmab cross-linked with methyl 3,4-bis(2-bromoacetamido)benzoate (5) showed a main peak at 74,528.03 Da, and expansion of the spectrum in the 74 kDa region showed main peaks at 74,528.03 Da and 74,689.24 Da (glycoform). Mass spectrometric analysis of the crude reaction product clarified that two linkers were attached and that one linker cross-linked the -HC-HC intradisulfide cysteine residue in the hinge region.

[0234] Figure 1(1) shows a schematic diagram of a half-antibody produced by two re-cross-linked disulfide bonds of intra-chain heavy chain-heavy chain and heavy chain-light chain (labeled HC-LC). Figure 1(5) is chemically identical to the structure shown in Figure 1(1) but represents the physical state that can be observed in the half-antibody. In size exclusion chromatography, this product would appear to have a molecular weight of 150 kDa, but each covalent structure has a mass of 75 kDa (half-antibody) as represented in Figure 1(1).

[0235] Analysis of the reaction product in the reaction with the non-functionalized linker compound 12 clarified that two linkers were bound, and in this case also, one linker cross-linked the -HC-HC internal cysteine residue in the hinge region.

[0236] Protein MS by deconvolution spectrum of Tmab cross-linked with N,N’-(1,2-phenylene)bis(2-bromoacetamide) (12). The MS spectrum of the cross-linked Tmab showed a main peak at 74,572.57 Da, while expansion of the spectrum in the 74 KDa region showed main peaks at 74,572.57 Da and 74,410.91 Da.

[0237] Example 5 Effect of pH on the selectivity of bis-(2-bromoacetamide)-linker The effect of pH in the reaction with bis-(2-bromoacetamide) that links compounds was investigated by performing SDS-PAGE analysis on the cross-linking of fully reduced Tmab (0.033 mM) in Tris.HCl buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 6, 7.5, or 8) incubated with bis-haloacetamide linker. Tmab (0.033 mM) was reduced at room temperature for 2 hours using 5 equivalents of TCEP.

[0238] This analysis and the resulting products are shown in Figure 5. L: Protein ladder, Lanes 1, 5, and 9: Tmab incubated with 5 equivalents of methyl 3,4-bis(2-bromoacetamide)benzoate (5), Lanes 2, 6, and 10: Tmab incubated with 5 equivalents of methyl 3,5-bis(2-bromoacetamide)benzoate (2), Lanes 3, 7, and 11: Tmab incubated with 5 equivalents of N,N’-(1,2-phenylene)bis(2-bromoacetamide) (12), Lanes 4, 8, and 12: Tmab incubated with 5 equivalents of N,N'-(1,3-phenylene)bis(2-bromoacetamide) (9), HC: heavy chain, LC: light chain, LC-LC: light chain homodimer, HC-HC: heavy chain homodimer. Protein samples were resolved by reducing SDS-PAGE (10% gel).

[0239] The highest level of conjugation was achieved at pH = 7.5. In all cases, ortho-(1,2- and 3,4-)substituted linker compounds were more selective (less between LC-LC and -HC-HC) than meta-(1,3- and 3,5-)modified linker compounds.

[0240] Example 6 Reaction of Excess Bis-(2-iodoacetamide) Linker with Trastuzumab Linker compounds 3, 6, 10, and 13 were reacted with an excess of trastuzumab, and the reaction product was analyzed using SDS-PAGE.

[0241]

Chemical formula

[0242] This reaction with the bis-iodoacetamide linker was carried out using fully reduced Tmab (0.033 mM) in Tris.HCl buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 7.5). Tmab (0.033 mM) was reduced with 5 equivalents of TCEP at room temperature for 2 hours.

[0243] This analysis and the resulting products are shown in Figure 7. L: Protein ladder, Lane 1: Tmab control (non-reducing dye), Lane 2: Tmab control (reducing dye), Lane 3: Tmab incubated with 5 equivalents of methyl 3,4-bis(2-iodoacetamide)benzoate (6), Lane 4: Tmab incubated with 5 equivalents of methyl 3,5-bis(2-iodoacetamido)benzoate (3), Lane 5: Tmab incubated with 5 equivalents of N,N'-(1,2-phenylene)bis(2-iodoacetamide) (13), Lane 6: Tmab incubated with 5 equivalents of N,N'-(1,3-phenylene)bis(2-bromoacetamide) (10), HC: Heavy chain, LC: Light chain, LC-LC: Light chain homodimer, HC-HC: Heavy chain homodimer. Protein samples were resolved by reducing SDS-PAGE (10% gel).

[0244] HC: Heavy chain, LC: Light chain, LC-LC: Light chain homodimer, HC-HC: Heavy chain homodimer. Protein samples were resolved by reducing SDS-PAGE (10% gel). Similar selectivity was observed as in the case of similar bromo linker compounds.

[0245] Example 7 Selectivity of the reaction of amide-substituted linkers with trastuzumab The reactions of amide-functionalized linker compounds 14, 15, 16, and 17 with trastuzumab were investigated.

[0246]

Chemical formula

[0247] SDS-PAGE analysis of the cross-linking of Tmab (0.013 mM) in Tris.HCl buffer (0.5 M, pH 7.5, 5 mM EDTA) reduced overnight at room temperature using 5 equivalents of TCEP is shown in Figure 8. L: Ladder, Lane 1: Tmab incubated with 5 equivalents of 16, Lane 2: Tmab incubated with 5 equivalents of 14, Lane 3: Tmab incubated with 5 equivalents of 17, Lane 4: Tmab incubated with 5 equivalents of 15.

[0248] Protein MS by deconvolution spectrum of Tmab re-crosslinked with compound 17 linker showed a main peak at 75,059.82 Da, clarifying the binding of two linkers to the half-antibody product.

[0249] Example 8 Selectivity with other monoclonal antibodies SDS-PAGE analysis of the crosslinking of fully reduced rituximab (0.035 mM) in Tris.HCl buffer using a bis-bromoacetamide linker is shown in Figure 9. Rituximab (0.035 mM) was reduced at room temperature for 2 hours using 5 equivalents of TCEP. L: Protein ladder, Lane 1: Rmab incubated with 5 equivalents of methyl 3,4-bis(2-bromoacetamido)benzoate (6), Lane 2: Rmab incubated with 5 equivalents of methyl 3,5-bis(2-bromoacetamido)benzoate (3), Lane 3: Rmab incubated with 5 equivalents of N,N'-(1,2-phenylene)bis(2-bromoacetamide) (12), Lane 4: Rmab incubated with 5 equivalents of N,N'-(1,3-phenylene)bis(2-bromoacetamide) (9).

[0250] HC: Heavy chain, LC: Light chain, LC-LC: Light chain homodimer, HC-HC: Heavy chain homodimer. Protein samples were resolved by reducing SDS-PAGE (10% gel). Similar selectivity and reaction products were observed for the reaction with trastuzumab. The 1,2-compound showed more efficient formation of HC-LC products at 75 kDa, with fewer LC-LC and HC-HC products. Therefore, the results observed using trastuzumab can be judged as a valid representative example for antibodies.

[0251] Example 9 Selective functionalization of antibodies The inventors have demonstrated that the selectivity of the linker can be utilized to selectively monofunctionalize antibodies such as trastuzumab and to hetero-bifunctionalize them.

[0252] The site-selective functionalization of trastuzumab was investigated. Figure 10 shows the SDS-PAGE analysis regarding the cross-linking of partially reduced Tmab in Tris.HCl buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 6, 7.5, or 8) using a bis-bromoacetamide linker. Tmab was reduced with 1.1 equivalents of TCEP at 4 °C for 2 hours and incubated overnight at room temperature with 1.1 equivalents of each linker. L: Protein ladder, Lanes 1, 9, and 17: 1 equivalent of methyl 3,4-bis(2-bromoacetamido)benzoate (5), Lanes 2, 10, and 18: 1.1 equivalents of methyl 3,5-bis(2-bromoacetamido)benzoate (2), Lanes 3, 11, and 19: 1.1 equivalents of N,N'-(1,2-phenylene)bis(2-bromoacetamide) (12), Lanes 4, 12, and 20: 1.1 equivalents of N,N'-(1,3-phenylene)bis(2-bromoacetamide) (9).

[0253] Lanes 5 - 8, 13 - 16, and 21 - 24 show the same reactions as above but performed under non-reduced conditions (non-reducing dye). HC: Heavy chain, LC: Light chain, LC-LC: Light chain homodimer, HC-HC: Heavy chain homodimer. The protein samples were resolved by reducing SDS-PAGE (10% gel).

[0254] The comparable intensities of the bands at 75 kDa and 50 kDa suggest that a single disulfide bridge was selectively functionalized to generate one HC-LC conjugate per mAb. In other words, it is monofunctionalized. The unfunctionalized linkers 12 and 9 suggest that the reaction proceeded significantly, as lower molecular weight bands were less visible under non-reducing conditions, and thus seem to give better results.

[0255] Selective hetero-bifunctionalization of trastuzumab was achieved via sequential partial reduction, incubation, further reduction, and incubation. Figure 11a shows SDS-PAGE for the bifunctional cross-linking of Tmab (0.033 mM) in Tris.HCl buffer (100 mM, 0.15 mM NaCl, 5 mM EDTA, pH 7.5) using the sequential method. Tmab (0.033 mM) was reduced with 2.2 equivalents of TCEP for 2 hours and incubated overnight at room temperature with 2.2 equivalents of methyl 3,4-bis(2-bromoacetamido)benzoate (5) (lane 1). Next, the functionalized Tmab (0.033 mM) was further reduced with 2.2 equivalents of TCEP for 2 hours and incubated overnight at room temperature with 4 equivalents of N,N’-(5-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-iodoacetamide) (15) (lane 2).

[0256] The protein sample was resolved by reducing SDS-PAGE (10% gel). Protein MS by deconvolution spectrum shows a major peak at 74,711.99 Da corresponding to the reaction of one molecule of linker 5 and one molecule of linker 15.

[0257] This is a demonstration of the ability to form hetero-bifunctionalized half-antibodies via sequential addition of two different linkers. This structure is formally represented in Figure 11b. Linker 5 shows the re-crosslinking of disulfides that connect the light and heavy chains of the antibody. Linker 15 is referred to herein as an "intra-chain" re-crosslinking and connects two thiols that form the hinge region of the antibody.

[0258] Example 10 Formation of Stable Thiol-Bridged Fab Derivatives and Selective Preparation of mAb-Protein Conjugates The linker compounds of the present invention are capable of forming stable thiol-bridged Fab derivatives. Subsequently, these can be conjugated to reduced or partially reduced antibodies or other proteins.

[0259] First, the IFab was buffer-exchanged into conjugation Tris.HCl buffer (pH 7.5) and diluted to (5 mg / mL, 0.1 μmol) using an Amicon® Ultra-0.5 mL (3KDa) centrifugal filter. A stock solution of TCEP (4.0 mg / mL, 0.042 mmol, 14 mM) was prepared in the same conjugation buffer. The IFab was reduced by incubating with TCEP (2 equivalents relative to IFab) for 1 hour at room temperature. Next, a 1-hour quenching step of TCEP using penta-PEG azide was subsequently performed.

[0260] The reduced IFab was incubated with bis-o-diiodoacetamide (PEG) 7 linker 18 (10 μL of stock solution (7 mg / mL), 5 equivalents) for 3 hours at room temperature. Excess reagents were removed using a rapid purification step using a Protein A column (1 mL HisTrap, FF) to remove excess linker from the functionalized IFab solution prior to conjugation with the partially reduced Tmab. The Fab fraction was recovered using binding buffer Tris.HCl (20 mM, 500 mM NaCl, pH 7.4), and then this recovered fraction was concentrated using an Amicon® Ultra-15 mL (3KDa) centrifugal filter and finally buffer-exchanged into conjugation buffer.

[0261] The deconvoluted protein MS spectrum of the IfAb control (non-reduced) showed a main peak at 47,636.32 Da, while the deconvoluted protein MS spectrum of the functionalized IfAb with a bis-o-diiodoacetamide (PEG) 7 linker (18) showed a main peak at 48,690.90 Da, clarifying that functionalization had occurred. It is shown as a schematic structure in Fig. 12a.

[0262] Tmab was buffer-exchanged into Tris.HCl (100 mM, 150 mM NaCl, 5 mM EDTA, pH 7.5) and diluted to (5.0 mg / mL, 0.03 μmol) using an Amicon® Ultra-0.5 mL (10 KDa) centrifugal filter. Tmab was reduced by incubating with TCEP (1.1 equivalents relative to Tmab) at 4 °C for 2 h. Next, functionalized IFab (4 equivalents relative to Tmab) was added to the reduced Tmab and left at room temperature overnight. Next, SEC was performed using a superdex column (HiLoad 16 / 600, Superdex 200 pg, GE Healthcare) for purification of the reaction product. Before loading onto the column, the sample was centrifuged at 20,000 g for 10 min. The recovered fractions containing the conjugate were buffer-exchanged into conjugation buffer and sterilized using a 0.45 μm filtration membrane.

[0263] Refer to Fig. 12b. L: Protein ladder Lane 1: Tmab incubated with 1.1 equivalents of methyl 3,4-bis(2-bromoacetamido)benzoate (5) as a control, Lane 2: IfAb conjugate, Lane 3: Tmab conjugation with IfAb performed overnight at room temperature, showing an approximate 125 KDa, the exact estimated mass of the conjugate, with a significant decrease in the 75 KDa band (half antibody) before performing size exclusion chromatography, Lane 4: Fractions (F6 - G2) recovered by size exclusion column purification, Lane 5: Fractions (G4 - G13) recovered by size - exclusion column purification, Lane 6: Fractions (H1 - H7) recovered by size - exclusion column purification.

[0264] Lanes 4 and 5 contain the desired mAb - fAb conjugate (lane 5 has some HC - HC impurities). A schematic of this antibody conjugate is shown in FIG. 12c. This is sometimes also called an mAb - protein conjugate or a trifunctional monoclonal antibody. The inventors demonstrated its selective preparation and stability towards AKTA purification on a Protein A column.

[0265] Example 11 The selectivity of thio - crosslinking compounds varies by their regiochemistry: ortho - substitution vs meta - substitution. The selectivity of compounds 15 and 17 was evaluated when fully reduced Tmab (4 equivalents) was incubated with only 2 equivalents each of bis - iodoacetamide linkers 15 and 17. The reaction products were resolved using SDS - PAGE analysis (shown in FIG. 13a) and further characterized by protein MS (shown in FIGS. 13b and c).

[0266] First, the Tmab was buffer-exchanged into Tris.HCl buffer (pH 7.5) and diluted to (5 mg / mL, 34 μM, 1 mL) using an Amicon® Ultra-0.5 mL (10 KDa) centrifugal filter. The Tmab was reduced by incubating with TCEP (4 equivalents relative to Tmab) at 4 °C for 2 hours. This reduced protein was aliquoted into 100 μL samples for each reaction. A stock solution of N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-iodoacetamide) (17) was prepared to a final concentration (1.0 mg / mL, 1.4 μmol, 1.4 mM) in DMF. A working solution of 17 (0.68 mg / mL) was prepared by serial dilution with DMF. A 2-equivalent working solution of 17 (7 μL, 2 equivalents relative to a fixed amount of Tmab) was added to the reduced protein and maintained at room temperature overnight. A similar procedure was followed for the reaction of compound 15 with Tmab.

[0267] The azide-modified bis-iodoacetamide compounds 15 and 17 showed interesting selectivity. SDS-PAGE analysis of the reaction with the meta-substituted compound 15 showed that the HC (internally cross-linked half-antibody) of Tmab was the major protein product present (Figure 13a, lane 2). Characterization using protein MS clarified that this HC protein product had undergone conjugation with one molecule of 15 via re-crosslinking of the heavy-chain-heavy-chain intradisulfide (Figure 13b).

[0268] In contrast, the ortho-substituted compound 17 showed a higher preference for cross-linking the heavy-chain-light-chain disulfide bond, with a significant amount of higher molecular weight product observed by SDS-PAGE analysis (Figure 13a, lane 1). Characterization using protein MS clarified that this protein product had undergone HC-LC disulfide re-crosslinking with one molecule of 17 (Figure 13c). Importantly, MS analysis also clarified that the remaining HC protein was unmodified and had not undergone any detectable conjugation with compound 17.

[0269] Figure 13a shows a 10% SDS-PAGE gel for evaluating the selectivity of bis-iodoacetamide linker in cross-linking Tmab (5 mg / mL, 34 μM) in Tris.HCl buffer (100 mM, pH 7.5) containing 150 mM NaCl and 5 mM EDTA, reduced with 4 equivalents of TCEP (136 μM) and incubated overnight at room temperature with each bis-iodoacetamide linker (6.8 μM, 2 equivalents). L: Protein ladder, Lane 1: Tmab incubated with 2 equivalents of N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-iodoacetamide) (17), Lane 2: Tmab incubated with 2 equivalents of N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-carbamoyl)-1,3-phenylene)bis(2-iodoacetamide) (15), Deconvoluted protein MS spectra of Tmab cross-linked with 2 equivalents of N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,3-phenylene)bis(2-iodoacetamide) (15) (Figure 13b) and N,N’-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-1,2-phenylene)bis(2-iodoacetamide) (17) (Figure 13c).

[0270] References Numerous publications have been cited above to more fully describe and disclose the present invention and the state of the art to which the present invention pertains. The complete citations of these references are presented below. The entire contents of each of these references are hereby incorporated by reference into this specification. EP333573 S M Andrew and J A Titus Current Protocols in Cell Biology (2000) 16.4.1-16.4.10. E J Smith, L Visai, S W Kerrigan, P Speziale, and T J Foster * Infect Immun. 2011, 79(9): 3801-3809. T Kantner, B Alkhawaja, A G Watts (2017) ACS Omega, 2, 5785-5791. For considerations regarding protecting groups and their synthesis and use, see Peter G.M. Wuts and Theodora W. Greene, Greene’s Protective Groups in Organic Synthesis, 4th Edition, 2006, Wiley-Blackwell.

[0271] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3rd Edition, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Claims

1. A method for generating a monofunctionalized antibody comprising a linker residue that crosslinks a heavy chain and a light chain, wherein the linker residue has the formula (I) 【Chemical 1】 (wherein each X is independently F, Cl, Br, or I, R １ is H, COOR Ａ , CONH ２ , CONHR Ａ , CONR Ａ ２ , CONHL, or CONR Ａ is L, L is a polyether or polythioether having a reactive group R ３ as a terminal group, R ３ is N ３ , a group containing a C≡C bond, NHBoc, NHFmoc, NHCbz, -I, -Br, -Cl, -OH, -OP, -O-aryl, -OTs, -OMs, -SP, -S-C １～４ alkyl, -OSO ２ -C １～４ alkyl, or formula (H) [Chemical 2] is a moiety, where P is a protecting group, Each R Ａ is independently selected from １～４ C alkyl n is 0, 1, 2, or 3, Each R ２ is independently selected from F, Cl, Me, CF ３ , OMe, and OCF ３ , or R ２ is a group as defined for R １ (as defined). is a moiety derived from a linker compound of or a salt thereof, and the method comprises (i) preparing a partially reduced antibody by reducing the antibody using about 1 to 1.1 equivalents of a reducing agent, and then (ii) treating the partially reduced antibody with a linker compound of formula (I) or a salt thereof to replace two Xs with thiol groups from the heavy and light chains to form two S---- moieties (---- represents the point of attachment to the heavy and light chains) to form a monofunctionalized antibody A method comprising.

2. The method according to claim 1 for generating a monofunctionalized antibody comprising a linker residue that crosslinks a heavy chain and a light chain, wherein the linker residue has the formula (Ia) [Chemical Formula 3] (wherein X is Br) is a moiety derived from a linker compound, and the method comprises treating the partially reduced antibody with about 1 to 1.1 equivalents of a linker compound of formula (Ia) to replace two Xs with thiol groups from the heavy and light chains to form two S---- moieties (---- represents the point of attachment to the heavy and light chains) to form a monofunctionalized antibody The said method.

3. The method according to claim 2, wherein n is 0.

4. A method for generating a half antibody comprising a linker residue that crosslinks a heavy chain-heavy chain intradisulfide residue in the hinge region and a linker residue that crosslinks a heavy chain and a light chain, wherein the linker residue has the formula (I) 【Chemical 4】 (wherein each X is independently F, Cl, Br, or I, R １ is H, COOR Ａ , CONH ２ , CONHR Ａ , CONR Ａ ２ , CONHL, or CONR Ａ L, and L is a polyether or polythioether having a reactive group R ３ as a terminal group, R ３ is N ３ , a group containing a C≡C bond, NHBoc, NHFmoc, NHCbz, -I, -Br, -Cl, -OH, -OP, -O-aryl, -OTs, -OMs, -SP, -S-C １～４ alkyl, -OSO ２ -C １～４ alkyl, or formula (H) 【Chemical Formula 5】 is a moiety, where P is a protecting group, Each R Ａ is independently selected from C １～４ alkyl, and n is 0, 1, 2, or 3, Each R ２ is independently selected from F, Cl, Me, CF ３ , OMe, and OCF ３ , or R ２ is a group as defined for R １ (as defined for R is a moiety derived from a linker compound of or a salt thereof, and the method comprises treating a fully reduced antibody with at least 4 equivalents of a linker compound of formula (I) or a salt thereof to replace two Xs with thiol groups from the heavy chain-heavy chain disulfide residue to form two S---- moieties (---- represents the point of attachment to the heavy chain-heavy chain disulfide residue) to form a half antibody, wherein the heavy chain-heavy chain disulfide residue is on the same heavy chain Method.

5. A method for generating a half-antibody in which a linker residue crosslinks a heavy-chain-heavy-chain intra-cysteine residue in a hinge region, wherein the linker residue has the formula (Ib) 【Chemical Formula 6】 (wherein X is I, R １ is H, COOR Ａ , CONH ２ , CONHR Ａ , CONR Ａ ２ , CONHL, or CONR Ａ L, and n is 0, 1, 2, or 3, Each R ２ is independently selected from F, Cl, Me, CF ３ , OMe, and OCF ３ , or R ２ is a group as defined for R １ (as defined). is a moiety derived from a linker compound of, the method comprising treating a fully reduced antibody with about 2 to 2.2 equivalents of a linker compound of formula (Ib) to replace two Xs with thiol groups from the heavy-chain-heavy-chain cysteine residues to form two S---- moieties (---- represents the point of attachment to the heavy-chain-heavy-chain cysteine residue) to form a half-antibody, the method comprising, after treatment of the fully reduced antibody with about 2 to 2.2 equivalents of a linker compound of formula (Ib), treating with an additional linker compound) method. **Claim 6** The method according to claim 5, wherein n is 0. **Claim 7** R １ The method according to claim 5 or 6, wherein the radical and the two haloacetamide groups are in the 1,3,5 arrangement. **Claim 8** A method for generating a hetero-bifunctionalized half-antibody having a first linker residue that crosslinks a light chain and a heavy chain in a hinge region and a second linker residue that crosslinks a heavy-chain-heavy-chain intra-cysteine residue in the hinge region, each linker residue having the formula (I) 【Chemical Formula 7】 (wherein each X is independently F, Cl, Br, or I, R １ is H, COOR Ａ , CONH ２ , CONHR Ａ , CONR Ａ ２ , CONHL, or CONR Ａ L, and L is a polyether or polythioether having a reactive group R ３ as a terminal group, R ３ is N ３ , a group containing a C≡C bond, NH Boc, NH Fmoc, NH Cbz, -I, -Br, -Cl, -OH, -OP, -O-aryl, -OTs, -OMs, -SP, -S-C １～４ alkyl, -OSO ２ -C １～４ alkyl, or formula (H) 【Chemical Formula 8】 is a moiety of, where P is a protecting group, Each R Ａ is independently selected from C １～４ alkyl, n is 0, 1, 2, or 3, Each R ２ is independently selected from F, Cl, Me, CF ３ , OMe, and OCF ３ , or R ２ is a group as defined for R １ (as defined above). is a moiety derived from a linker compound of or a salt thereof, the method comprising (i) treating a partially reduced antibody with a first linker compound of formula (I) or a salt thereof to replace two Xs of the first linker compound with thiol groups from the light chain and the heavy chain to form two S---- moieties (---- represents the point of attachment to the light chain and the heavy chain) to generate a first conjugate, and then (ii) further reducing the first conjugate to generate a reduced conjugate, and then (iii) treating the reduced conjugate with a second linker compound of formula (I) or a salt thereof to replace two Xs of the second linker compound with thiol groups from the heavy-chain-heavy-chain cysteine residues to form two S---- moieties (---- represents the point of attachment to the heavy-chain-heavy-chain cysteine residue) to generate the hetero-bifunctionalized half-antibody conjugate, wherein the first and second linker compounds are different) method. **Claim 9** The method according to any one of claims 1 to 8, wherein at least one of the linker compounds comprises L, and the method comprises the step of conjugating an additional moiety selected from an antibody, a half-antibody, an antibody fragment, a protein, a polypeptide, a drug, or a fluorescent or radioactive label.

10. A method for generating a Fab-Mab or Fab-protein conjugate, comprising the step of treating a reduced or partially reduced antibody or protein with a thiol-bridged fab antibody fragment containing a linker residue, said linker residue being: (i) of formula (I) 【Chemical Formula 9】 (wherein each X is independently F, Cl, Br, or I, R １ is COOR Ａ , CONH ２ , CONHR Ａ , CONR Ａ ２ , CONHL, or CONR Ａ L, and L is a polyether or polythioether having a reactive group R ３ as a terminal group, R ３ is N ３ , a group containing a C≡C bond, NHBoc, NHFmoc, NHCbz, -I, -Br, -Cl, -OH, -OP, -O-aryl, -OTs, -OMs, -SP, -S-C １～４ alkyl, -OSO ２ -C １～４ alkyl, or formula (H) 【Chemical Formula 10】 is a moiety, where P is a protecting group, Each R Ａ is independently selected from １～４ C alkyl n is 0, 1, 2, or 3, Each R ２ is independently selected from F, Cl, Me, CF ３ , OMe, and OCF ３ , or R ２ is a group as defined for R １ (as defined) a moiety derived from a linker compound of or a salt thereof, wherein two Xs are replaced by thiol groups in the fab antibody fragment to form two S---- moieties (---- represents the binding point to the fab antibody fragment); or (ii) of formula (IXa), (IXb), (IXc), or (IXd) 【Chemical Formula 11-1】 【Chemical Formula 11-2】 (wherein X is Br or I, m is selected from 3, 4, 5, 6, 7, 8, 9, and 10, and ---- represents the binding point to the protein chain of the fab antibody fragment) is a moiety of method.

11. Formula (I): 【Chemical Formula 12】 (wherein each X is independently F, Cl, Br, or I, R １ is CONH ２ , CONHR Ａ , CONR Ａ ２ , CONHL, or CONR Ａ L, and L is a polyether or polythioether chain having a reactive group R ３ as a terminal end, R ３ is N ３ , a group containing a C≡C bond, NH Boc, NH Fmoc, NH Cbz, -I, -Br, -Cl, -OH, -OP, -O-aryl, -OTs, -OMs, -SP, -S-C １～４ alkyl, -OSO ２ -C １～４ alkyl, or formula (H) 【Chemical 13】 is a moiety, where P is a protecting group, Each R Ａ is independently selected from C １～４ alkyl, n is 0, 1, 2, or 3, Each R ２ is independently selected from F, Cl, Me, CF ３ , OMe, and OCF ３ , or R ２ is CONH₂, CONRₐ₂, CONHL, or CONRₐL). a linker compound of or a salt thereof.

12. The compound according to claim 11, wherein n is 0.

13. The compound according to claim 11 or 12, wherein X is Br or I.

14. Formula (IIa), (IIb), or (IIc): 【Chemical Formula 14】 (wherein X is Br or I, R １ is CONH ２ or CONHL) is a compound according to any one of claims 11 to 13.

15. Formula (IVa), (IVb), or (IVc) 【Chemical Formula 15】 (wherein m is selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10) is a compound according to any one of claims 11 to 14.

16. Wherein each X is Br, or each X is I, or two Xs on one arylene are Br and two Xs on the other arylene are I, the compound according to claim 15.

17. Examples (1) to (3), (7), and (14) to (18): 【Chemical 16】 A compound selected from

18. A half antibody comprising a linker residue that crosslinks the heavy chain-heavy chain cysteine residues in the hinge region, wherein the linker residue is such that two Xs are replaced by thiol groups from the heavy chain-heavy chain cysteine residues to form two S---- moieties (---- represents the point of attachment to the heavy chain-heavy chain cysteine residue), which is the moiety defined in any one of claims 11 to 17. A half antibody.

19. A monofunctionalized antibody comprising a linker residue that crosslinks the heavy chain and the light chain, wherein the linker residue is such that two Xs are replaced by thiol groups from the heavy chain and the light chain to form two S---- moieties (---- represents the point of attachment to the heavy chain and the light chain), which is the moiety defined in any of claims 11 to 17. A monofunctionalized antibody.

20. A thiol-crosslinked fab antibody fragment comprising a linker residue that is such that two Xs are replaced by thiol groups from the fab antibody fragment to form two S---- moieties (---- represents the point of attachment to the fab antibody fragment), which is the moiety defined in any one of claims 11 to 17.

21. The linker residue has the formula (IXa), (IXb), (IXc), or (IXd): 【Chemical Formula 17-1】 【Chemical Formula 17-2】 (wherein X is Br or I, m is selected from 3, 4, 5, 6, 7, 8, 9, and 10, and ---- represents the point of attachment to the protein chain of the fab antibody fragment). The thiol-crosslinked fab antibody fragment according to claim 20.

22. R １ is H, COOMe, CONH ２ or CONHL, and n is 0, the method according to any one of claims 1 to 9.

23. R １ is COOMe, CONH ２ or CONHL, and n is 0, the method according to claim 10.

24. R １ is CONH ２ or CONHL, and n is 0, the antibody, half-antibody, or antibody fragment according to any one of claims 18 to 21.

Images