Novel compound and therapeutic use of the same

Abstract

To provide a novel compound having an ability to linking immune response to a defined therapeutic target, use of the compound in treatment of cancer and infectious disease, a composition containing the compound, a process for preparing them, and a novel intermediate used in the process.SOLUTION: An immunoconjugate includes an antibody connected to a saccharide molecule capable of connecting to a human anti-α-galactosyl antibody via a linker, or an antigen-binding fragment thereof, where the linker includes at least one phenyl ring capable of presenting one or more saccharides epitope capable of connecting to the human anti-α-galactosyl antibody.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention provides novel compounds capable of linking immune responses to defined therapeutic targets, including cancer and the use of said compounds in the treatment of infectious diseases, compositions containing said compounds, and their preparation. The present invention relates to a process for the preparation of benzophenone-3, as well as novel intermediates used in the process. [Background technology]

[0002] BACKGROUND OF THE INVENTION It is necessary to find new ways to mobilize an individual's immune system to fight disease. The human immune system fights off potentially harmful pathogens or mutated human cells (which can cause cancerous growths). Constantly surveying the body for foreign signals to identify and target them for elimination They are recruited against the pathogen or mutated human cells, triggering the immune system to fight the threat. There are natural antibodies that can eliminate

[0003] Cancer is a group of diseases involving abnormal cell growth that can invade or spread to other parts of the body. In 2012, cancer occurred in approximately 14.1 million people, which resulted in approximately 8.2 million deaths. It is the most common type of cancer in men, accounting for 14.6% of all human deaths. The most common cancers in women are lung, prostate, colorectal, and stomach. The types are breast cancer, colorectal cancer, lung cancer, and cervical cancer. The immune response It is well established that HIV-1 plays a vital role in the identification and elimination of cancer. There are drugs that fight cancer by boosting an individual's immune system to help fight it. This allows for better targeting of specific immune responses to cancer cells and a broader range of It is necessary to be able to generate the patient's own tumor-associated antigens. Targeting pre-existing natural antibodies against tumors meets this need. There is an urgent need to identify new methods for treating bacterial, viral, and fungal infections. Antimicrobial resistance is becoming a major global health threat. For example: Over 2 million people in the United States are infected each year with bacteria that are resistant to certain classes of antibiotics It is estimated that (US Centers for Disease Control and Prevention, 2013).

[0004] A novel approach to treat infectious diseases or cancer involves the use of immunoglobulins containing two binding sites. The first binding site is a binding site of an individual's immune system. The second binding site is capable of binding to an immune response component of an antigen, pathogen, or chemical. Any compound or foreign substance, such as a leukemia virus, or an internal cell, such as an altered cell found in cancer. The resulting effect of the immunity linker molecule is to bind to an external substance. Diversion of pre-existing immune responses to targets, i.e., cancer cells or specific pathogens An example of the first binding site is one that is recognized as foreign by the individual's immune system and therefore is Contains compounds or substances that may trigger an immune response

[0005] Typical examples of first binding sites are the small molecule haptens dinitrophenyl (DNP), rhamnose, or A further example of a first binding site is the human serum antibody anti-α-galactosidase. Carbohydrate molecules that can be linked to galactosyl (i.e., galactosyl-α-1,3-galactosyl) Anti-Gal (anti-β-1,4-N-acetylglucosamine; "anti-Gal").

[0006] Because immune function depends on multivalency, anti-Gal mobilization depends not only on the concentration of anti-Gal but also on the target. It will also depend on the affinity of the antibody for it.

[0007] Examples of the second binding moiety include an antibody or a fragment thereof that binds to a specific target molecule. Further examples of two binding moieties include established therapeutic antibodies or functional fragments thereof. The cells or pathogens targeted in this way are recognized as foreign by the immune system. Thus, natural antibodies can attack these They can be recruited against tumor cells or pathogens and harness the immune system to eliminate the threat.

[0008] WO98 / 34957 describes a method for stimulating immune responses using antibodies labeled with α-galactosyl epitopes. The document describes the stimulation of the immune system by the α-galactosyl epitope, the antibody Embodiments that rely on the incorporation of engineered glycosylation sites within the constant region are described. The appropriately engineered α-1,3-galactosyltransferase-expressing cell line The production of antibodies against this antigen has been reported to result in the addition of α-galactosyl epitopes. In this case, the number of incorporated epitopes depends on the number of residues engineered for glycosylation. The disadvantage of this approach is the limited number of α-galactosyl residues that can be introduced. (limited by the number of site-specific amino acid modifications). The advantage is that α-1,3-galactosyltransferase delivers a collection of carbohydrate derivatives. This may not be optimal for anti-galactosyl antibody mobilization, and may contribute to immune mobilization. This further limits the number of α-galactosyl epitopes available for antibody sites. Linkers that allow optimized loading and presentation of the peptide to maximize immune response A molecule is needed.

[0009] Thus, the first binding site is located relative to the second binding site (i.e., antibody or antigen-binding fragment). binding site (i.e., carbohydrate molecule capable of binding to human anti-α-galactosyl antibody) Linker molecules containing spacer groups that are optimized to control the number and position are highly Such linker molecules are needed to optimize the efficacy of immune recruitment while minimizing the risk of infection. It is designed to attract natural antibodies so that potential side effects can be minimized. and therefore are highly useful in providing effective anti-cancer and anti-infectious agent therapies. do. Summary of the Invention

[0010] (Summary of the Invention) According to the first aspect of the invention, a human anti-α-galactosyl antibody is capable of binding to the antibody. Immunoconjugates containing antibodies or antigen-binding fragments thereof linked to carbohydrate molecules via linkers a conjugate, wherein the linker is capable of binding to a human anti-α-galactosyl antibody. at least one phenyl ring capable of presenting one or more carbohydrate epitopes. The immunoconjugate is characterized by comprising:

[0011] According to a second aspect of the present invention, there is provided an immunosuppressant comprising a compound of formula (A) or a pharmaceutically acceptable salt thereof: Conjugates are provided: [ka] where F is a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody; y is phenyl, biphenyl, or triphenyl; S A and S B is the optimal distance between F and L m represents an integer selected from 1 to 5; z represents an integer selected from 1 to 30; and L is an antibody or an antigen-binding fragment thereof).

[0012] According to a further aspect of the present invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof To be: [ka] (In the formula, L represents a binding site selected from an antibody or antigen-binding fragment thereof; S1 is a group in which 1 to 10 of the -CH2- groups are -O-, -S-, ═N(H)-, -C(═O)-, -C(O)NH-, or -NHC(O)-. , cyclohexyl, or pyrrolidine-2,5-dione; optionally substituted, -(CH2) a -or-(CH2) b -(CH2-CH2-O) c -(CH2) d A spacer selected from the - group represents; a represents an integer selected from 1 to 35; b represents an integer selected from 0 to 5; c represents an integer selected from 1 to 20; d represents an integer selected from 1 to 20; S2 is a group in which 1 to 3 of the -CH2- groups are one selected from -N(H)-, -C(O)NH-, and -NHC(O)-. -(CH2) optionally substituted with the above groups; e -or-(CH2) f -(CH2-CH2-O)g -(CH2) h -Moto represents a spacer selected from the group consisting of: e represents an integer selected from 1 to 15; f represents an integer selected from 1 to 10; g represents an integer selected from 1 to 20; h represents an integer selected from 1 to 5; z represents an integer selected from 1 to 30; X1 represents the antibody or antigen-binding fragment attachment site; Y1 and Y2 independently represent a bond, -O-, -S-, -NH-, -NHC(O)-, -C(O)NH-, -OC(O)-, or -C(O)O-. , -SC(O)-, -C(O)S-, -NHSO2-, -SO2NH-, or -NHC(O)NH- group; F represents a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody; m represents an integer selected from 1 to 5; and Cy represents phenyl, biphenyl, or triphenyl; When -Y1-S1-X1-L represents a phenyl ring, the -Y1-S1-X1-L group may be located on any of the phenyl rings. , one or more of the [F-S2-Y2] m The - group may be present on any of the phenyl rings. ). [Brief explanation of the drawings]

[0013] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 is a reducing SDS PAGE analysis of Examples 1-4. [Figure 2] FIG. 2 is a reducing SDS PAGE analysis of Examples 5-8. [Figure 3] FIG. 3 shows the SEC analysis of the monomer content of Examples 1 to 8. [Figure 4] FIG. 4 shows M86 IgM data for the compounds of Examples 1-8. [Figure 5] FIG. 5 shows M86 IgG data for the compounds of Examples 1 to 4. [Figure 6] Figure 6 shows the capture of anti-α galactosyl IgM antibodies to the cell surface using Example 5 (Figure 6A), Example 6 (Figure 6B), Example 7 (Figure 6C), and Example 8 (Figure 6D) at 10 nM compared to 10 nM cetuximab. [Figure 7] FIG. 7 shows the dose titration of compounds of Examples 18-23 for mobilizing hIVIG anti-α-galactosyl IgG into A431 cells. [Figure 8] FIG. 8 shows the dose setting of the compounds of Examples 9 to 24 for recruiting anti-galactosyl M86 IgM antibodies to A431 cells. [Figure 9] FIG. 9 shows C3b deposition data for compounds of Examples 13-17, 20, 22, 23, and 24 on A431 cells using 20% ​​human serum (HS) or heat-inactivated human serum (HI HS)+25 μg / ml M86 IgM. [Figure 10] FIG. 10 shows the phagocytosis data for the compounds of Examples 20 and 23. [Figure 11] FIG. 11 shows the dose-setting of compounds of Examples 25 and 26 to recruit anti-galactosyl M86 IgM antibodies to Raji cells. [Figure 12] FIG. 12 is a reducing SDS-PAGE gel analysis of cetuximab conjugates (Examples 9-17). [Figure 13] FIG. 13 is a reducing SDS-PAGE gel analysis of cetuximab-Fab conjugates (Examples 18-24). [Figure 14] Figure 14 shows MS analyses of Examples 20 (Figure 14A), 22 (Figure 14B), and 23 (Figure 14C). [Figure 15] FIG. 15 shows a reducing SDS-PAGE (FIG. 15A) and SEC analysis of rituximab and rituximab-Fab conjugates (Examples 25 (FIG. 15B) and 26 (FIG. 15C)). [Figure 16]Figure 16 shows SDS-PAGE (Figure 16A), MS (Figure 16B), and SEC (Figure 16C) analyses of cetuximab-Fab. [Figure 17] Figure 17 shows SDS-PAGE (Figure 17A) and SEC analysis (Figure 17B) of rituximab and SEC analysis of rituximab-Fab (Figure 17C). DETAILED DESCRIPTION OF THE INVENTION

[0014] (Detailed Description of the Invention) According to the first aspect of the invention, a human anti-α-galactosyl antibody is capable of binding to the antibody. Immunoconjugates containing antibodies or antigen-binding fragments thereof linked to carbohydrate molecules via linkers a conjugate, wherein the linker is capable of binding to a human anti-α-galactosyl antibody. at least one phenyl ring capable of presenting one or more carbohydrate epitopes. The immunoconjugate is characterized by comprising:

[0015] In one embodiment of the first aspect of the invention, the linker is phenyl, biphenyl, or In a further embodiment of the first aspect of the present invention, the linker contains a biphenyl group.

[0016] According to a second aspect of the present invention, there is provided an immunosuppressant comprising a compound of formula (A) or a pharmaceutically acceptable salt thereof: Conjugates are provided: [ka] where F is a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody; y is phenyl, biphenyl, or triphenyl; S A and S B is the optimal distance between F and L m represents an integer selected from 1 to 5; z represents an integer selected from 1 to 30; and L is an antibody or an antigen-binding fragment thereof).

[0017] According to a second aspect of the invention that may be mentioned, there is provided a compound of formula (A) or a pharmaceutically acceptable salt thereof Immunoconjugates are provided which are: [ka] where F is a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody; y is phenyl, biphenyl, or triphenyl; S A and S B is the optimal distance between F and L m represents an integer selected from 1 to 5; z represents an integer selected from 1 to 10; and L is an antibody or an antigen-binding fragment thereof).

[0018] According to a further aspect of the present invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof To be: [ka] (In the formula, L represents a binding site selected from an antibody or antigen-binding fragment thereof; S1 is a group in which 1 to 10 of the -CH2- groups are -O-, -S-, ═N(H)-, -C(═O)-, -C(O)NH-, or -NHC(O)-. , cyclohexyl, or pyrrolidine-2,5-dione; optionally substituted, -(CH2) a -or-(CH2) b -(CH2-CH2-O) c -(CH2) d A spacer selected from the - group represents; a represents an integer selected from 1 to 35; b represents an integer selected from 0 to 5; c represents an integer selected from 1 to 20; d represents an integer selected from 1 to 20; S2 is a group in which 1 to 3 of the -CH2- groups are one selected from -N(H)-, -C(O)NH-, and -NHC(O)-. -(CH2) optionally substituted with the above groups; e -or-(CH2) f -(CH2-CH2-O) g -(CH2) h -Moto represents a spacer selected from the group consisting of: e represents an integer selected from 1 to 15; f represents an integer selected from 1 to 10; g represents an integer selected from 1 to 20; h represents an integer selected from 1 to 5; z represents an integer selected from 1 to 30; X1 represents the antibody or antigen-binding fragment attachment site; Y1 and Y2 independently represent a bond, -O-, -S-, -NH-, -NHC(O)-, -C(O)NH-, -OC(O)-, or -C(O)O-. , -SC(O)-, -C(O)S-, -NHSO2-, -SO2NH-, or -NHC(O)NH- group; F represents a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody; m represents an integer selected from 1 to 5; and Cy represents phenyl, biphenyl, or triphenyl; When -Y1-S1-X1-L represents a phenyl ring, the -Y1-S1-X1-L group may be located on any of the phenyl rings. , one or more of the [F-S2-Y2] m The - group may be present on any of the phenyl rings. ).

[0019] According to a further aspect of the invention which may be mentioned, a compound of formula (I) or a pharmaceutically acceptable salt thereof The salts provided are: [ka] (In the formula, L represents a binding site selected from an antibody or antigen-binding fragment thereof; S1 is a group in which 1 to 10 of the -CH2- groups are -O-, -S-, ═N(H)-, -C(O)NH-, -NHC(O)-, or cyclohexyl optionally substituted with one or more groups selected from: methyl, ... Good, -(CH2) a -or-(CH2) b -(CH2-CH2-O) c -(CH2) d represents a spacer selected from the group: a represents an integer selected from 1 to 35; b represents an integer selected from 0 to 5; c represents an integer selected from 1 to 20; d represents an integer selected from 1 to 20; S2 is a group in which 1 to 3 of the -CH2- groups are one selected from -N(H)-, -C(O)NH-, and -NHC(O)-. -(CH2) optionally substituted with the above groups; e -or-(CH2) f -(CH2-CH2-O) g -(CH2) h -Moto represents a spacer selected from the group consisting of: e represents an integer selected from 1 to 15; f represents an integer selected from 1 to 10; g represents an integer selected from 1 to 20; h represents an integer selected from 1 to 5; z represents an integer selected from 1 to 10; X1 represents the antibody or antigen-binding fragment attachment site; Y1 and Y2 independently represent a bond, -O-, -S-, -NH-, -NHC(O)-, -C(O)NH-, -OC(O)-, or -C(O)O-. , -SC(O)-, -C(O)S-, -NHSO2-, -SO2NH-, or -NHC(O)NH- group; F represents a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody; m represents an integer selected from 1 to 5; and Cy represents phenyl, biphenyl, or triphenyl; When -Y1-S1-X1-L represents a phenyl ring, the -Y1-S1-X1-L group may be located on any of the phenyl rings. , one or more of the [F-S2-Y2] m The - group may be present on any of the phenyl rings. ).

[0020] The present invention provides conjugation to single or multiple sites on a selected antibody or fragment thereof. In addition to the target peptide, it allows the ability to present one or more carbohydrate epitopes, thus Novel immunoconjugates that allow optimal recruitment of natural antibodies while retaining binding efficacy The present invention includes a description and use of conjugate linkers, which will enable those skilled in the art to determine the optimal anti-Gal mobilization. Fine-tune the optimal number of carbohydrates per antibody or its fragment selected for targeting Provided to maintain validity.

[0021] Monoclonal antibodies have greatly improved outcomes for patients with cancer; however, However, certain patient populations exhibit inherent resistance to these therapies and have been shown to have favorable outcomes. Although some patients have reported side effects, these may be short-lived and may not respond to mAb therapy. Resistance continues to be a problem and increased antibody efficacy is desirable. Tumors have increased receptivity. Anti-inflammatory effects, such as through alterations to immune expression or signaling pathways, or a diminished immune response. may exhibit or develop mechanisms that result in resistance or a reduced response to systemic treatment. (Reslan, L. Reference Mabs 2009, 3, 222). For example, patients may develop EGFR signaling-affected Intrinsic resistance to cetuximab may arise from the expression of KRAS mutations that affect (Lievre, A.: J. Clin. Oncol. 2008, 26, 374). In addition, if the patient initially Even if patients respond well to simam, most will eventually develop resistance. (Bianco, R., Endocr. Relat. Cancer 2005, S159; Brand, T.M., Cancer Bi ol. Ther. 2011, 11, 777).

[0022] A further example of resistance to therapeutic antibodies is rituximab (an anti-CD20 monoclonal antibody). Resistance to rituximab has been observed in patients with non-Hodgkin's lymphoma who were treated with It is observed in about half of treatment-naive patients. Patients who initially respond to rituximab therapy Resistance often develops. The mechanisms of resistance are complex, and strategies to overcome resistance are difficult. has shown limited success in patients (Best Pract. Res. Clin. Haematol. 2011 , 203-216), thus improving the activity of therapeutic antibodies and increasing and prolonging patient responses. This is still desperately needed.

[0023] Many approaches have been developed using antibody-drug conjugates (ADCs), antibody-toxin conjugates (immunotoxins), and enhanced effector mechanisms, e.g., increased antibody-dependent cellular cytotoxicity (ADCC) have been employed to improve the efficacy of therapeutic antibodies, including engineered antibodies with Despite these efforts, few therapies have achieved clinical success and many have been associated with toxicity and other problems. Side effects of steroids remain a significant problem (Beck, A.: Nat. Rev. Drug. Discov. 2 017). Therefore, new strategies to modify mAbs to enhance efficacy and improve patient outcomes are needed. It is being done.

[0024] The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) is naturally found on glycolipids and glycoproteins. It is a unique carbohydrate that displays multiple epitopes on branched oligosaccharides (J. Immunol. Nology (2007), 178 (7), 4676-87). This α-Gal epitope is found in, for example, multiple glycosylated Transferrin, an enzyme known to catalyze the synthesis of Galα-1,3-Gal at the cleavage site, It is synthesized by the enzyme α1,3-GT (WO98 / 34957). Although this method is efficient, This synthesis relies on existing or engineered glycosylation sites and allows for one conjugation step. This only allows for one α-Gal unit per binding site.

[0025] Therefore, one or more of the α-Gal trisaccharide units are used to optimally utilize the natural immune system. There is an interesting need for a modular approach that is suitable for either presentation.

[0026] The compounds of the present invention are characterized in that they have an F group (i.e., the number and location of carbohydrate molecules that can bind to human anti-α-galactosyl antibodies For example, rigid linker molecules that are optimized for controlling and presenting The cyclic group has the advantage of providing a scaffold for optimal placement of one or more F groups relative to L. The exact number and orientation of F groups relative to L varies depending on the nature of the L group. It will be understood that the term "phenyl ring" is used interchangeably with "biphenyl ring" and "triphenyl ring." The presence of a cyclic group containing a ring allows for the incorporation of multiple F groups (i.e., that is, presenting carbohydrate molecules capable of binding to human anti-α-galactosyl antibodies. The chemical presentation of multiple binding groups has not been previously described in the art. was known in the art, which contained a single six-membered ring system (i.e., phenyl), two bonded rings a six-membered ring system (biphenyl) or a three-membered ring system (triphenyl) connected by two bonds In contrast to the present invention, which uses either one or more amino acid groups (e.g., WO2014 / 178878 ) or branched linker groups (see, e.g., US 2014 / 0112975) The technical effect of this difference is that the compounds of the present invention are They can be prepared more easily than previously known linkers and advantageously do not contain chiral centers. This means that the presence of proteases can be avoided, making it less susceptible to protease degradation. The synthesis of the compounds of the present invention is also resin-free and therefore suitable for large-scale pharmaceutical manufacturing. Thus, the compounds of the present invention offer the advantage of being suitable for therapeutic expansion. Not only does it have the advantage of enhancing the immune response from the host, but it also has the advantage of being scalable and high yielding. In addition, the linkers of the present invention are not unstable and can be easily and efficiently synthesized. Thus, the "cleavable" cleavage sites required by many compounds previously known in the art are not They typically do not include a "linker" moiety (see, e.g., US 8,828,956). The linkers of the present invention are well suited to the skilled artisan's ability to select specific left- and right-handed groups, along with ease and efficiency of synthesis. Allows for the selection of right hand combinations to provide antibody or fragment thereof conjugation sites or This allows for an optimal number of F groups.

[0027] Fab, Fab', Fab'2, Fab2, Fab3, F(ab)2, Fv, scFv, diabody, triabody, Monoclonal antibody fragments, such as tetrabodies and nanobodies, are also known in the field of oncology. Increased tumor penetration resulting from their smaller size, easier manufacturing (which They can be expressed in E. coli or yeast, allowing for increased convenience and greater efficiency. advantages over full-length mAbs, including reduced immunogenicity (resulting in efficient scale-up) and reduced immunogenicity However, fragments such as scFvs or Fab fragments have been reported to have several advantages. This may result in reduced efficacy compared to full-length mAbs. For example, the presence of F on the Fab fragment Lack of the c domain often results in ADCC-driven efficacy, which is a component of the antitumor response. (Nelson, AL Reference Nelson 2009, 2, 77).

[0028] Thus, the benefits of antibody fragments are often offset by the loss of function associated with them. The optimal number of α-Gal sites provides new constructs conjugated to antibody fragments. The use of phenyl-containing linkers of the present invention to enhance the antitumor activity of antibody fragments. It offers a new approach to

[0029] For both mAbs and their fragments, the ability to differentiate mAbs from their host cells can be improved, for example, by incorporating additional cysteine ​​residues. Such controlled, site-specific conjugation is well known in the art. Such an approach is particularly useful for the detection of the mAb or fragment without destroying the target binding epitope. By allowing derivatization and controlled loading, It offers some advantages, namely, that constant stoichiometry is achieved (Shen, BQ: Nat. Biotechnol. 2012, 30, 184). One limitation of site-specific conjugation is the This can result in low loading of conjugated sites, which can affect efficacy. This may limit the number of α-Ga moieties that can be displayed per conjugation site. The use of phenyl-containing linkers of the present invention that allow for the number of conjugation sites to be increased Provides a method to achieve high α-Gal / antibody ratios (high loading) even with low amounts .

[0030] (Linker definition) In one embodiment, S1 is: 1 to 5 (e.g., 2, 3, or 5) of the -CH2- groups are -S-, ═N(H)-, -C(═O)-, or -NHC(O)- , cyclohexyl, or pyrrolidine-2,5-dione; optionally substituted, -(CH2) a -(e.g., -(CH2)2-NHCO-cyclohexyl-CH2-3-pyrrolidine-2, 5-dione-, -(CH2)2-NHCO-cyclohexyl-CH2-3-pyrrolidine-2,5-dione-S-(CH2)3-C(=N H)-, or -(CH2)2-NHCO-(CH2)3-CO, etc.); or One to five (e.g., two) of the -CH2- groups are -NHC(O)- or pyrrolidine-2,5-dione. -(CH2) optionally substituted with one or more groups selected from b -(CH2-CH2-O) c -(CH2) d -( For example, -(CH2)2-NHCO-(CH2CH2O)4-(CH2)2-3-pyrrolidine-2,5-dione- represents a spacer selected from

[0031] In a further embodiment, S1 is: 1 to 10 of the -CH2- groups are -O-, -S-, ═N(H)-, -C(O)NH-, -NHC(O)-, or cyclohexyl or pyrrolidine-2,5-dione, -(CH2) a -(e.g., -(CH2)2-NHCO-cyclohexyl-CH2-pyrrolidine-2,5-dione-, or -(C H2) 2-NHCO-cyclohexyl-CH2-pyrrolidine-2,5-dione-S-(CH2)3-C(=NH)-, etc. represents a spacer selected from

[0032] a, b, c, d, e, f, g, and h are selected to maintain a suitable linker length between groups F and L. It will be understood that the linker length between F and L is, for example, about 5 Å to about 50 Å. Lengths of about 6 Å to about 45 Å, about 7 Å to about 40 Å, about 8 Å to about 35 Å, about 9 Å to about 30 Å, about 10 Å The range is about 25 Å, about 11 Å to about 20 Å, or about 12 Å to about 15 Å. , b, c, d, e, f, g, and h are integers totaling no more than 45, for example, from 5 to 45, for example, from 7 to 42; For example, it represents 30 or less, for example, 5 to 30, for example, 7 to 29.

[0033] In one embodiment, a represents an integer selected from 1 to 30. , a represents an integer selected from 2 to 30. In a further embodiment, a is 2, 4, 6, 9, 1 represents an integer selected from 1, 18, or 30. In a further embodiment, a is an integer selected from 6 to 30. In a further embodiment, a represents an integer selected from 6, 11, 18, or 30. In a further embodiment, a represents an integer selected from 5 to 15. In embodiments, a represents an integer selected from 6 to 11. In further embodiments, a is In still further embodiments, a represents an integer selected from 6, 7, or 11. In an alternative embodiment, a represents an integer selected from 7. In another embodiment, a represents an integer selected from 11.

[0034] In one embodiment, b represents an integer selected from 0 to 3. , b represents an integer selected from 0 or 3. In a further embodiment, b is selected from 1 to 3 In a further embodiment, b represents an integer selected from 2 or 3. In still further embodiments, b represents an integer selected from 3.

[0035] In one embodiment, c represents an integer selected from 1 to 15. , c represents an integer selected from 1 to 12. In a further embodiment, c is selected from 4 to 12. In still further embodiments, c represents an integer selected from 4 or 12. In still further embodiments, c represents an integer selected from 4.

[0036] In one embodiment, d represents an integer selected from 1 to 15. and d represents an integer selected from 2 to 13. In further embodiments, d is 2, 5, or 13. In a further embodiment, d represents an integer selected from 13. In an alternative embodiment, d represents an integer selected from 3.

[0037] In one embodiment, Y1 represents a bond, -C(O)NH-, or -O-. , Y1 represents -C(O)NH-.

[0038] In one embodiment, S2 is One or two of the -CH2- groups are replaced by one or two groups selected from -N(H)-, -C(O)NH- and -NHC(O)-. optionally substituted with -(CH2) e -(e.g., -(CH2)3-NHCO-CH2-, -(CH2)3-, -(CH2)3-NH CO-(CH2)4-CONH-CH2-, -(CH2)3-NH-CH2- or -(CH2)3-NHCO-(CH2)3-NHCO-CH2-, etc.); or 1 to 3 of the -CH2- groups are optionally replaced by 1 to 3 -NHC(O)- groups, -(CH2) f -(CH2-CH 2-O) g -(CH2) h -(e.g., -(CH2)3-NHCO-(CH2CH2O)4-(CH2)2-NHCO-CH2-, -(CH2)3-NHCO-(CH 2CH2O) 12-(CH2)2-NHCO-CH2- or -(CH2)3-NHCO-(CH2)3-NHCO-(CH2CH2O)4-(CH2)2-NHCO -CH2- etc.): represents a spacer selected from

[0039] In a further embodiment, S2 is: one or two of the -CH2- groups are optionally replaced by a -C(O)NH- or -NHC(O)- group, -(CH2) e -(e.g., -(CH2)3-NHCO-CH2-, -(CH2)3-NHCO-, -(CH2)3-, -(CH2)3-NHCO-(CH2)4-CONH-C H2- or -(CH2)3-NH-CH2-, etc.); or one or two of the -CH2- groups are optionally replaced by a -C(O)NH- or -NHC(O)- group, -(CH2) f -(CH2-CH2-O) g -(CH2) h -(e.g., -(CH2)3-NHCO-(CH2)2-(OCH2CH2)4-NHCO-CH2- or -( CH2)4-NHCO-(CH2)2-(OCH2CH2)4-NHCO-CH2- etc.) represents a spacer selected from

[0040] In still further embodiments, S2 is: one or two of the -CH2- groups are optionally replaced by one or two -NHC(O)- groups, -(CH2) e -(example For example, -(CH2)3-NHCO-CH2- or -(CH2)3-NHCO-(CH2)3-NHCO-CH2-; or 1 to 3 of the -CH2- groups are optionally replaced by 1 to 3 -NHC(O)- groups, -(CH2) f -(CH2-CH 2-O) g -(CH2) h-(e.g., -(CH2)3-NHCO-(CH2CH2O)4-(CH2)2-NHCO-CH2-, -(CH2)3-NHCO-(CH 2CH2O) 12 -(CH2)2-NHCO-CH2- or -(CH2)3-NHCO-(CH2)3-NHCO-(CH2CH2O)4-(CH2)2-NHCO -CH2-, etc.) represents a spacer selected from

[0041] In still yet further embodiments, S2 is: One or two of the -CH2- groups are one or more selected from -N(H)-, -C(O)NH-, and -NHC(O)-. is optionally substituted with two groups, -(CH2) e - (e.g., -(CH2)3-NHCO-CH2-, etc.); or 1 to 3 of the -CH2- groups are optionally replaced by 1 to 3 -NHC(O)- groups, -(CH2) f -(CH2-CH2-O) g -(CH2) h - (e.g., -(CH2)3-NHCO-(CH2CH2O)4-(CH2)2-NHCO-CH2-, etc.) represents a spacer selected from

[0042] In still yet further embodiments, S2 is: One or two, for example one, of the -CH2- groups are selected from -N(H)-, -C(O)NH-, and -NHC(O)-. optionally substituted with one or two, for example one, group selected from the group —(CH) e -(for example, -(CH2)3-NHCO-CH2-, etc.) represents a spacer selected from

[0043] In still yet further embodiments, S2 is: one of the -CH2- groups is optionally replaced by a -NHC(O)- group, -(CH2) e -(for example,- (CH2)3-NHCO-CH2-, etc.); or two of the -CH2- groups are optionally replaced by -NHC(O)- groups, -(CH2) f -(CH2-CH2-O ) g -(CH2) h - (e.g., -(CH2)3-NHCO-(CH2CH2O)4-(CH2)2-NHCO-CH2-, etc.) represents a spacer selected from

[0044] In one embodiment, e represents an integer selected from 1 to 10. , e represents an integer selected from 3 to 10. In further embodiments, e is 3, 5, 9, or In a further embodiment, e represents an integer selected from 5 to 9. In a further embodiment, e represents an integer selected from 5 or 9. In an embodiment, e represents an integer selected from 4 to 10. In still a further embodiment, e represents an integer selected from 4, 5, or 10. In still yet further embodiments, e is 5 Represents an integer selected from

[0045] In one embodiment, f represents an integer selected from 1 to 8. and f represents an integer selected from 2 to 8. In a further embodiment, f is selected from 2 to 6. In still further embodiments, f represents an integer selected from 4 to 8. In still further embodiments, f represents an integer selected from 4 or 8. In an embodiment, f represents an integer selected from 4.

[0046] In one embodiment, g represents an integer selected from 1 to 15. In a further embodiment, g represents an integer selected from 4 to 12. In a further embodiment, g represents an integer selected from 1 to 5. In a further embodiment, g represents an integer selected from 1 to 4. wherein g represents an integer selected from 4.

[0047] In one embodiment, h represents an integer selected from 1 to 4. In a further embodiment, h represents an integer selected from 4.

[0048] In one embodiment, Y2 represents a bond, -O-, or -NHC(O)-. wherein Y2 represents a bond or -O- In a still further embodiment, Y2 represents -O-.

[0049] In one embodiment, m represents an integer selected from 1 to 4. and m represents an integer selected from 1 to 3. In still further embodiments, m is an integer selected from 1 or 3. In still further embodiments, m represents an integer selected from 2 or 3. In still further embodiments, m represents an integer selected from 1 or 2. In yet further embodiments, m represents an integer selected from 1. and m represents an integer selected from 2. In still further embodiments, m is selected from 3 In still further embodiments, m represents an integer selected from 4.

[0050] In a further embodiment, z represents an integer selected from 1 to 25. In a further embodiment, z represents an integer selected from 2 to 20. Represents a selected integer (e.g., 2, 4, 9, 5, 7, 8, 10, 11, 14, 15, 17, or 20). In some embodiments, z represents an integer selected from 1 to 8. represents an integer selected from 1 to 5. In still further embodiments, z is selected from 2 to 5. In still further embodiments, z represents an integer selected from 2 or 5. In still further embodiments, z represents an integer selected from 2. In the above formula, z represents an integer selected from 5.

[0051] In one embodiment, Cy represents phenyl or biphenyl. In yet a further embodiment, Cy represents biphenyl or triphenyl. In still yet further embodiments, Cy represents biphenyl or triphenyl. vinegar.

[0052] According to a further aspect of the present invention, a compound of formula (I) a or a pharmaceutically acceptable salt thereof To be: [ka] (In the formula, L represents a binding site selected from an antibody or antigen-binding fragment thereof; S1 is: Two, three or five of the -CH2- groups are -S-, =N(H)-, -C(=O)-, -NHC(O)-, cyclohexyl and optionally substituted with one or more groups selected from: silyl, methylsilyl, methylpyrrolidine-2,5-dione ... i, -(CH2) a -or two of the -CH2- groups are one or more selected from -NHC(O)- or pyrrolidine-2,5-dione; optionally substituted by a -(CH) group; b -(CH2-CH2-O) c -(CH2) d - represents a spacer selected from: a represents an integer selected from 6, 7, or 11; b represents an integer selected from 3; c represents an integer selected from 4; d represents an integer selected from 3; S2 is: one of the -CH2- groups is optionally replaced by a -NHC(O)- group, -(CH2) e -or two of the -CH2- groups are optionally replaced by -NHC(O)- groups, -(CH2) f -(CH2-CH2-O ) g -(CH2) h - represents a spacer selected from: e represents an integer selected from 5; f represents an integer selected from 4; g represents an integer selected from 4; h represents an integer selected from 4; z represents an integer selected from 2 to 20; X1 represents -S- or -N(H)-; Y1 represents -C(O)NH-; Y2 represents -O-; F represents a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody; m represents an integer selected from 1 or 3; and Cy represents biphenyl, and the -Y1-S1-X1-L group is present on either of the phenyl rings. may be present, and one or more of the [F-S2-Y2] m The - group may be on either of the phenyl rings. may be present in

[0053] According to a further aspect of the present invention which may be mentioned, a compound of formula (I) a or a pharmaceutically acceptable salt thereof The salts provided are: [ka] (In the formula, L represents a binding site selected from an antibody or antigen-binding fragment thereof; S1 is a group in which 1 to 5 of the -CH2- groups are -O-, -S-, =N(H)-, -C(O)NH-, -NHC(O)-, or cyclohexyl optionally substituted with one or more groups selected from: methyl, ... Good, -(CH2) a represents a spacer selected from the group: a represents an integer selected from 6 or 11; S2 is a group in which 1 to 3 of the -CH2- groups are one selected from -N(H)-, -C(O)NH-, and -NHC(O)-. -(CH2) optionally substituted with the above groups; e represents a spacer selected from the group: e represents an integer selected from 5; z represents an integer selected from 2 to 5; X1 represents -S- or -N(H)-; Y1 represents -C(O)NH-; Y2 represents -O-; F represents a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody; m represents an integer selected from 1 or 3; and Cy represents biphenyl, and the -Y1-S1-X1-L group is present on either of the phenyl rings. may be present, and one or more of the [F-S2-Y2] m The - group may be on either of the phenyl rings. may be present in

[0054] In a further embodiment, the present invention provides the compounds of Examples 1 to 26 or pharmaceutically acceptable salts thereof. The present invention provides compounds of formula (I) including salts thereof.

[0055] In a further embodiment, the present invention provides compounds of formula (I) which are the free bases of the compounds of Examples 1 to 26. Provide a mixture.

[0056] In a further embodiment, the present invention provides the compounds of Examples 1 to 8 or pharmaceutically acceptable salts thereof. The present invention provides compounds of formula (I), including salts thereof.

[0057] In a further embodiment, the present invention provides compounds of formula (I) which are the free bases of the compounds of Examples 1 to 8. Provide a mixture.

[0058] (αGal) As used herein, the term "carbohydrate capable of binding to a human anti-α-galactosyl antibody" refers to a carbohydrate that is capable of binding to a human anti-α-galactosyl antibody. Reference to a "molecule" refers to a molecule that binds to a component of the human immune response (i.e., an anti-α-galactosyl antibody). and containing a sugar (i.e., carbohydrate) moiety that can result in eliciting an immune response in humans. In one embodiment, the anti-α-galactosyl antibody is an anti-α-galactosyl IgG antibody or Examples of such carbohydrate molecules include α-galactosyl compounds and Further examples of suitable carbohydrate molecules include those capable of selectively targeting tumor cells. α-gal epitopes listed in US2012 / 0003251 suitable for use in targeting and killing and these epitopes are incorporated herein by reference. In the formula, F is galactosyl-α-1,3-galactosyl-β-1,4-N-acetylglucosamine, α1-3 galactobiose, α1-3-β1-4 galactotriose or galilipentasaccharide ( galilipentasaccharide).

[0059] In one particular embodiment, F has the formula: [ka] where S2 refers to the point of attachment to the S2 group.

[0060] In one particular embodiment, F has the formula: [ka] where S2 refers to the point of attachment to the S2 group.

[0061] (Antibodies and their antigen-binding fragments) Reference herein to the term "antibody" or "antibodies" refers to antibodies that bind to a known antigen. Molecules or active fragments of molecules that bind to immunoglobulins, particularly immunoglobulin molecules and immunoglobulin molecules. Refers to an epidemiologically active moiety, i.e., a molecule that contains a binding site that immunospecifically binds to an antigen The immunoglobulins according to the present invention may be of any class (IgG, IgM, IgD, IgE, IgA, and IgY), or subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or immunoglobulins The subclass (isotype) of the phosphomolecule (e.g., IgG in IgG1, IgG2, IgG3, and IgG4) , or IgA in IgA1 and IgA2).

[0062] Within the scope of the present invention, the term "antibody" or "antibodies" refers to a monoclonal antibody. antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, bispecific antibodies, human antibodies, and human Examples of active fragments of molecules that bind to known antigens include: The antibodies may be derived from the products of a Fab immunoglobulin expression library and any of the antibodies and fragments mentioned above. Examples of such fragments include Fab, F(ab')2, scFv, and Fv fragments that contain an epitope-binding fragment.

[0063] As used herein, the term "monoclonal antibody" refers to a single clone produced in the laboratory. Monoclonal antibodies are mass-produced antibodies that recognize only one antigen. Typically, antibody-producing B cells, which are normally short-lived, are transformed into fast-growing cells ("immortal" cells), such as cancer cells. The resulting hybrid cells are produced by fusing the cells with the host. The hybridoma, or hybridoma, grows rapidly and gives rise to clones that produce large amounts of antibody. For purposes of this invention, a "monoclonal antibody" also refers to a antibody that is not yet completely monoclonal. It is also understood to include antibodies produced by mother clones that have not yet reached maturity.

[0064] As used herein, the term "chimeric antibody" refers to an antibody that is produced by recombinant DNA techniques, typically The variable regions, i.e., binding regions, of mouse origin and those of different origins or species are prepared. It refers to a monoclonal antibody containing at least a portion of the constant region obtained by mouse variable region and Chimeric antibodies comprising a mouse / human constant region are exemplary embodiments. Chimeric antibodies are composed of DNA segments encoding mouse immunoglobulin variable regions and human immunoglobulins. Expressed immunoglobulin genes containing DNA segments encoding immunoglobulin constant regions Another form of "chimeric antibody" encompassed by the present disclosure is a chimeric antibody of any class or subclass. The "texture" of the antibody is modified or changed from that of the original antibody. "Class-switched" antibodies are also called "class-switched antibodies." Methods for producing chimeric antibodies include: Conventional recombinant DNA techniques and gene transfection techniques now well known in the art can be used to For example, Morrison, SL et al., Proc. Natl. Acad Sci. USA 81 (1 984) 6851-6855; U.S. Patent Nos. 5,202,238 and 5,204,244.

[0065] As used herein, the term "humanized antibody" or "humanized version of an antibody" refers to a , the framework or "complementarity determining regions" (CDRs) are different from those of the parent immunoglobulin. It refers to an antibody that has been modified to contain the CDRs of an immunoglobulin of different specificity. In one embodiment, the CDRs of the VH and VL are grafted into the framework regions of a human antibody. For example, Riechmann, L. et al., Nature 332 (1988) 3 23-327; and Neuberger, MS et al., Nature 314 (1985) 268-270. The variable framework regions of the high and light chains may be derived from the same or different human antibody sequences. The human antibody sequence can be the sequence of a naturally occurring human antibody. Heavy and light chain variable framework regions are described, for example, in Lefranc, M.-P.: Current Protocols in Molecular Biology. are listed in Protocols in Immunology (2000)-Appendix 1P A.1P.1-A.1P.37, and See, for example, IMGT, the international ImMunoGeneTics information System (registered trademark) (http: / / imgt.org / immunogenetics / information / systems / immunogenetics ... Available via http: / / imgt.cines.fr or http: / / vbase.mrc-cpe.cam.ac.uk. Optional Additionally, the framework regions can be modified by further mutations. The sequences correspond to those representing the antigen-recognizing sequences described above for the chimeric antibody. In embodiments, such humanized versions are chimerized with human constant regions. As used herein, the term "humanized antibody" also refers to antibodies that have been "class switched", i.e. Specifically, changes or mutations in the Fc portion (e.g., IgG1 to IgG4 and / or IgG1 / IgG4 mutations) and modified in the constant region to produce properties according to the present disclosure with respect to C1q binding and / or FcR binding. The present invention includes such antibodies that cause the same reaction.

[0066] As used herein, the term "human antibody" refers to an antibody that is based on human germline immunoglobulin sequences. Human antibodies are intended to include antibodies having variable and constant regions derived from: It is well known in the state of the art (van Dijk, MA and van de Winkel, JG: Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies are produced in the absence of endogenous immunoglobulin production. In the presence of The gene can also be produced in a transgenic animal (e.g., a mouse) that can reproduce the gene. The human germline immunoglobulin gene array in such germline mutant mice is The introduction results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A. et al., Proc. c. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A. et al.: Nature 362 (1993) 255-258; Brueggemann, MD et al., Year Immunol. 7 (1993) 33-40. Human antibodies can also be produced in phage display libraries ( Hoogenboom, H.R. and Winter, G.: J. Mol. Biol. 227 (1992) 381-388; Marks, J. D. et al., J. Mol. Biol. 222 (1991) 581-597). Cole, A. et al. and Boerner, P. et al. Techniques are also available for the preparation of human monoclonal antibodies (Cole, A. et al., Monoclonal Antibodies, 2004). Onal Antibodies and Cancer Therapy, Liss, AR (1985) p. 77; and Boerner, P. et al. Reference: J. Immunol. 147 (1991) 86-95). As already mentioned, according to the present disclosure, The term "human antibody" as used herein also refers to antibodies that have been "class switched", i.e. i.e., by changes or mutations in the Fc portion (e.g., IgG1 to IgG4 and / or IgG1 / IgG4 mutations). For example, the constant region may be modified to produce the properties according to the present disclosure with respect to C1q binding and / or FcR binding. The present invention also includes such antibodies modified with a region.

[0067] As used herein, a "single chain antibody" refers to an antibody in which the VH domain and the VL domain are separated into two domains. are linked by a peptide linker that allows them to cooperate to form an antigen-binding site. Single-chain Fv molecules (scFv) (Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Science 242: 423-426) 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883), or bispecific single-chain Fv (WO03 / 1 1161).

[0068] As used herein, the term "bispecific antibody" refers to an antibody that binds two (or more) different It refers to an antibody that binds to an antigen.

[0069] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, e.g., By "antibody fragment" it is meant a variable domain thereof, or at least an antigen-binding portion thereof. Examples of antibody fragments are: Examples include diabodies formed from antibody fragments, single-chain antibody molecules, and multispecific antibodies. The scFv antibody can be produced by, for example, the method described in Huston, JS: Methods in Enzymol. 203 (1991) Antibody fragments can be prepared from the antibodies of the present invention by several known techniques. For example, purified monoclonal antibodies can be derived from enzymes such as pepsin. The fragments containing the Fab fragments can then be digested with HPLC gel filtration. The appropriate fractions are collected and concentrated by membrane filtration or the like. For further description of typical techniques, see, for example, Khaw, BA et al., J. Nucl. Med. 23: 1011-1019 (1982); Rousseaux et al., Methods Enzymology, 121:663-69, Academic Press See ess, 1986.

[0070] As used herein, the terms "specific" and "specifically" are used interchangeably. The antibody specifically binds to the biomolecule of interest, preventing other biomolecules from significantly binding to the antibody. In some embodiments, a peptide other than the peptide containing the epitope within the peptide is used. The level of binding to the body molecule is consequently negligible by ELISA or affinity determination (e.g. The binding affinity is so low that it cannot be determined.

[0071] "Negligible binding" refers to binding to peptides containing epitopes within the peptide. than at least about 85%, particularly at least about 90%, and more particularly at least about 95%, even more particularly at least about 98%, but especially at least about 99% and Up to 100% less binding is meant.

[0072] As used herein, the term "epitope" refers to a region of interest that is ... an antigen-binding molecule (e.g., an antibody It refers to the site on a target molecule (e.g., an antigen such as a protein) to which a specific antibody (or antibody fragment) binds. A pitope may consist of both contiguous or adjacent non-contiguous residues (e.g., amino acid residues) of a target molecule. Epitopes formed from consecutive residues (e.g., amino acid residues) can be formed from Epitopes are usually also called linear epitopes. Epitopes usually consist of at least five and It contains a maximum of about 12 residues, and often 6 to 10 residues (eg, amino acid residues).

[0073] As used herein, the term "CDR" refers to a hypervariable region of an antibody. The terms "HVR" or "HV" as used herein refer to a region in which a sequence is hypervariable. It refers to the regions of an antibody variable domain that are present and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Several hypervariable region delineations are in use and are encompassed herein. Kabat complement The sex-determining region is based on sequence variability and is the most commonly used (Kabat et al., 2004). Sequences of Proteins of Immunological Significance rest), 5th edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) The letters "HC" and "LC" before the term "CDR" refer to the heavy and light chain CDRs, respectively. Refers to CDR.

[0074] As used herein, the terms "homology" and "identity" are used interchangeably. Calculations of sequence homology or identity between sequences are performed as follows.

[0075] To determine the percent (%) identity of two amino acid sequences or two nucleic acid sequences, the sequences are The columns are aligned for optimal comparison purposes (e.g., the first and second arrays are aligned for optimal alignment). Gaps can be introduced into either or both of the amino acid or nucleic acid sequences and can be added for comparison purposes. In a preferred embodiment, for comparison purposes, non-homologous sequences can be ignored. The length of the reference sequence to be aligned for this purpose is at least 30% of the length of the reference sequence, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 %. Then, the amino acid residue or nucleotide at the corresponding amino acid position or nucleotide position is The sequences are compared. If a position in the first sequence has the same amino acid sequence as the corresponding position in the second sequence, If the position is occupied by an acid residue or a nucleotide, the molecules are identical at that position (as defined herein). As used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology." The percent identity between two sequences is calculated based on optimal alignment of the two sequences. The number of gaps that need to be inserted and the length of each gap must be taken into account. It is a function of the number of identical positions that are present.

[0076] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, this can be achieved by using a percentage between two amino acid sequences. The identity of the target gene was determined using either the BLOSUM 62 matrix or the PAM250 matrix. Gap weights of 16, 14, 12, 10, 8, 6, or 4 and 1, 2, 3, 4, 5, or The GCG software package (http: / / www Needleman et al. (1970), which is incorporated into the GAP program of the GCG (available at gcg.com) J. Mol. Biol. 48:444-453. In an embodiment, the percent identity between two nucleotide sequences is determined by the NWSgapdna.CMP matrix. Uses a gap weighting of 40, 50, 60, 70, or 80 and a gap weighting of 1, 2, 3, 4, 5, or using a length weighting of 6, and the GCG software package (available at http: / / www.gcg.com A particularly preferred set of parameters (and and practitioners will be able to determine which parameters determine whether a molecule falls within the sequence identity or homology limits of the present invention. (which should be used when you are unsure whether it applies to determining whether it is Gap penalty of 12, gap extension penalty of 4, and frameshift gap BLOSUM 62 scoring matrix with a penalty of 5.

[0077] Alternatively, the percent identity between two amino acid or nucleotide sequences can be calculated using the PAM120 weights: PAM120 weight residue table, gap length penalty of 12, and gap Using the cap penalty, the Me The determination can be made using the algorithm of Yers et al. (1989) CABIOS 4:11-17. .

[0078] As used herein, the term "conservative amino acid substitution" refers to a substitution of a similar amino acid residue. It refers to the replacement of amino acid residues with amino acid residues having similar side chains. These families include those with basic side chains. amino acids with an acidic side chain (e.g., lysine, arginine, histidine), (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains amino acids (e.g., threonine, valine, isoleucine), as well as amino acids with aromatic side chains acids (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0079] In one embodiment, the antibody is a polyclonal antibody. The antibody is a humanized antibody, a human antibody, a murine antibody, or a chimeric antibody.

[0080] In one embodiment, the antigen-binding fragment thereof is an antigen-binding fragment (Fab) or single chain variable In a further embodiment, the fragment is a Fab, Fab', F(ab)2, F(ab')2 fragment. X1 is selected from the group consisting of any suitable antibody or antigen-binding fragment attached thereto. It is also possible to represent a site, and the selection of the group depends on the amino acid selected as the point of attachment within the antibody. It will be appreciated that the degree of cleavage will depend on the acid residue.

[0081] In one embodiment, X1 represents -S- or -N(H)-. In this embodiment, L represents three conjugated to the compound of formula (I) by one of the following structures: [ka] where S1 and z are as defined herein, and Ab is a reactive thiol or lysine group. (This refers to an antibody or antigen-binding fragment thereof terminated with a thiol or cis-amino group.) Reacting the tein groups may be available on the antibody or antigen-binding fragment thereof, or These are introduced by site-specific modifications to the peptide chain, resulting in engineered amino acids. This would create additional conjugation points via the amino acid residues (Nat. Biotechnol. 2013). 08, 925; Nat. Biotechnol. 2012, 184). Alternatively, site-specific amino acids can be added to the desired peptide. Orthogonal chemistries for site-specific conjugation incorporated into peptide chains It is possible to achieve orthogonal reactivity (OPRD (2016), 20, 852-866). Examples of amino acids include p-acetylphenylalanine (pAcPhe, J. Mol. Biol. 2011, 595), para-azidomethyl-l-phenylalanine (pAMF, Bioconjug. Chem 2014, 351), N6-((2-azidomethyl-l-phenylalanine) (Didoethoxy)carbonyl)-L-lysine (Bioconjug. Chem. 2015, 2249).

[0082] The antibody or antigen-binding fragment of the present invention can be used to treat either cancer cells or specific pathogens. It will be appreciated that the antibody will be configured to bind to a target.

[0083] In one embodiment, the antibody or antigen-binding fragment is configured to bind to a cancer cell. In a further embodiment, the antibody or antigen-binding fragment binds to its cell surface expression on tumor cells. The expression specifically binds to a tumor-associated antigen that differs from its expression on healthy cells.

[0084] In a preferred embodiment, the antibody or antigen-binding fragment binds to a target on a cancer cell or pathogen. Preferred targets include: EGFR, HER2, HER3, CD22, EpCAM, PSMA, PSCA, FL T-3, CD30, CD20, CD33, CD23, CD2, CD37, CD25, CD73, CD47, LGR-5, CD80, CD86, CD7 0, CD74, CD40, CD19, CD79b, CA-125, c-met, CXCR4, DR5, PD-1, PD1L, LeY, MUC1, MU C2, MUC3, MUC4, MUC5ac, MIP-1A, MIP-1B, KIT, TRAIL receptor (R1 and R2), CXCR4, CEACA M, IGF-1R, carbonic anhydrase IX, PDGFRa, CD137, CD276, mesothelin, VEGFR, P-cadherin , CD56, bacterial Psl, bacterial lipopolysaccharide, galactan-III epitope of bacterial LPS, bacterial PcrV, RSV F protein is an example.

[0085] (Anti-EGFR antibody) In a further embodiment, the antibody or antigen-binding fragment binds to epidermal growth factor receptor (EGFR) EGFR is a binding epitope that is overexpressed in several types of human cancer. High expression on certain cancer cells makes EGFR attractive for new therapies. In one embodiment, the EGFR-binding antibody or antigen-binding fragment is directed against: EGFR (Erb EGFR subunits selected from ErbB-1), HER2 / c-neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). Examples of suitable EGFR-binding antibodies include: These include cetuximab, nimotuzumab, matuzumab, zalutumumab, and panitumumab. However, it is not limited to these. [Table 1]

[0086] In a further embodiment, the EGFR antibody is selected from the group consisting of: (a) Li et al. (2005) Cancer Cell 7, 3 01-11; (b) Dubois et al. (2008) Anal. Chem. 80, 1737-45; and (c) available at www.imgt.org. the IMGT database available at www.drugbank.ca; or the heavy chains disclosed in WO2016 / 196682 Cetuximab is a hybrid mouse / human chimeric antibody containing both human and light chain sequences. .

[0087] In a further embodiment, the cetuximab antibody or antigen-binding fragment thereof inhibits EGFR. Recognizes and specifically binds to SEQ ID NOs: 1 and 2 and has at least 80%, 82%, 84%, 85%, %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% sequence identity between heavy and light chain variable domains.

[0088] In further embodiments, a fragment of an EGFR-binding antibody may be selected as an example of L. Examples of these include cetuximab Fab (Li, S. et al. (2005) Cancer Cell 7, 301-311); Tuximab scFv (US 7,060,808); panitumumab Fab (Sickmier EA et al. (2016) PLoS One 11, 9, e0163366); panitumumab scFv (US 6,235,883), and D2C7 scFv (US 2013 / 002259 8; Clin Cancer Res 2013, 19(17), 4717-4727) .

[0089] (anti-CD20 antibody) In a further embodiment, the antibody or antigen-binding fragment is a CD20-binding epitope. Depletion of CD20-expressing cells (e.g., B cells) has been shown to be effective in treating blood cancers such as leukemia and lymphoma. It is well known that CD20 binding antibodies have therapeutic benefits in the treatment of rheumatoid arthritis. Examples of suitable CD20 binding antibodies include: These include rituximab, ocrelizumab, ofatumumab, and obinutuzumab. Not limited to these. [Table 2]

[0090] In a further embodiment, the CD20 antibody is a antibody described in (a) US Pat. No. 5,736,137; (b) Wang, B. et al. 2013) Analyst 138, 3058-3065, and (c) the IMGT database available at www.imgt.org; Hybrid mouse / human containing both heavy and light chain sequences disclosed at www.drugbank.ca. The chimeric antibody is rituximab.

[0091] In a further embodiment, the rituximab antibody or antigen-binding fragment thereof recognizes CD20. and specifically bind to SEQ ID NOs: 3 and 4 with at least 80%, 82%, 84%, 85%, and 86% ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or have heavy and light chain variable domains with 100% sequence identity.

[0092] In a further embodiment, a fragment of a CD20 binding antibody may be selected as an example of L. An example of this type is rituximab Fab (Du, J. et al. (2007) J. Biological Chem. 282, 15 073-15080), but are not limited to these.

[0093] In a further embodiment, the CD20 antibody or fragment thereof is rituximab or rituximab. It is selected from the Fab.

[0094] (pathogen-specific antibody target) In an alternative embodiment, the antibody or antigen-binding fragment binds to a specific pathogen. In a further embodiment, the antibody or antigen-binding fragment is It is configured to bind to S. aureus and Pseudomonas aeruginosa. Examples of suitable antibodies include those reported in WO2015 / 196011, WO2012 / 170807, and WO2014 / 074528. Examples of such compounds include, but are not limited to, those that are known to be

[0095] (conjugate) Any of the antibodies or fragments thereof disclosed herein may be used in combination with the antibodies of the present invention having one or more α-Gal. The antibody may be conjugated to a cyclic spacer linker as described herein. or a fragment thereof, while retaining the efficacy of The optimal number of α-Gal units can be selected. The type of the linker depends on the functionality at the end of the linker and the surface reactivity of the antibody or fragment thereof or the selected Based on the reactive sites, the skilled artisan will select the best possible yield and purity of the isolated material. In some embodiments, conjugation can be performed at a maleimide moiety. Alternatively, conjugation can be achieved by the addition of an amino group This can be easily achieved by amide bond formation between .

[0096] The conjugate contains several reactive sites that lead to multiple conjugation reactions. If the ranges are based on the actual or average number of linker molecules per antibody or fragment thereof, It should be understood that this may refer to the number of

[0097] As used herein, the term "LAR" (linker:antibody ratio) refers to the ratio of a linker to an antibody or its derivatives. LAR refers to the actual or average number of linker molecules conjugated to a fragment of the This is equivalent to the integer value defined as "z" in the documentation. Various methods are known in the art. In one embodiment, the LAR is Determined by the reaction of Mal-vc-PAB-MMAE as an alternative payload for Car and using HIC Be analyzed.

[0098] (Properties of the conjugate) In any of the various embodiments described herein, the antibody or fragment thereof The fragment allows optimal recruitment of anti-Gal while retaining its binding efficacy to its target. In one embodiment, cetuximab may be administered in an average of up to 5, e.g., 2 0, especially up to 30 linker molecules, while still binding to EGFR. Retains the ability to.

[0099] In certain embodiments, the conjugates of the invention have the following properties: a) Conjugation of a linker to an antibody or fragment thereof is should not significantly alter the binding efficacy to the target compared to the reactant; b) Conjugation of α-Gal-containing linkers is due to the hydrophilic nature of oligosaccharides. may allow high levels of LAR without concomitant high levels of aggregation; c) The addition of a linker containing α-Gal may allow the antibody or fragment thereof to be more stable. ru; d) Heavy or light chain conjugates depending on the selected linker and / or conjugation technique. gation priority; e) conjugation of one selected linker containing a high multiplicity of α-Gal units; Compared to several conjugation sites using equal loading of α-Gal may exhibit optimal properties; f) Conjugation of a linker to an antibody or fragment thereof may result in an enhanced pharmacokinetic profile. Can link to files may indicate one or more of:

[0100] (Salts and their derivatives) Reference to compounds of formula (I) and subgroups thereof also refers to compounds that are These ionic forms, salts, solvates, isomers (geometric and stereochemical isomers) tautomers, N-oxides, esters, isotopes and protected forms are also included. preferably salts or tautomers or isomers or N-oxides or solvates thereof; and more preferably salts or tautomers or N-oxides or solvates thereof, and More preferably, the present invention includes salts, tautomers, or solvates thereof. Compounds as defined in any of the above aspects, as well as their ionic forms, salts, solutions, etc. Solvates, isomers (including geometric and stereochemical isomers), tautomers, N-oxides, esters ter, isotopes and protected forms (excluding intermediate compounds in chemical processes) are included in the These compounds are called "compounds."

[0101] The compounds of formula (I) may be in the form of salts, such as acid addition salts or, in certain cases, carboxylate salts. They can be present as salts of organic and inorganic bases such as sulfonates and phosphates. All such salts are within the scope of the present invention and references to compounds of formula (I) include references to these compounds. In one embodiment, the compound of formula (I) is present as a phosphate salt.

[0102] The salts of the present invention are described in "Pharmaceutical Salts: Properties, Selection and Uses" Selection, and Use,” P. Heinrich Stahl (editor), Camille G. Wermuth (editor), I SBN: 3-90639-026-8, hardcover, 388 pages, August 2002. They can be synthesized from parent compounds containing basic moieties by chemical methods such as: The salts are prepared by dissolving the base forms of these compounds in water or organic solvents, or in mixtures of the two. can be prepared by reaction with a suitable base or acid in water; generally in ether, acetic acid, Non-aqueous media such as ethyl acetate, ethanol, isopropanol, or acetonitrile are used. It is used.

[0103] Acid addition salts (simple or double salts) may be formed with a variety of acids, both inorganic and organic. Examples of acid addition salts are acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid ( For example, L-ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetyl Amidobenzoic acid, butanoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor Phar-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexyl methyl ester Laminic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy Ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid) acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g., bromide hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-D L-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL -Mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid nicotinic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, Acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid , salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, citric acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid, and an acylated amino acid selected from the group consisting of an acylated amino acid and a cation exchange resin. This includes simple or double salts formed with acids such as benzophenone, benzotriazole ...

[0104] One particular group of salts is acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, Lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid , toluenesulfonic acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalene Sulfonic acid, valeric acid, acetic acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid and lactate It comprises a salt formed from biotic acid. One particular salt is the hydrochloride salt. Another particular salt is , also known as the hemisulfate, is the hydrogen sulfate salt.

[0105] Salts include, by way of example only, sodium, potassium, calcium, magnesium, ammonium ammonium, tetraalkylammonium, and the like.

[0106] When compounds of formula (I) contain amine functions, these may be reacted, for example, with amine groups by methods well known to those skilled in the art. Subsequent reaction with an alkylating agent can form a quaternary ammonium salt. Quaternary ammonium compounds are within the scope of formula (I).

[0107] The compounds of the present invention may be used as single or double salts depending on the pKa of the acid from which the salt is formed. It may exist.

[0108] The salt forms of the compounds of the present invention are typically pharmaceutically acceptable salts and are compatible with pharmaceuticals. Examples of pharmaceutically acceptable salts are described in Berge et al., 1977, "Pharmaceutically Acceptable Salts." "(Usually Acceptable Salts)," J. Pharm. Sci., Vol. 66, pp. 1-19. However, pharmaceutically unacceptable salts may also be subsequently converted to pharmaceutically acceptable salts. For example, in the purification or separation of the compounds of the invention. Such non-pharmaceutically acceptable salt forms which may be useful also form part of the present invention. There are.

[0109] Those skilled in the art of organic chemistry will recognize that many organic compounds are suitable for use in the synthesis of various compounds, whether they are reacted in or out of solution. are capable of forming complexes with the solvent from which they are precipitated or crystallized; These complexes are known as "solvates." Complexes of the formula (I) are known as "hydrates." Pharmaceutically acceptable solvents for the compounds of the present invention Solvates are within the scope of the present invention.

[0110] Compounds of formula (I) containing an amine function can also form N-oxides. References herein to compounds of formula (I) that contain an N-functional group also include the N-oxide.

[0111] If the compound contains several amine functional groups, one or more nitrogen atoms may be oxidized. Specific examples of N-oxides are tertiary amines or nitrogen-containing heterocycles. It is the N-oxide of the nitrogen atom in the ring.

[0112] N-oxides can be converted to hydroxy groups by oxidizing agents such as hydrogen peroxide or peracids (e.g., peroxycarboxylic acids). These can be formed by treatment of the corresponding amines, as described, for example, in "March's Latest Advanced Organic Chemistry by Jerry March, 4th Edition, Wiley Interscience See page 1 of the Synthetic Chemistry of the American Chemical Society. More specifically, N-oxides can be prepared by the procedure of LW Deady (Synthetic Chemistry of the American Chemical Society). m. 1977, 7, 509-514), where the amine compound is This is reacted with mCPBA in an inert solvent such as dichloromethane.

[0113] Certain protected derivatives of compounds of formula (I) that may be produced prior to the final deprotection step are They do not have any pharmaceutical activity themselves, but in certain cases, they are administered orally or parenterally and subsequently It is well known in the art that the compounds of the present invention can be metabolized in vivo to form the pharmacologically active compounds of the present invention. Such derivatives are therefore described as "prodrugs." All such prodrugs of the compounds of the present invention are included within the scope of the present invention. Examples of suitable prodrug functionalities are given in Drugs of Today, Vol. 19, No. 9, 1983, 499 -538, and Topics in Chemistry, Chapter 31, pp. 306-316, and H. Bundgaard, "Design of Prodrugs," Elsevier, 1985, Chapter 1 (the disclosure of which is incorporated herein by reference) See, e.g., H. Bundgaard, "Prodrug Design" (the disclosure of which is incorporated herein by reference). (which is incorporated herein by reference) and known by those skilled in the art as "pro-moieties." The particular moieties shown are suitable functional groups when such functional groups are present in the compounds of the invention. It will further be understood by those skilled in the art that the functional groups may be disposed on the surface of the polymer.

[0114] These polymorphs are also included within the scope of the compounds and various salts of the present invention.

[0115] Compounds of formula (I) may exist in a number of different geometric isomeric and tautomeric forms; and references to compounds of formula (I) include all such forms. The compound may exist in one of several geometric isomers or tautomers, and If only one is specifically described or shown, all the others are nevertheless included. are encompassed by formula (I).

[0116] The present invention includes all pharmaceutically acceptable isotopically labeled compounds of the present invention, i.e., More than one atom may have the same atomic number, but the atomic mass or mass number is usually different in nature. of formula (I) replaced by atoms different from the atomic masses or mass numbers recognized. Contains compounds.

[0117] Examples of isotopes suitable for inclusion in compounds of the invention include isotopes of hydrogen, e.g. 2 H(D) and 3 H(T), an isotope of carbon, e.g. 11 C. 13 C and 14 C, isotopes of fluorine, e.g. 18 F, an isotope of nitrogen Body, e.g. 13 N and 15 N, isotopes of oxygen, e.g. 15 O. 17 O and 18 Includes O.

[0118] Certain isotopically labeled compounds of formula (I), for example those incorporating radioactive isotopes. The compounds of formula (I) are also useful in tissue distribution studies of drugs and / or substrates. Complexation between these labeled compounds and other molecules, peptides, proteins, enzymes or receptors It has valuable diagnostic properties in that it can be used to detect or identify the formation of coalescence. The detection or identification method may include radioisotopes, enzymes, fluorescent substances, chemiluminescence, etc. Substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase) Compounds labeled with a labeling substance such as a radioactive enzyme can be used. The radioactive isotope tritium, i.e. 3 H(T), and carbon-14, i.e.14 C is a built-in It is particularly useful for this purpose in view of its ease of use and rapid means of detection.

[0119] Deuterium, i.e. 2 Substitution with heavier isotopes, such as H(D), results in greater metabolic stability. Certain therapeutic benefits may result, for example, from increased in vivo half-life or reduced dosage requirements. This may provide advantages and may even be preferable in some circumstances.

[0120] 11 C. 18 F, 15 O and 13 Substitution with positron-emitting isotopes, such as N, tests target occupancy This may be useful in positron emission tomography (PET) studies for

[0121] Isotopically labeled compounds of formula (I) are generally prepared by conventional techniques known to those skilled in the art, or Concomitant use of suitable isotopically labeled reagents in place of previously utilized unlabeled reagents. It may be prepared by processes similar to those described in the examples and preparations. can.

[0122] (Method for preparing compounds of formula (I)) In this section, unless the context indicates otherwise, Additionally, reference to formula (I) also includes all other subgroups and examples thereof as defined herein. include.

[0123] The compounds related to the invention described herein can be prepared in the following "Processes and Schemes" These syntheses may be prepared in a stepwise synthetic sequence, as illustrated. These then involve the preparation of suitable core structures, which in turn form the linkers that connect the two binding sites. The branching and length can be selected. Compounds of formula (I) can be prepared by the synthesis methods well known to those skilled in the art. For example, one skilled in the art will appreciate that the choice of chemical steps and protecting groups will depend on the synthesis. It will be appreciated that the data may be administered in any order as desired.

[0124] In certain embodiments, the compound of Formula (A) may be a linker (S B ) Exemplary linker components include 6-maleimidocaproyl (MC), maleimide propanoyl (MP), valine-citrulline (vc), alanine-phenylalanine (ala-phe), 4-( 2-pyridylthio)pentanoic acid N-succinimidyl (SPP) and 4-(n-maleimidomethyl)cyclo Examples of suitable hexane-1-carboxylate include, but are not limited to, hexane 1-carboxylate (SMCC).

[0125] In certain embodiments, the compound of Formula (A) reacts with a free thiol on an antibody to form a covalent bond. A linker (S B The compounds of the present invention may contain the following phosphorus Carr reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMC C, SMPB, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, Although antibody conjugates prepared using sulfo-SMPB and SVSB are expressly contemplated, Examples include, but are not limited to, pyrrolidine-2,5-dione (macromethylphenyl ether), which is reactive with thiol groups on antibodies. Other functional groups besides iodoacetamide, bromocetamide, amide), vinylpyridine disulfide, pyridyl disulfide, isocyanate, isothiocyanate Examples include thiocyanates, activated esters, sulfonyl halides, and acid chlorides.

[0126] In certain embodiments, the linker is capable of reacting with an electrophilic group on an antibody. Exemplary electrophilic groups include aldehyde groups, ketone groups, and carbonyl groups. Furthermore, the reactive functional group of the linker may be heterocyclic. The atom can react with electrophilic groups on the antibody. Typical examples are hydrazines, oximes, and amines. amines, hydrazides, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazines. These include, but are not limited to, drazides.

[0127] In certain embodiments, a compound of Formula (A) reacts with a free amine on an antibody to form a covalent bond. A linker (S B The compounds of the present invention may contain: N-hydroxycobalamin. Succinimide (NHS), 2-succinimido-1,1,3,3-tetra-methyluronium tetrafluoride benzotriazol-1-yl-oxytripyrrolidinophosphonium Antibodies prepared using carboxylic acid activators such as hexafluorophosphate (PyBOP) Conjugates are expressly contemplated, but are not limited to these.

[0128] According to a further aspect of the present invention, a method for preparing a compound of formula (I) as defined above A process is provided, the process comprising:

[0129] (a) The antibody or antigen-binding fragment has at least one reactive thiol group represented by the formula (III) ) with a compound of formula (II) in which S1 is terminated with maleimide. , preparing a compound of formula (IA) wherein X1 represents -S-: [ka] wherein F, S2, Y2, m, z, Cy, Y1, and S1 are as defined above; or

[0130] (b) the antibody or antigen-binding fragment has at least one reactive thiol group represented by the formula (III) A) with a compound of formula (II) in which S1 is terminated with a maleimide. To prepare a compound of formula (IC) in which X1 represents -NH2 and S1 contains -S-CH2-CH2-CH2-C(=NH)- To: [ka] wherein F, S2, Y2, m, z, Cy, Y1, S1, and L are as defined above; or teeth

[0131] (c) A compound of formula (IIB) in which S1 is terminated with an N-hydroxysuccinimide group is added to the antibody or an antigen-binding fragment thereof, which reacts with a compound of formula (IIIB) having at least one reactive amino group. preparing a compound of formula (IB) in which X1 represents -NH2 by reacting: [ka] (wherein F, S2, Y2, m, z, Cy, Y1, S1, and L are as defined above); and and / or

[0132] (d) a compound of formula (I) or a protected derivative thereof, followed by a further compound of formula (I) or a protected derivative thereof; Interconversion to selected derivatives.

[0133] Processes (a) and (b) are typically thiol-maleimide reactions; α,β-unsaturated thiols such as maleimides; It involves the Michael addition reaction of a reactive thiol group with a saturated ketone.

[0134] Preferred conditions include reaction at room temperature in a suitable buffer as described herein. Incubation of the linker-maleimide intermediate with an antibody or fragment thereof bearing a reactive thiol. A typical linker:antibody ratio (LAR) is determined by the amount of free radicals present on the antibody or its fragment. Although it depends on the number of thiol groups, it is typically in the range of 2 to 8.

[0135] Process (c) typically involves an amide bond forming reaction in the presence of an activated ester. Typical conditions are incubation with an antibody or fragment thereof having a reactive amino acid in a suitable buffer at room temperature. Incubation of the linker-NHS ester is included. A typical linker:antibody ratio (LAR) is: Although it depends on the number of free amino groups present on the antibody or fragment thereof, it is typically in the range of 2 to 20. It is an enclosure.

[0136] Process (d) typically involves interconversion procedures known to those skilled in the art. For example, the compound of formula (I) In the compound, the first substituent can be converted to a second alternative substituent by methods known to those skilled in the art. A wide range of well-known functional group interconversions may be used to convert precursor compounds to compounds of formula (I). For details, see "March's Modern Organic Chemistry," 4th Edition, John Wiley & Sons, ns, 1992. For example, organotin reagents (Stille reaction), Grignard reagents Possible metal-catalyzed functionalization and reactions with nitrogen nucleophiles, such as those used "Palladium Reagents and Catalysts" [Jiro Tsuji, Wiley , ISBN 0-470-85032-9], and "Handbook of Organopalladium Chemistry for Organic Synthesis" of Organo Palladium Chemistry for Organic Synthesis) [Vol. 1, edited by Eiichi Negishi Collection, Wiley, ISBN 0-471-31506-0].

[0137] Where appropriate, the reactions described above in processes (a), (b), and (c) may be carried out by one skilled in the art. may be preceded or followed by one or more reactions known in the art and provide other compounds of formula (I). are performed in the order appropriate to achieve the necessary substitutions defined above. Non-limiting examples of such reactions, the cases of which can be found in the literature, include: Protection of reactive functional groups, Deprotection of reactive functional groups, Halogenation, Dehalogenation, dealkylation, Alkylation and arylation of amines, anilines, alcohols, and phenols, Mitsunobu reaction of hydroxyl groups, Cycloaddition reactions of suitable groups, Reduction of nitro, ester, cyano, aldehyde, transition metal-catalyzed coupling reactions, acylation, Sulfonylation / introduction of sulfonyl groups, Saponification / hydrolysis of ester groups, amidation or transesterification of ester groups, esterification or amidation of carboxyl groups, Halogen exchange, nucleophilic substitution by amines, thiols or alcohols, reductive amination, Oxime formation of carbonyl and hydroxylamine groups, S-oxidation, N-oxidation, chloride.

[0138] The compound of formula (III) has at least one reactive thiol group available for reaction. Reactive thiol groups may be generated by reduction of the antibody or antigen-binding fragment with TCEP. This may be achieved by:

[0139] Preferred conditions include either 1.1 eq TCEP:Ab, 4.2 eq TCEP:Ab, or 8 eq TCEP:Ab. nothing.

[0140] Alternatively, the generation of a reactive thiol group can be achieved by cleaving at least one thiol group on the antibody or antigen-binding fragment. Reaction of lysine residues with thiolating agents such as Traut's reagent (2-iminothiolane) This may be achieved.

[0141] The preferred conditions were 12.2 eq of 2-iminothiolane:Ab for low LAR and 12.2 eq of 2-iminothiolane:Ab for high LAR. containing 30.5 eq of 2-iminothiolane:Ab.

[0142] Compounds of formula (II) and (IIB) can be prepared from compounds of formula (V) and (VI) by the synthesis of succinimidyl 4-(N-maleimidomethyl)propanol. Imidomethyl)cyclohexane-1-carboxylate (SMCC) or di(N-succinimidyl)glycerol Following reaction with diethyl stearate (DSG), it may be prepared by the method described in Scheme 1: [ka] where PG is a protecting group such as monomethoxytrityl; S1 is a maleimide or NHS-terminated Y1 is CONH, and S2, Y2, m, Cy, and F are as defined above. (That is why.)

[0143] Compounds of formula (II) may be prepared from compounds of formula (IV) by the addition of reactive maleates to the terminal amino groups. The preferred conditions are the conversion of the terminal amino group to a succinyl group in DMSO at room temperature. Reaction with N-maleimidomethyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) Alternatively, the compound of formula (IIB) can be obtained by interconversion of the terminal amino group into a reactive NHS group. The compound of formula (IV) may be prepared from the compound of formula (IV). Preferred conditions are DMF and DMSO or a combination thereof. with the crosslinker di-(N-succinimidyl) glutarate in a suitable anhydrous organic solvent such as This includes the reaction of

[0144] The compound of formula (IV) can be prepared by process steps (iii) and (iv) and the amide bond formation step and subsequent steps. Following appropriate deprotection steps, they may be prepared from compounds of formula (V) and (VI). The amide bond formation step is carried out by reacting a phosphate-containing reagent, thiamin, in an organic solvent in the presence of an organic base. Carbodiimide-based reagents or carbodiimide-containing reagents The preferred conditions involve activation of triethylamine in DMF or a mixture of DMF and DMSO. HATU((1-[bis(dimethylamino)methyl]methyl)methyl) with either methylamine or diisopropylethylamine [ethylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphine When PG contains monomethoxytrityl, the deprotection reaction is acid-mediated. Preferred conditions include 0.2M aqueous HCl at room temperature.

[0145] Alternatively, a compound of formula (II) can be prepared from a compound of formula (VA) by the synthesis of succinimidyl 4-(N-maleimide) The reaction with methylcyclohexane-1-carboxylate (SMCC) is described in Scheme 1A. It may be prepared by the following method: [ka] where PG is a protecting group such as monomethoxytrityl; S1 is a maleimide-terminated wherein Y1 is CONH, and S2, Y2, m, Cy, and F are as defined above. ).

[0146] Compounds of formula (II) can be prepared by process steps (iii) and (iv) as previously described in Scheme 1. and may be prepared from compounds of formula (VA) and (VI). The interconversion of This can be achieved as follows.

[0147] Compounds of formula (V) and (VA) (wherein S2 terminates with -NHCO-CH2-) can be prepared by the reaction of compounds of formula (VIII), (IX) and (IXA) may be prepared by the method described in Scheme 2: [ka] wherein m, Cy, S1, S2, and F are as defined above, and PG1 is tert-butyl. PG2 is a protecting group containing any one of methyl, ethyl, or benzyl, and PG3 is a protecting group containing methyl , ethyl, or tert-butyl).

[0148] Compounds of formula (V), (IXA), and (VA) can be prepared by process steps (iii) and (iv), the amide bond forming step. The compound of formula (IX) may be prepared by the steps of the above-mentioned step (1) and the appropriate deprotection reaction. In the case of benzyl, the deprotection reaction is mediated by catalytic hydrogenation. In MeOH / EtOH or water or any combination thereof under nitrogen atmosphere (15-70 psi (103-483 kPa)). Alternatively, deprotection may be mediated by a phase transfer reaction. Suitable conditions include TEA and water at room temperature for 16 hours.

[0149] When PG contains methyl, ethyl, or tert-butyl, the acid required by the protecting group Alternatively, a base-mediated deprotection reaction may be employed. Acid-mediated deprotection conditions may be required. When used, preferred conditions are TFA, dioxane, optionally with a co-solvent of DCM or water. 4M HCl, or 37% HCl in water. When base-mediated conditions are required, preferred Suitable conditions include sodium hydroxide or hydroxide in an aqueous medium such as methanol or THF containing water. lithium chloride.

[0150] Compounds of formula (IX) can be prepared from compounds of formula (XI) and formula (XII) by the method described in Scheme 3. It may be prepared. [ka] wherein m and Cy are as defined above, and PG1 is tert-butyl, methyl, ethyl, PG2 is a protecting group containing either methyl, ethyl, or tert- butyl, and X is Cl, Br, or I).

[0151] The compound of formula (IX) can be converted to compounds of formula (XI) and formula (XII) by alkylation reaction in process step (v). Typical conditions include an inorganic base in a polar organic solvent at room temperature. Preferred conditions include potassium carbonate in DMF.

[0152] When Cy is biphenyl or triphenyl, the compound of formula (XI) is a bi / tri-phenyl The compound may be prepared by utilizing the Suzuki reaction to construct the unit. The preferred conditions are 100-1 Sodium carbonate, potassium acetate, or sodium bicarbonate in dioxane and water at 10°C Tetrakistriphenylphosphinepalladium(0) or [1,1'-bis(diphenyl) (phosphino)ferrocene]dichloropalladium(II) dichloromethane complex. If suitable protecting groups are utilized, such protecting groups may be fluoride-mediated. The compound may be deprotected using the following deprotection conditions: Preferred conditions include TBAF in THF at room temperature.

[0153] Alternatively, when Cy is bi / tri-phenyl, the compound of formula (XI) may be any of the compounds described above and herein. Suzuki reaction to construct the bi / tri-phenyl unit using conditions as described in It may be prepared directly by use of

[0154] Compounds of formula (III), (IIIA), (IIIB), (VI), (VIII), (XII), (VII), and (X) are commercially available. or prepared according to the methods described herein.

[0155] Certain intermediates described herein are novel compounds not previously known in the art. It will be understood that the term "represents" refers to a product of the invention. an intermediate selected from compounds of formula (II), (IIB), (V), (VA), (IX), or (XI) as defined above; A compound is provided.

[0156] Those skilled in the art will readily appreciate that the methods described herein may be used to produce the best yields for the Examples and Preparations described herein. It will be appreciated that any suitable combination of the above steps may be selected.

[0157] (Pharmaceutical composition) While it is possible for a compound of formula (I) to be administered alone, it may also be administered in the form of a pharmaceutical composition (e.g. It is preferred that the compound is present as a pharmaceutical agent (e.g., a formulation).

[0158] Thus, according to a further aspect, the present invention provides pharmaceutical compositions, as well as compositions comprising the compounds of the present invention, as described herein. At least one of the compounds of the present invention is combined with one or more pharmaceutically acceptable excipients and, optionally, other therapeutic agents. The present invention provides a method for producing a pharmaceutical composition containing (e.g., mixing) a therapeutic or prophylactic agent. When the pharmaceutical composition contains one or more additional therapeutic agents, the agents may be It will be appreciated that other compounds of formula (I) may be included.

[0159] The pharmaceutically acceptable excipient(s) can be, for example, a carrier (e.g., a solid, liquid, or semi-solid carriers), adjuvants, diluents, fillers or extenders, granulating agents, coating agents, Release control agents, binders, disintegrants, lubricants, preservatives, antioxidants, buffers, suspending agents, thickeners, Flavoring agents, sweeteners, taste masking agents, stabilizers, or any commonly used in pharmaceutical compositions. Other excipients may be selected from the following: This is shown in more detail below.

[0160] As used herein, the term "pharmaceutically acceptable" means any substance that is pharmaceutically acceptable within the bounds of sound medical judgment. within a reasonable benefit / risk ratio and without undue toxicity (i.e., Generally Recognized As Safe (GRAS)) and has not been shown to cause irritation, allergic reactions, or other problems or A compound that is suitable for use in contact with the tissue of a subject (e.g., a human) without complications. Each carrier, excipient, etc. may also refer to other components of the formulation. It must be "acceptable" in the sense of being compatible with the components of the

[0161] Pharmaceutical compositions containing the compounds of the present invention can be formulated according to known techniques, e.g. For example, Remington's Pharmaceutical Sciences, Mack Publishing See g Company, Easton, PA, USA.

[0162] The pharmaceutical composition may be administered parenterally, intranasally, intrabronchially, sublingually, intraocularly, intraauricularly, rectally, intravaginally, or intravenously. It can be in any form suitable for transdermal administration. Where illustrated, these may be for intravenous, intramuscular, intraperitoneal, subcutaneous administration, or by injection or infusion. or other delivery means to the target organ or tissue. This delivery can be by bolus injection, short-term infusion, or long-term infusion. and can be administered via passive delivery or with the aid of a suitable infusion pump or syringe driver. It can be through use.

[0163] Pharmaceutical formulations adapted for parenteral administration are, inter alia, formulated to stabilize the active ingredient in dissolved form. and antioxidants, buffers, etc., to render the formulation isotonic with the blood of the intended recipient. , bacteriostatic agents, cosolvents, surfactants, organic solvent mixtures, cyclodextrin complexing agents, emulsifiers agents (for the formation and stabilization of emulsion formulations), liposome components for the formation of liposomes, high The present invention includes a gelling polymer for forming a molecular gel, a lyoprotectant, and a combination of these agents. Pharmaceutical formulations for parenteral administration also include aqueous and non-aqueous sterile injection solutions, which may be used in the form of a liquid or a pharmaceutical preparation. and the like. (RG Strickly, "Solubilizing Excipients for Oral and Injectable Formulations" xcipients in oral and injectable formulations)”, Pharmaceutical Research, Vol 2 1(2) 2004, p 201-230).

[0164] The formulations may be packaged in single containers, such as sealed ampoules, vials and pre-filled syringes. It may be presented in a unit-dose or multi-dose container and immediately prior to use may be added to a sterile liquid carrier, e.g. For example, it may be stored in a freeze-dried (lyophilized) form that requires only the addition of water for injection. It may be controlled.

[0165] The pharmaceutical formulation can be prepared by lyophilizing the compound of the present invention. Drying refers to the procedure of freeze-drying a composition. Thus, freeze-drying and lyophilization are , are used interchangeably herein.

[0166] Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0167] The pharmaceutical compositions of the present invention for parenteral injection may also be prepared in a pharmaceutically acceptable sterile aqueous or non-aqueous solution. Aqueous solutions, dispersions, suspensions or emulsions, as well as sterile injectable solutions or dispersions immediately before use It may contain a sterile powder for reconstitution into the formulation.

[0168] Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles are water, ethanol, polyethylene glycol, polyethylene glycol diluents, polyethylene glycol esters ... diluents, polyethylene glycol esters, polyethylene glycol diluents, polyethylene glycol diluents, polyethylene glycol diluents, polyethylene Polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) , carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (e.g., sunflower oil , safflower oil, corn oil, or olive oil), as well as other oils such as ethyl oleate. Use of thickening or coating substances such as lecithin, including organic esters for injection. By maintaining the required particle size in the case of dispersants, and by By using it, the appropriate fluidity can be maintained.

[0169] The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The prevention of the action of microorganisms can include various antibacterial and antifungal agents, for example, parabens, This can be ensured by the inclusion of chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include agents to adjust tonicity, such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical dosage forms is achieved by using ingredients such as aluminum monostearate and gelatin. This can be achieved by including an agent that delays absorption, such as:

[0170] In one preferred embodiment of the present invention, the pharmaceutical composition is administered, for example, by injection or infusion. For intravenous or subcutaneous administration, the solution is It can be administered in a drip bag (0.9% saline or 5% dextrose) prior to administration. The drug can be injected into a blood vessel containing a pharmaceutically acceptable excipient such as

[0171] In another preferred embodiment, the pharmaceutical composition is in a form suitable for subcutaneous (sc) administration. .

[0172] The compounds of the present invention may be formulated with a carrier and administered in the form of nanoparticles. The increased surface area of ​​nanoparticles aids their absorption. In addition, nanoparticles can be directly absorbed into cells. Nanoparticle drug delivery systems offer the possibility of penetration. Nanoparticle Technology for Drug Delivery, Ram B. Gupta and Uday B. Kompella Edited by Informa Healthcare, ISBN 9781574448573, 13th Edition, March 2006 Nanoparticles for drug delivery have also been reported in J. Control. Release, 2003, 91 (1-2), 167-1 72 and Sinha et al., Mol. Cancer Ther. August 1, (2006) 5, 1909. do.

[0173] The pharmaceutical compositions typically contain from about 1% (w / w) to about 95% (w / w) active ingredient and 99% (w / w) Preferably, the composition contains from 1% to 5% (w / w) of a pharmaceutically acceptable excipient or combination of excipients. The composition preferably contains from about 20% (w / w) to about 90% (w / w) of the active ingredient and from 80% (w / w) to 10% The pharmaceutical composition contains a pharmaceutically acceptable excipient or combination of excipients of approximately It contains 1% to about 95%, preferably about 20% to about 90%, of the active ingredient. Pharmaceutical compositions according to the present invention may be packaged, for example, in ampoules, vials, suppositories, pre-filled syringes, drug packages, etc. The composition may be in unit dosage form, such as in the form of a tablet or capsule.

[0174] The pharmaceutically acceptable excipient(s) are selected according to the desired physical form of the formulation. and, for example, a diluent (e.g., a solid diluent such as a filler or bulking agent) ; and liquid diluents such as solvents and co-solvents), disintegrants, buffers, lubricants, flow aids, Release-controlling agents (e.g., polymers or waxes that inhibit or delay release), binders, granulating agents, Dyes, plasticizers, antioxidants, preservatives, flavoring agents, taste masking agents, tonicity adjusting agents, and coating agents The anti-inflammatory agent may be selected from the group consisting of anti-inflammatory agents, ...

[0175] Those skilled in the art have the expertise to select appropriate amounts of components for use in a formulation. For example, tablets and capsules typically contain 0-20% (depending on drug dose). of disintegrant, 0-5% of lubricant, 0-5% of flow aid, and / or 0-99% (w / w) of filler / if These also contain 0-10% (w / w) polymer binders, 0-5% (w / w) antioxidants, The sustained-release tablets also contain 0-99% (w / w) of controlled release (e.g. The film coating of the tablet or capsule may contain a (depending on the dose) retarding polymer. typically contains 0-10% (w / w) polymer, 0-3% (w / w) dye, and / or 0-2% (w / w) Contains plasticizers.

[0176] Parenteral or subcutaneous formulations typically contain 0-20% (w / w) buffer, 0-50% (w / w) cosolvent, and and / or 0-99% (w / w) water for injection (WFI) (depending on the dosage and if freeze-dried) Formulations for intramuscular depots also contain 0-99% (w / w) oil.

[0177] The compounds of the present invention may also be formulated as solid dispersions. Solid dispersions may comprise two or more A homogeneous, extremely finely dispersed phase of solid matter. A solid solution (molecular Dispersions of hydroxybenzoates (Chiou and Riegelman, J. Pharmacol. 2002, 13, 111-115, 1999) are well known for use in the pharmaceutical arts (Chiou and Riegelman, J. Pharmacol. 2002, 13, 111-115, 1999). Pharm. Sci., 60, 1281-1300 (1971)), and dissolution of poorly water-soluble drugs. It is useful for increasing the rate and bioavailability.

[0178] The pharmaceutical preparation is a "patient package" that contains the entire course of treatment in a single package, usually a blister pack. The patient pack may be provided to the patient in a "patient pack." Patient packs offer the following advantages over traditional prescriptions: wherein the pharmacist divides a patient's supply of the drug from the bulk supply, Patients should always have access to the package inserts included in their patient packs, which are not usually included on their prescriptions. The inclusion of a package insert has been shown to improve patient compliance with physician instructions. An example of a patient pack includes a pre-filled syringe. The tip of a pre-filled syringe with a needle attached already contains the drug substance. Before injection, remove the nozzle cap from the tip. The plunger rod is then pushed into the tip. This causes the gasket to slide, resulting in the release of the drug.

[0179] Compositions for nasal delivery include ointments, creams, sprays, patches, gels, drops, and Such compositions may be formulated according to known methods. This can be done.

[0180] Examples of formulations for rectal or vaginal administration include pessaries and suppositories, which can be used, for example, in , formed from a shaped moldable or waxy material containing an active compound. Solutions of the active compounds may also be used for rectal administration.

[0181] Compositions for administration by inhalation may be inhalable powder compositions or liquid or powder sprays. and can be administered using a powder inhaler or aerosol dispensing device. Such devices are well known. To achieve this, powdered formulations typically involve dissolving the active compound in an inert solid powder such as lactose. It contains the compounded diluent.

[0182] The compounds of the invention are generally presented in unit dosage form and thus typically provide a desired level of production. For example, a formulation may contain 1 ng to 2 g of active ingredient, e.g., For example, 1 ng to 2 mg of the active ingredient. Within these ranges, specific subranges of the compound may be: 0.1 mg to 2 g of active ingredient (more usually 10 mg to 1 g, e.g. 50 mg to 500 mg), or 1 μg to 20 mg ( For example, 1 μg to 10 mg, for example, 0.1 mg to 2 mg of active ingredient).

[0183] The active compounds are administered to a patient in need thereof in an amount sufficient to achieve the desired therapeutic effect. The compound will be administered to a subject (e.g., a human or animal patient).

[0184] Therapeutic Use According to a further aspect of the present invention there is provided a compound of formula (I) as defined herein for use in therapy. ) is provided.

[0185] The therapeutic use of the compounds of the present invention is determined by the selection of the antibody or antigen-binding fragment thereof. It will be recognized that

[0186] For example, the antibody or antigen-binding fragment thereof may be an EGFR antibody (e.g., cetuximab or nivolumab). In embodiments where the compound of Formula (I) is motuzumab) or a fragment thereof, the compound of Formula (I) is It is for use in

[0187] Thus, according to a further aspect of the present invention, the antibody or antigen-binding fragment thereof is an EGFR antibody (e.g., cetuximab or nimotuzumab) for use in the treatment of There is provided a compound of formula (I) as defined herein that is a fragment thereof.

[0188] According to a further aspect of the invention, the antibody or its antigen binding site is administered to an individual in need thereof. The synthetic fragment is an EGFR antibody (e.g., cetuximab or nimotuzumab) or a fragment thereof. a method of treating cancer comprising administering a compound of formula (I) as defined herein is provided.

[0189] Furthermore, when the antibody or antigen-binding fragment thereof is a pathogen-specific antibody or a fragment thereof, In an embodiment, the compounds of formula (I) are for use in the treatment of bacterial infections. do.

[0190] Thus, according to a further aspect of the present invention, the antibody or antigen-binding fragment thereof is a pathogen-specific antibody or fragment thereof, as defined herein, for use in the treatment of an infectious disease. Compounds of formula (I) as described above are provided.

[0191] According to a further aspect of the invention, the antibody or its antigen binding site is administered to an individual in need thereof. The antibody of formula (I) as defined herein, wherein the synthetic fragment is a pathogen-specific antibody or a fragment thereof. Methods for treating bacterial infections are provided that include administering the compounds.

[0192] The compounds of the present invention are generally administered to, for example, a human or animal patient, preferably a human. The compound is administered to a subject in need thereof.

[0193] The compounds of the present invention are typically therapeutically or prophylactically useful and generally non-toxic. However, in certain circumstances (e.g., life-threatening illnesses), In some cases, the benefits of administering the compounds of the present invention are achieved without any toxic or adverse effects. In such cases, the compounds of the present invention are administered in amounts that are associated with some degree of toxicity. It is considered desirable to provide

[0194] The compounds of the invention may be administered over an extended period of time to maintain beneficial therapeutic effects. (i.e., chronic administration) or may be administered for only a short period of time (i.e., acute administration). These may be administered in a continuous manner or in a manner that provides intermittent dosing (e.g., in a pulsatile manner). It is okay to do so.

[0195] Typical daily doses of the compounds of the invention are in the range of 100 pg to 100 mg / kg body weight, more typically 5 ng to 25 mg / kg body weight, and more commonly 10 ng to 15 mg per kg body weight (e.g., 10 ng to 10 mg, and more commonly Typically, the dose can be 1 μg / kg to 20 mg / kg, for example, 1 μg to 10 mg / kg, but if necessary Higher or lower doses may be administered if desired. The compounds of the invention may be administered on a daily basis or For example, every 2 days, or every 3 days, or every 4 days, or every 5 days, or every 6 days, or every 7 days, or every 10 days It can be administered on a repeat basis, either every 14 days, every 21 days, or every 28 days. Alternatively, the compounds of the invention can be administered by infusion, multiple times per day.

[0196] The compound of the present invention may be administered in an amount of, for example, 1 to 1500 mg, 2 to 800 mg, or 5 to 500 mg, for example, 2 to 200 mg or may be administered in a range of dosages from 10 to 1000 mg, with specific examples of dosages being 10, 20, 50 and The compounds of the present invention may be administered once or more than once each day. Can be administered continuously (i.e., every day without a break for the duration of the treatment regimen) Alternatively, the compounds of the present invention can be administered intermittently (i.e., weekly). Take the medicine continuously for a specified period of time, then take a break for a period of time such as one week, and then take it again for a period of time such as one week. (These drugs should be taken consecutively for separate periods and throughout the duration of the treatment regimen.) An example of a treatment regimen for administration is one in which administration is repeated for one or more cycles, e.g., 2, 3, 4, 1 week on, 1 week off for 5, 6, 7, 8, 9, or 10 or more cycles; or 2 weeks on, 1 week off; or 3 weeks on, 1 week off; or 2 weeks on, 2 weeks off or a cycle of 4 weeks on, 2 weeks off; or a cycle of 1 week on, 3 weeks off. Includes:

[0197] In one particular dosing schedule, patients should receive the drug for up to 10 days, typically up to 5 days per week. A one-hour infusion of a compound of the present invention is given daily for a period of 2 days, and the treatment is continued for 2 days. It may be repeated at desirable intervals such as every 4 weeks, preferably every 3 weeks.

[0198] More specifically, patients receive a compound of the invention via intravenous infusion for a period of one hour daily for five days. This treatment may be given every three weeks.

[0199] In another specific dosing schedule, patients receive an infusion over 30 minutes to an hour, followed by A maintenance infusion is then given of variable duration, for example 1 to 5 hours, for example 3 hours.

[0200] In a further specific dosing schedule, patients receive a continuous infusion over a period of 12 hours to 5 days. However, continuous infusion is typically given for 24 to 72 hours.

[0201] Ultimately, however, the amount of compound of the invention administered and the composition used will determine The type will correspond to the nature of the disease or physiological condition being treated and will be determined at the discretion of the physician. This may be due to the following reasons.

[0202] It is recognized that the compounds of the present invention can be used as single agents or in combination with other therapeutic agents. Combination studies are likely to be conducted as described, for example, in the article by Chou TC, Talalay P., "Dose-effect relationships" Quantitative analysis of dose-effect relative ationships: the combined effects of multiple drugs or enzyme inhibitors)”, Adv This can be carried out as described in Enzyme Regulat, 1984;22:27-55.

[0203] The compounds of the invention may be used in combination with one, two, three, four or more therapeutic agents (preferably one or two, more preferably When administered in combination therapy with other drugs (more preferably one), these drugs may be administered simultaneously or consecutively. In the latter case, the two or more drugs may be administered sequentially, with or without a beneficial or synergistic effect over a period of time. The compound will be administered in an amount and manner sufficient to ensure that the intended effect is achieved. If administered sequentially, these should be administered at closely spaced intervals (e.g., over a period of 5 to 10 minutes). or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart, or as needed) The exact dosing regimen can be determined by These doses are commensurate with the properties of the therapeutic agent(s). The doses may be administered, for example, once, twice or more times per course of treatment, which may be repeated daily. That's fine.

[0204] The preferred method and order of administration of each component of the combination, as well as the respective dosages and regimens, are described in the accompanying drawings. Certain other medicinal agents and compounds of the present invention, their routes of administration, and the particular tumor being treated It will be appreciated that the optimal dosage will depend on the individual patient and the particular host being treated. The method and sequence, as well as the dosages and regimens, may be determined using conventional methods and as described herein. This can be readily determined by one of ordinary skill in the art given the information.

[0205] Weight ratio of compounds of the invention to one or more other therapeutic agent(s) when given in combination The ratio and the exact dose and frequency of administration are well known to those skilled in the art. As such, the specific compound of the invention and other therapeutic agent(s) used, the specific the condition of the patient, the severity of the condition being treated, the age, weight, sex, diet, time of administration, and The dose will depend on the individual's general physiological state, the mode of administration, and other medications the individual is taking. The effective daily dose is determined depending on the response of the treated subject and / or the compound of the present invention is prescribed. Obviously, this may be decreased or increased depending on the physician's assessment. A specific weight ratio of the therapeutic agents is 1 / 10 to 10 / 1, more specifically 1 / 5 to 5 / 1, and even more specifically It may be in the range of 1 / 3 to 3 / 1.

[0206] (Anti-cancer treatment) Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, for example, bladder and urinary tract cancers. tract, breast, digestive tract (esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder bladder and biliary system, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal bronchial alveolar carcinoma and mesothelioma), head and neck (e.g. tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, saliva) gland, nasal cavity and sinus cancer), ovaries, fallopian tubes, peritoneum, vagina, vulva, penis, cervix the uterus, myometrium, endometrium, thyroid gland (e.g., follicular thyroid carcinoma), adrenal glands, prostate, skin and Carcinoma of the genital organs (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi) Tumors of epithelial origin (various types including adenocarcinoma, squamous carcinoma, transitional cell carcinoma, and other carcinomas), such as adenomas and carcinomas); hematologic malignancies of the lymphatic system and related conditions (e.g., acute lymphocytic leukemia) B-cell lymphomas such as ALL, chronic lymphocytic leukemia (CLL), and diffuse large B-cell lymphoma (DLBCL) cell lymphoma, follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma Lymphoma and leukemia, natural killer (NK) cell lymphoma, Hodgkin's lymphoma, hairy cell lymphoma cytoma, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), hematologic malignancies of the myeloid system and related conditions (e.g., acute bone marrow Myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], eosinophilia syndromes, such as polycythemia vera, essential thrombocythemia, and myeloproliferative disorders such as primary myelofibrosis; Hematology, including reproductive disorders, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia malignancies (i.e., leukemia, lymphoma), as well as premalignant hematological disorders and borderline malignancies tumor disorders; tumors of mesenchymal origin, e.g., sarcomas of soft tissue, bone, or cartilage, e.g., osteosarcoma, fibrosarcoma, Fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, and U. sarcoma, synovial sarcoma, epithelioid granuloma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans, etc.; tumors of the central or peripheral nervous system (e.g., astrocytoma, neuroblastoma) gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors, and schwannomas); endocrine tumors (e.g., For example, pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and thyroid tumors. medullary carcinoma; ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., for example, teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma; and pediatric and Embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumors) tumor); or a condition, congenital or otherwise, that predisposes the patient to malignancy. Groups of disorders include, but are not limited to, rheumatoid arthritis (e.g., xeroderma pigmentosum).

[0207] In one embodiment, the cancer is a solid tumor. Cancers include breast cancer, ovarian cancer, cervical cancer, colorectal cancer, liver cancer, prostate cancer, and lung cancer. That's it.

[0208] In one embodiment, the cancer comprises a hematological malignancy. The hematological malignancy is selected from the group consisting of myeloma, non-Hodgkin's lymphoma, and chronic lymphocytic leukemia. It's one of them.

[0209] Other drugs that may be administered together (either simultaneously or at different time intervals) with the compounds of the present invention Examples of anti-cancer therapeutic agents or treatments include, but are not limited to: : Topoisomerase I inhibitors; ·Antimetabolites; ·Tubulin-targeting drugs; DNA binders and topoisomerase II inhibitors; · Alkylating agents; · Monoclonal antibodies; Anti-hormonal drugs; Signal transduction inhibitors; · Proteasome inhibitors; ·DNA methyltransferase; · Cytokines and retinoids; · Chromatin-targeted therapy; Radiation therapy; and Other therapeutic and prophylactic medications, such as immunotherapeutic medications.

[0210] The compounds of the present invention also have therapeutic effects on the immune system, such as radiation therapy, photodynamic therapy, gene therapy; surgery and dietary restrictions. Such a treatment may be administered in combination with a non-chemotherapeutic treatment.

[0211] The compounds of the present invention and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, One or more other therapeutic agents may be included, e.g., two, three, four or more therapeutic agents. Formulated together in a dosage form with all the ingredients, i.e., in a single pharmaceutical composition containing all the ingredients In an alternative embodiment, the individual therapeutic agents are formulated separately and The compounds may be provided together in the form of a kit, optionally accompanied by instructions for their use.

[0212] (Anti-infective therapy) Examples of infectious agents include any pathogen, such as a bacterium, fungus, parasite, or virus. Thus, in one embodiment, a disease or condition transmitted and / or caused by an infectious agent is The disorder is a bacterial infection.

[0213] Examples of such bacterial infections include the following bacteria: Staphylococcus aureus (methicillin-resistant Staphylococcus aureus) Staphylococcus species, such as Staphylococcus aureus (including MRSA); Clostridium species (e.g., Clostridium Clostridium difficile, Clostridium tetani and and Clostridium botulinum; Enterobacter species, Mycobacterium tuberculosis tuberculosis); Shigella species such as Shigella dysenteriae; Campylobacter Campylobacter species, such as Campylobacter jejuni; Enterococcus furunculosis Enterococcus species, such as Enterococcus faecalis; Bacillus anthracis hracis); Yersinia pestis, Bordetella pertussis, Streptococcus spp. treptococcal species); Salmonella typhimurium, intestinal Salmonella enterica; Chlamydia species, Treponema pallidum, Gonorrhea Neisseria gonorrhoeae, Borrelia burgdorferi, Vibrio cholerae, Corynebacterium diphtheriae, Helicobacter Helicobacter pylori; Acinetobacter baumannii annii, Pseudomonas aeruginosa, Klebsiella pneumoniae ), and Escherichia coli (as well as one or more classes of antibiotics) This includes infections caused by strains that are resistant to ras, especially multidrug-resistant (MDR) strains.

[0214] (vaccine therapy) According to a further aspect of the present invention, there is provided a method for treating a leukemia comprising administering to a subject the immunoconjugate as defined herein. A vaccine containing the virus will be provided.

[0215] According to a further aspect of the present invention, there is provided a method for treating a leukemia comprising administering to a subject the immunoconjugate as defined herein. Adjuvants that are effective against the inflammatory bowel disease are provided. [Example]

[0216] (Example) The present invention is now exemplified by reference to specific embodiments described in the following examples. Compounds may be named using automated nomenclature such as AutoNom (MDL) or ChemDraw. It is named using the packaging or by the chemical supplier.

[0217] The following synthetic procedures are provided to illustrate the methods used; Therefore, the precursors used are those required for the individual batches synthesized according to the steps in the given description. It is not necessarily derived from

[0218] (Analysis method) LCMS System 1 LCMS Agilent 1100 (quaternary pump); mass spectrometer: Waters Micromass ZQ. Column: XBridge C18, 4.6 x 50 mm, 5 μm. Solvents: A = water; B = acetonitrile; C = 10 mM ammonium formate in water; D = in acetonitrile 0.05% formic acid. Column temperature: 25°C, injection volume: 5 μL.

[0219] (LCMS Method A: 4.5 minute acidic run) [Table 3] (LCMS Method B: 4.5 minute buffer run) [Table 4] (LCMS Method C: 8 minute acidic run) [Table 5]

[0220] (System 2) LCMS: Agilent 1100 (quaternary pump); mass spectrometer: PE SCIEX API 2000 MS / MS. Column: Agilent Poroshell 120 column, SB-C18, 4.6 mm x 30 mm, 2.7 μm Solvents: A = water; B = 0.1% formic acid in acetonitrile. Column temperature: 20°C, injection volume: 5 μL.

[0221] (LCMS Method D: 4.5 min acidic run) [Table 6]

[0222] (NMR) NMR spectrographs were recorded on either an Oxford Instruments AS400.

[0223] (abbreviation) Where the following abbreviations are used, the following meanings apply: AcOH is acetic acid; aq. is aqueous; br is broad singlet; δ is the chemical shift in ppm; d is a double line; dd is a double line of double lines; ddd is a double line of a double line; DCM is dichloromethane; DIPEA is diisopropylethylamine; DMF is dimethylformamide; DMSO is dimethyl sulfoxide; DMSO-d6 is perdeuterated dimethyl sulfoxide NMR solvent; DSG is di-(N-succinimidyl) glutarate; EtOH is ethanol; EtOAc is ethyl acetate; HATU is a derivative of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium. hexafluorophosphate; HPLC is high pressure liquid chromatography; IMS is industrial denatured spirits (typically 5%-10% MeOH in EtOH); μ is micro; m is a multiplet; Mal is maleimide; MeCN is acetonitrile; MeOH is methanol; mins is minutes; mL is milliliters; MMTr is monomethoxytrityl; MS is mass spectrometry; NH3 is ammonia or ammonium hydroxide (28% aqueous solution); NHS is N-hydroxysuccinimide; or N-hydroxysuccinimidyl; NMR is nuclear magnetic resonance; Pd / C is a palladium on carbon (typically 5%-10%) hydrogenation catalyst (water-wet); Pd(PPh3)4 is tetrakistriphenylphosphinepalladium(0); ppm is parts per million; q is a quartet; Rt is retention time; s is a singlet; SMCC is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. can be; t is the triple line; TBAF is tetra-n-butylammonium fluoride; TBME is tert-butyl methyl ether; TEA is triethylamine; TBS is tert-butyldimethylsilyloxy; TFA is trifluoroacetic acid; and THF is tetrahydrofuran.

[0224] When α-Gal is mentioned, the following intermediates apply: 3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxybenzoate) hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydrofuran tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-4-hydro 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxypropylamine [ka] This intermediate is described in the literature by Bovin et al. (Mendeleev Communications (2002), (4), 143-145) It can be prepared by the method described above.

[0225] Preparations 1-19 can be prepared by processes (a)-(d) as previously described and Schemes 1, 1A, 2, and 3. The key linker molecules required for conjugation to the examples are as illustrated by The present invention describes methods used to prepare intermediates from

[0226] (Preparation 1 4'-(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy 6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) Methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy )-4-Hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl )amino)-2-oxoethoxy)-N-(2-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) (methyl)cyclohexane-1-carboxamido)ethyl)-[1,1'-biphenyl]-3-carboxamide Samid) [ka] 4'-(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6 R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyra (4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy )oxy)propyl)amino)-2-oxoethoxy)-N-(2-aminoethyl)-[1,1'-biphenyl]- A solution of 3-carboxamide (Preparation 3, 5.0 mg, 2.26 μmol) was added to SMCC (2.2 mg) in DMSO (100 μL). A solution of 1,2-dimethyl-3,4-trimethyl-2,4-trimethyl-1 ...1,4-trimethyl-2,4-tri The crude residue was used directly in the next step. LCMS method B: Rt=1.97 min, ES + MS m / z 1118.5 [M+H] +

[0227] (Preparation 2 2,2',2''-((5'-((2-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl (1,1'-biphenyl)-3,3',5-trimethyl-2,3'-biphenyl-1-carboxyamidoethylcarbamoyl yl)tris(oxy))tris(N-(3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S, 5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)tetrahydro-2H- Pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)propyl)acetamide)) [ka] 2,2',2''-((5'-((2-aminoethyl)carbamoyl)-[1,1'-biphenyl] in DMSO (400 μL) -3,3',5-triyl)tris(oxy))tris(N-(3-(((2R,3R,4R,5S,6R)-3-acetamido-5- (((2S,3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3, 4,5-Trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro (2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro- Solution of 2H-pyran-2-yl)oxy)propyl)acetamide) (Preparation 4, 5.0 mg, 2.26 μmol) To the solution was added a solution of SMCC (0.69 mg, 2.05 μmol) in DMSO (100 μL). The resulting solution was cooled to room temperature. The mixture was stirred at rt for 18 h and then dried under vacuum. The crude residue was used directly in the next step. LCMS method B: Rt=1.57 min, ES + MS m / z 1218.5 [M+2H] + / 2, theoretical mass: 2435.4

[0228] (Preparation 3 4'-(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy 6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) Methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy )-4-Hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl )Amino)-2-oxoethoxy)-N-(2-aminoethyl)-[1,1'-biphenyl]-3-carboxamide ) [ka] (Process 1) 4'-(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R) in DMF (0.5 mL) -3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy -6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran -2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl) (oxy)propyl)amino)-2-oxoethoxy)-[1,1'-biphenyl]-3-carboxylic acid (Preparation 6 To a solution of EtN (21.8 μL, 156.4 μmol), followed by DMF (0 N in 0.5mL 1 -((4-methoxyphenyl)diphenylmethyl)ethane-1,2-diamine (Preparation 19, A solution of 19.3 mg (58.1 μmol) of HCl was added and the reaction was stirred at room temperature for 1.5 hours.

[0229] (Process 2) 0.2 M HCl (aq) was added dropwise until the pH was 3-4, and the solution was stirred at room temperature for 18 hours. Concentrate and run on a reversed-phase column eluted with 5-40% MeCN in water containing 0.1% ammonia. Purification using chromatography gave the title compound as a colorless solid (12.4 mg, 28%). This was obtained. LCMS method B: Rt=1.32 min, ES + MS m / z 899.3 [M+H] +

[0230] (Preparation 4 2,2',2''-((5'-((2-aminoethyl)carbamoyl)-[1,1'-biphenyl]-3,3',5-triyl) Tris(oxy)tris(N-(3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R )-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyra (4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy )Oxy)propyl)acetamide)) [ka] (Process 1) 3',5,5'-tris(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3 R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4 ... Trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro 2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran 2-( ... To a solution of carboxylic acid (Preparation 5, 66.0 mg, 30.4 μmol), Et3N (14.8 μL, 106.4 μmol) was added, and followed by N in DMF (0.5 mL) 1 -((4-methoxyphenyl)diphenylmethyl)ethane-1,2-diamine A solution of (Preparation 19, 13.1 mg, 39.5 μmol) was added and the reaction was stirred at room temperature for 1.5 hours.

[0231] (Process 2) 0.2 M HCl (aq) was added dropwise to pH 3-4 and the solution was stirred at room temperature for 18 hours. Concentrate under vacuum and use a reversed-phase column chromatograph eluting with 5-40% MeCN in water containing 0.1% ammonia. Purification using HPLC gave the title compound as a colorless solid (47.0 ml). g, 70%). LCMS method B: Rt=1.57 min, ES + MS m / z 1106.9 [M+2H] + / 2, theoretical mass: 2216.1

[0232] (Preparation 5 3',5,5'-tris(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5- Dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-( Hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2- yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy (Propyl)amino)-2-oxoethoxy)-[1,1'-biphenyl]-3-carboxylic acid [ka] (Method A) Benzyl 3',5,5'-tris(2-((3-(((2R,3R,4R,5S)) dissolved in MeOH / water (1:1 v / v, 7.1 mL) ,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4 -(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pi tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydro Oxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)amino)-2-oxoethoxy 1,1'-biphenyl]-3-carboxylate (Preparation 7, 71.2 mg, 31.4 μmol) was added to 10% Pd / C (7.1 mg) was added. The reaction was hydrogenated at 50 psi (345 kPa) with stirring at room temperature for 3 hours. The reaction mixture was filtered through Dicalite and concentrated in vacuo. The residue was dissolved in water (2 mL) and purified by SiliCycle D After stirring with MT resin (50 mg) for 30 min and filtering, a colorless solution was obtained. The solution was concentrated in vacuo. This afforded the title compound as a colorless solid (61.2 mg, 89%). LCMS method B: Rt=1.27 min, ES + MS m / z 1088.4 [M+2H] + / 2, theoretical mass: 2174.4 MALDI-ToF 2195.8 [M-H+Na] +

[0233] Preparation 3 may be prepared by the following method:

[0234] (Method B) Benzyl 3',5,5'-tris(2-((3-(((2R,3R,4R,5S,6R)-3-acetate) dissolved in water (7 mL) Mido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R ,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy (Ci) tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl Hydro-2H-pyran-2-yl)oxy)propyl)amino)-2-oxoethoxy)-[1,1'-biphenyl To the hydroxybenzoate (Preparation 7, 278 mg, 123 μmol), TEA (7 mL) was added, and the reaction mixture was heated at room temperature. The reaction mixture was concentrated in vacuo and diluted with 5-40% MeCN / water containing 0.1% NH Purification using reverse phase column chromatography eluting with 1000 r.p.m. afforded the title compound as a colorless of 1,2-dimethyl-2,4-trimethyl-1,3 ...

[0235] (Preparation 6 4'-(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy 6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) Methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy )-4-Hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl )amino)-2-oxoethoxy)-[1,1'-biphenyl]-3-carboxylic acid) [ka] Benzyl 4'-(2-((3-(((2R,3R,4R,5S,6R)-3-acetate) dissolved in MeOH / water (1:1 v / v, 5 mL) Amido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S, 5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)o oxy)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl Hydro-2H-pyran-2-yloxypropylamino-2-oxoethoxy-[1,1'-biphenyl] To the [Nyl]-3-carboxylate (Preparation 8, 93.5 mg, 98.7 μmol) was added Pd / C (10%, 10 mg). The reaction was placed under an atmosphere of hydrogen (50 psi (345 kPa)) and stirred at room temperature for 3 hours. The solvent was removed under reduced pressure to give the crude product. The mixture was then subjected to reversed-phase column chromatography eluting with 5-40% MeCN / water containing 0.1% NH3. Purification using a cyclohexane afforded the title compound as a colorless solid (71.6 mg 84%). . LCMS method A: Rt=1.83 min, ES + MS m / z 857.57 [M+H] +

[0236] (Preparation 7 Benzyl 3',5,5'-tris(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S ,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydro Oxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran (2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl (2-hydroxypropyl)amino)-2-oxoethoxy)-[1,1'-biphenyl]-3-carboxylate to) [ka] α-Gal (100 mg, 166 μmol) dissolved in DMSO (1.25 mL) and DMF (3.75 mL) was added to triethylamine. amine (52.1 μL, 374 μmol) and 2,2',2''-((5'-((benzyloxy)carbonyl)-[1,1'-biphenyl]- (phenyl-3,3',5-triyl)tris(oxy)triacetic acid (Preparation 9, 21.2 mg, 41.5 μmol) was added. A solution of HATU (63.1 mg, 166 μmol) in DMF (1.25 mL) was added and the reaction was purged with nitrogen. The reaction mixture was concentrated under vacuum and then resuspended in 10-40% MeOH in water containing 0.1% NH3. Purification using reverse phase column chromatography eluting with CN gave the title compound. This was obtained as a colored solid (71.2 mg, 76%). LCMS method B: Rt=1.80 min, ES + MS m / z 1313.3 [M+2H] + / 2, theoretical mass: 2624.3

[0237] (Preparation 8 Benzyl 4'-(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-di Hydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl (hydroxymethyl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy )propyl)amino)-2-oxoethoxy)-[1,1'-biphenyl]-3-carboxylate) [ka] 2-((3'-((benzyloxy)carbonyl)-[1,1'-biphenyl]-4-yl)o To (hydroxy)acetic acid (Preparation 10, 55.0 mg, 152 μmol), TEA (63.4 μL, 455 μmol) was added, followed by α-Gal (119 mg, 197 μmol) in DMSO (500 μL) was added. HATU (86.6 mg, 228 μmol) was dissolved in DMF (5 00 μL) and the reaction was left stirring at room temperature under nitrogen for 16 hours. The extract was concentrated under vacuum and subjected to reversed-phase column chromatography eluting with 7-60% MeCN / water containing 0.1% NH3. Purification using chromatography gave the title compound as a colorless solid (93.5 mg , 65%). LCMS method B: Rt=2.54 min, ES + MS m / z 947.62 [M+H] +

[0238] (Preparation 9 2,2',2''-((5'-((benzyloxy)carbonyl)-[1,1'-biphenyl]-3,3',5-triyl) Tris(oxy)triacetic acid [ka] Tri-tert-butyl 2,2',2''-((5'-((benzyl 2,2',2''-)-methyl) ester dissolved in DCM / TFA / water (10 / 10 / 1 v / v / v, 5 mL) (1,1'-biphenyl)-3,3',5-triyl)tris(oxy)triphenyl A solution of acetate (Preparation 11, 100 mg, 147 μmol) was stirred at room temperature for 16 hours. It was concentrated in vacuo, dissolved in MeOH (1 mL) and precipitated with water (10 mL). The precipitate was filtered off. The product was collected from the column, washed with water and dried under vacuum to give the title compound as a colorless solid (57.8 mg, 77%). was obtained as. LCMS method A: Rt=2.48 min, ES - MS m / z 509.3 [M−H] - [ka]

[0239] (Preparation 10 2-((3'-((benzyloxy)carbonyl)-[1,1'-biphenyl]-4-yl)oxy)acetic acid) [ka] The title compound was prepared by the method described for Preparation 9 using Preparation 12, 0.1% Purified using reversed-phase column chromatography eluting with 5-40% MeCN / water containing NH3 did. LCMS method B: Rt=2.43 min, ES + MS m / z 363.2 [M+H] + [ka]

[0240] (Preparation 11 Tri-tert-butyl 2,2',2''-((5'-((benzyloxy)carbonyl)-[1,1'-biphenyl]-3, 3',5-triyl)tris(oxy))triacetate [ka] Benzyl 3',5,5'-trihydroxy-[1,1'-biphenyl]-3-carboxylate dissolved in DMF (10 mL) To the carboxylate (Preparation 13, 356 mg, 1.06 mmol) was added tert-butyl bromoacetate (625 μL, 4.23 mmol). 1) and potassium carbonate (1.17 g, 8.47 mmol) were added. The resulting suspension was stirred under nitrogen for 16 hours. The mixture was stirred and then concentrated in vacuo. The residue was dissolved in water (10 mL) and extracted with EtOAc (2×10 mL). The combined organic layers were washed with brine (10 mL), 2M aqueous NaOH (10 mL), dried over MgSO4, and evaporated in vacuo. The residue was purified by silica gel column chromatography eluting with 7-60% EtOAc in heptane. Purification using chromatography gave the title compound as a clear, colourless gum (618 mg, 86 %). LCMS Method C: Rt = 4.34 min, no mass ion observed. [ka]

[0241] (Preparation 12 Benzyl 4'-(2-(tert-butoxy)-2-oxoethoxy)-[1,1'-biphenyl]-3-carboxy rate) [ka] Benzyl 4'-hydroxy-[1,1'-biphenyl]-3-carboxylate (preparation) in DMF (5 mL) To a solution of 14 (368 mg, 1.21 mmol) and tert-butyl bromoacetate (178 μL, 1.21 mmol), carbonate was added. Sodium (200 mg, 1.45 mmol) was added and the reaction was stirred at room temperature for 20 hours followed by 2 hours at 50°C. The reaction mixture was concentrated in vacuo and the resulting residue was dissolved in water (20 mL) and DCM (20 mL). The organic layer was separated and the aqueous layer was extracted again with DCM (20 mL). The extract was washed with water (10 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with 0-15% EtOAc in hexane. The title compound was obtained as an oil (510 mg, 100%). LCMS Method D: Rt = 3.85 min, no mass ions observed [ka]

[0242] (Preparation 13 Benzyl 3',5,5'-trihydroxy-[1,1'-biphenyl]-3-carboxylate [ka] Crude benzyl 3',5'-bis((tert-butyldimethylsilyl)oxy)acetate dissolved in THF (12 mL) )-5-hydroxy-[1,1'-biphenyl]-3-carboxylate (Preparation 16, 1.27 g, 2.46 mmol) To the solution was added dropwise a solution of TBAF (1 M in THF, 6.15 mL, 6.15 mmol). The reaction was stirred at room temperature under nitrogen for 9 min. The mixture was stirred for 10 minutes and then diluted with EtOAc (100 mL). The organic phase was washed with water (2 x 50 mL) and diluted with MgSO4 The residue was purified by silica gel column eluting with 5% MeOH in DCM. The title compound was purified using column chromatography to give a light brown solid (356 mg, 3 steps) The result was 43% (throughout the experiment). LCMS method A: Rt=2.66 min, ES - MS m / z 335.3 [M−H] - [ka]

[0243] (Preparation 14 Benzyl 4'-hydroxy-[1,1'-biphenyl]-3-carboxylate [ka] To a solution of benzyl chloride (295 μL, 2.56 mmol) in DMF (5 mL) was added 4'-hydroxybiphenyl-3- Carboxylic acid (500 mg, 2.33 mmol) and potassium carbonate (322 mg, 2.33 mmol) were added to this reaction. The mixture was stirred at room temperature for 20 hours and then concentrated in vacuo. The residue was dissolved in water (20 mL) and diethyl ether. The organic layer was separated and the aqueous layer was diluted with diethyl ether (20 mL). The combined organic extracts were washed with water (10 mL) and dried over sodium sulfate. The residue was purified by silica gel column eluting with 0-30% EtOAc in hexanes. The product was purified by column chromatography to give the title compound as a white solid (378 mg, 53%). I got it. LCMS method D: Rt=3.48 min, ES + MS m / z 305.0 [M+H] + [ka]

[0244] (Preparation 15 Benzyl 3-bromo-5-hydroxybenzoate [ka] To a solution of 3-bromo-5-hydroxybenzoic acid (4.08 g, 18.8 mmol) dissolved in DMF (25 mL) After 5 min, K2CO3 (2.60 g, 18.8 mmol) was added, and after 5 min, benzyl bromide (2.24 mL, 18.8 mmol) was added for 10 min. The reaction was stirred at room temperature under nitrogen for 16 hours. Additional K2CO3 (520 mg, 3.7 6 mmol) and benzyl bromide (450 μL, 3.79 mmol) were added and the reaction was stirred for 3 hours. The reaction mixture was concentrated in vacuo and the residue was partitioned between EtOAc (30 mL) and water (30 mL). The mixture was extracted with EtOAc (2×20 mL) and the combined organic layers were washed with brine (30 mL). The residue was dried over MgSO4 and concentrated in vacuo. The residue was eluted with 5% EtOAc in heptane. The title compound was purified by silica gel column chromatography to give a colorless solid (3 0.88g, 67%). LCMS method A: Rt=3.36 min, ES - MS m / z 307.2 [M−H] - [ka]

[0245] (Preparation 16 Benzyl 3',5'-bis((tert-butyldimethylsilyl)oxy)-5-hydroxy-[1,1'-biphenyl] [Nyl]-3-carboxylate) [ka] Benzyl 3-bromo-5-hydroxybenzoate dissolved in dioxane / water (30 mL, 5:1 v / v) 1,2-Dimethyl-2,4-diol (Preparation 15, 755 mg, 2.46 mmol), sodium carbonate (912 mg, 8.60 mmol), and ((5-(4,4,5,5- Tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-phenylene)bis(oxy))bis(ter A mixture of 1.87 g of tert-butyldimethylsilane (Preparation 17, 2.95 mmol) and 1.87 g of tert-butyldimethylsilane was degassed with nitrogen for 30 minutes. Pd(PPh3)4 (284 mg, 246 μmol) was added and the reaction was heated under nitrogen at 100 °C for 90 min. After cooling to room temperature, EtOAc (100 mL) and water (50 mL) were added. The layers were separated. The aqueous phase was back-washed with EtOAc (2 x 25 mL). The combined organic phases were dried over MgSO4 and purified. The residue was treated with heptane (100 mL) and the resulting mixture was heated in an oven for 5 minutes. The filtrate was concentrated in vacuo to give the crude title compound. was obtained as a clear brown oil (1.27 g) which was used directly in the next step. LCMS Method C: Rt=5.47 min, ES + MS m / z 565.4 [M+H] +

[0246] (Preparation 17 ((5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-phenylene)bis(o Bis(tert-butyldimethylsilane) [ka] 1,3-bis((tert-butyldimethylsilyl)oxy)benzyl ether dissolved in isohexane (15 mL) of benzene (Preparation 18, 1.00 g, 2.95 mmol) and bis(pinacolato)diboron (750 mg, 2.95 mmol). The solution was degassed with nitrogen for 1 hour. [Ir(OMe)(COD)] (19.6 mg, 59.1 μmol) and 4,4'-dichloromethane were added. tert-butyl-2,2'-bipyridine (15.9 mg, 59.0 μmol) was added, the reaction mixture was sealed, and the mixture was heated to 110°C. Heated for 16 hours. The reaction was cooled, concentrated in vacuo and used directly in the next step (1.87 g). . LCMS Method C: Rt=6.19 min, ES + MS m / z 465.4 [M+H] + [ka]

[0247] (Preparation 18 1,3-bis((tert-butyldimethylsilyl)oxy)benzene) [ka] Resorcinol (2.00 g, 18.2 mmol) and imidazole (3.71 g, To the resulting solution (54.5 mmol), tert-butyldimethylchlorosilane (8.21 g, 54.5 mmol) was added. More DCM (40 mL) was added and the reaction was then stirred at room temperature under nitrogen for 16 hours. Filter and concentrate the filtrate in vacuo. Elute the residue with 0-10% EtOAc in heptane. Purification using silica gel column chromatography gave the title compound as a colorless oil (6.1 8g, >99%). LCMS Method C: Rt=5.39 min, ES + MS m / z 339.3 [M+H] + [ka]

[0248] (Preparation 19 N 1 -((4-methoxyphenyl)diphenylmethyl)ethane-1,2-diamine [ka] A solution of ethylenediamine (54.0 mL, 809.6 mmol) in DCM (175 mL) was added to MMT crystals in DCM (175 mL). A solution of 25.0 g (81.0 mmol) of methyl chloride was added slowly over 1 hour with stirring. The solution was stirred for 16 hours at room temperature. The reaction mixture was diluted with 10% aqueous potassium carbonate (400 mL) and brine. The organic layer was separated, dried over magnesium sulfate, and concentrated in vacuo to give The title compound was obtained as a viscous pale yellow oil (26.1 g, 97%). LCMS Method B: Rt = 2.73 min, no ionization observed. [ka]

[0249] (Preparation 20 4'-(2-((2-(3-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxybenzoate Hydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) (oxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl) Oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)prop N-(2-(propyl)amino)-3-oxopropoxy)ethyl)amino)-2-oxoethoxy)-N-(2-(3-(2-(2 ,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)ethyl)-[1,1 '-biphenyl]-3-carboxamide) [ka] A solution of preparation 24 in DMF (100 mM) was added to an equal volume of Mal-PEG4-NHS (80 mM, 0.8 eq) in DMF. The reaction mixture was left standing at room temperature for 2 hours. The reaction mixture was stored at a final concentration of 50 mM and used for conjugation. It was used directly in the filtration process.

[0250] (Preparation 21 3,3',3''-((((2,2',2''-((5'-((2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) (I)ethoxy)propanamido)ethyl)carbamoyl)-[1,1'-biphenyl]-3,3',5-triyl tris(oxy))tris(acetyl))tris(azanediyl))tris(ethane-2,1-diyl ))tris(oxy))tris(N-(3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S, 6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydro Oxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran (2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl (Oxypropyl)propanamide [ka] A solution of preparation 25 in DMF:DMSO (3:1, 25 mM) was diluted with an equal volume of Mal-PEG4-NHS (80 mM, 0.8 The reaction mixture was added to the PBS solution (eq.) and left to stand at room temperature for 2 hours. The reaction mixture was stored at a final concentration of 20 mM. It was used directly in the jugation process.

[0251] (Preparation 22 2,5-Dioxopyrrolidin-1-yl 5-((2-(4'-(2-((3-(((2R,3R,4R,5S,6R)-3-acetamide -5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R) -3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy Tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro 2H-pyran-2-yloxypropylamino-2-oxoethoxy-[1,1'-biphenyl] ]-3-Carboxamido)ethyl)amino)-5-oxopentanoate) [ka] 4'-(2-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydro Oxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) Dimethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy (4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl (2-aminoethyl)-2-oxoethoxy)-N-(2-aminoethyl)-[1,1'-biphenyl]-3-carboxamide The acetone (Preparation 3, 21.76 mg, 24 mmol) was dissolved in 1:1 anhydrous DMF:DMSO (1062 μL) with stirring. Di-(N-succinimidyl) glutarate (39.49 mg, 120 mmol) was dissolved in 1:1 anhydrous DMF:DMSO. (400 μL) of HCl. The reaction was stirred at room temperature under a positive nitrogen atmosphere for 1 hour. The reaction mixture was purified by reversed-phase column chromatography ( The desired fraction was evaporated to dryness using a 10 x 250 mm Hichrom ACE 10 column. The mixture was stirred for 1 hour at rt and lyophilized to give the title compound as a colorless glass (23 mg, 88%). MS m / z 1110.3 [M+H] +

[0252] (Preparation 23 2,5-Dioxopyrrolidin-1-yl 5-oxo-5-((2-(3',5,5'-tris(2-((2-(3-((3-(((2R, 3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxy methyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6- (Hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)amino)-3-oxo Propoxy)ethyl)amino)-2-oxoethoxy)-[1,1'-biphenyl]-3-carboxamide )Ethyl)amino)pentanoate) [ka] The title compound was prepared by the method described for Preparation 22 using Preparation 25. It was directly adopted in the injecting process.

[0253] (Preparation 24 4'-(2-((2-(3-((3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxybenzoate Hydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) (oxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl) Oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)prop (propyl)amino)-3-oxopropoxy)ethyl)amino)-2-oxoethoxy)-N-(2-aminoethoxy)- (ethyl)-[1,1'-biphenyl]-3-carboxamide [ka] The title compound was synthesized by the reaction of 4'((2,2-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)- 3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran- 2-yl)oxy)-2-18-dioxo-6,9,12,15-tetraoxa-3,19-diazadocosyl)oxy)- Preparation 3 was described using [1,1'-biphenyl]-3-carboxylic acid (WO2017060729) It was prepared by the method. LCMS (Method B): Rt=1.66 min; ES + MS m / z 1145.4 [M+H] +

[0254] (Preparation 25 3,3',3''-((((2,2',2''-((5'-((2-aminoethyl)carbamoyl)-[1,1'-biphenyl]-3,3' ,5-triyl)tris(oxy))tris(acetyl))tris(azanediyl))tris(ethane-2 ,1-diyl))tris(oxy))tris(N-(3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S, 3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4 ... Trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro 2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran 2-Propan-2-yloxypropylpropanamide [ka] The title compound was synthesized by the reaction of 3',5,5'-tris((2,2-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S, 3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4 ... Trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro 2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran 2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazadocosyl)oxy Preparation 3 is described using 1,1'-biphenyl-3-carboxylic acid (WO2017060729). It was prepared by the method described above. LCMS (Method B): Rt=1.52 min; ES + MS m / z 1479.0 [M+2H] + / 2;Theoretical mass: 2958.0

[0255] (Example) Materials and Methods vcMMAE (vcE): ADCB TOX001, 10mM in DMA TCEP-Biovectra Cat 1300 Lot: 42359 N-Acetylcysteine ​​- Sigma A7250 Lot: WXBC3104V Dimethylacetamide - Sigma-Aldrich 271012 Lot: STBF9638V KNE buffer: 50mM KPI, 50mM NaCl, 2mM EDTA, pH 7.5 Reconstituted using PBS: Sigma, P5368 ♯SLBQ7495, WFI: Sigma W3500 ♯RNBF6963 thing 1.76 M HEPES, pH 10.8 2-Iminothiolane HCl: Sigma I6256 #SLBS1775V Polysorbate 80: Sigma-Aldrich P8074 #BCBG4547V LAR is the linker:antibody ratio. DAR is the drug:antibody ratio. SEC is size exclusion chromatography.

[0256] (Monomer content by size exclusion HPLC (SEC)) The total content of each conjugate was adjusted to 10% IPA, 0.2M potassium phosphate, 0.25M potassium chloride. Chromatography on a TOSOH TSK gel G3000SWXL 7.8 mm x 30 cm, 5 μm column at 0.5 mL / min in pH 6.95. The samples were loaded neat and the data were collected at 214, 252, and 28 All data reported is at 280 nm.

[0257] (Hydrophobic Interaction Chromatography (HIC)) HIC was performed using mobile phase A - 1.5 M (NH4)2SO4, 25 mM NaPi, pH 6.95 ± 0.05 and mobile phase B - 75% 25 mM Na A 12-minute linear gradient between Pi, pH 6.95 ± 0.05, and 25% IPA was used to measure 0.8 mL / min. The sample was collected in 10 μL of the highest concentration on a TOSOH Butyl-NPR 4.6 mm x 3.5 cm, 2.5 μm column. Loaded neat to capacity, data collected at 280, 252, and 214 nm; reported data All at 214 nm.

[0258] (Polymer-based reversed-phase chromatography (PLRP)) The reverse phase was analyzed by a 25-minute linear gradient between 0.1% TFA 25% MeCN and 0.1% TFA 50% MeCN. Polymer Labs PLRP-S 2.1 mm x 50 mm, 5 μm, 1000 Å, at 0.25 mL / min at 80°C. 10 μg of the eluate was mixed with 5 μL of 0.1 M DTT and made up to 50 μL in 0.5 M Tris / Cl, pH 8.0. The samples were reduced by incubating at 49°C for 15 minutes at 37°C. The reduction was stopped by diluting 1:1 with 49% acetonitrile, 49% water, and 2% formic acid, and the PLRP fraction was then purified. The sample was allowed to stabilize until analysis. 20 μL of the sample was loaded onto the analytical column. All data is reported at 214 nm.

[0259] NOTE: In HIC and PLRP, separate individual linker loadings due to the hydrophilicity of α-Gal. The linker:Ab ratio (LAR) can be adjusted to the ratio of the initial thiol-reactive antibody to the completed reaction. The vcE was determined by vcE chase of an aliquot of the mixture.

[0260] Data for each example are illustrated in Figures 1-3 and 12-17.

[0261] (Antibody conjugate (cetuximab))

[0262] Cetuximab (Merck Serono; Lot No: 223155, exp: 09 / 202) with a molecular weight of 152,000 Da 0 was used for the following conjugations. The calculation was 1.45 cm -1 mg / mL -1 Abs 0.1% Based on UV analysis at 280 nm, 4.7 mg / mL, and a calibration curve by SEC at 214 nm.

[0263] Reduction of cetuximab and subsequent conjugation to a maleimide-containing linker Outline of the method for (Examples 1 to 4) Add cetuximab (4.7 mg / mL) to 6% 0.5 M Tris-Cl, 0.025 M EDTA, pH 8.5, and dilute to 1.1 eq. of TCEP:mAb (for an average LAR of 2) or 4.2 eq. of TCEP:mAb (for an average LAR of 5) at room temperature. The reduced samples were incubated for 90 and 120 minutes, respectively. An aliquot of the sample was conjugated with a molar excess of the surrogate payload Mal-vc-PAB-MMAE to obtain D The AR was determined (using HIC analysis). 8 eq of linker-maleimide was added to this reduced sample. The reaction was incubated at room temperature for 60 minutes. The sample was quenched by the addition of acetylcysteine ​​(10 mM in water) for 30 minutes. The remaining unbound linker was purified and dialyzed into PBS using a Vivaspin6 device for 10 min. The serum was removed by membrane diafiltration in 1000 DIA volumes.

[0264] Example 1 [ka] Average LAR: 2; Average total number of α-Gal units: 2 % Monomer [SEC]: 98.8% (see Figure 3) Precursor: Preparation 1

[0265] Example 2 [ka] Average LAR: 5; Average total number of α-Gal units: 5 % Monomer [SEC]: 99.4% (see Figure 3) Precursor: Preparation 1

[0266] Example 3 [ka] Average LAR: 2; average total number of α-Gal units: 6 % Monomer [SEC]: 99.5% (see Figure 3) Precursor: Preparation 2

[0267] Example 4 [ka] Average LAR: 5; average total number of α-Gal units: 15 % Monomer [SEC]: 99.0% (see Figure 3) Precursor: Preparation 2

[0268] (Interconversion of cetuximabridin to a thiol and subsequent maleimide-containing linker General Methods for Conjugation to (Examples 5-8) Cetuximab (4.7 mg / mL) was incubated with Protein A resin (GE Healthcare, HiTrap MabSelect SURE 1 mL), and then the column was washed with 50 mM KPI, 50 mM NaCl, 2 mM EDTA, pH 7.5. The mAb was eluted with 4 CV of 0.1 M glycine at pH 3. The eluted fractions containing the target protein (UV280 To the smeared samples (pooled by absorbance), 20% 1.76M HEPES at pH 10.8 was added. The antibody was diluted with 12.2 eq of 2-iminothiolane:mAb (for low LAR samples) or 30.5 eq of 2-iminothiolane:mAb. The samples were incubated with mAb (against high LAR samples) for 120 min at 23°C. Aliquots of the samples were diluted to 5 ml. Buffer exchanged into 50 mM His, 50 mM trehalose pH 6.0, and a molar excess of the surrogate payload Mal-v The DAR was determined by conjugation with c-PAB-MMAE. The lysed mixture was buffer exchanged into 5 mM His, 50 mM trehalose, pH 6.0 with NAP25. The linker-maleimide conjugation was quenched using a final 5% DMA. Incubation was performed at 23°C for 120 minutes using 4 eq of linker-maleimide:thiol. Immediately after loading, the sample was buffer exchanged into PBS using G25 resin. The amount of free thiol was monitored throughout the experiment. , can be monitored using Ellman's assay.

[0269] Example 5 [ka] Average LAR: 2; Average total number of α-Gal units: 2 % Monomer [SEC]: 95.0% (see Figure 3) Precursor: Preparation 1

[0270] Example 6 [ka] Average LAR: 4.9; average total number of α-Gal units: 4.9 % Monomer [SEC]: 90.3% (see Figure 3) Precursor: Preparation 1

[0271] Example 7 [ka] Average LAR: 2; average total number of α-Gal units: 6 % Monomer [SEC]: 90.4% (see Figure 3) Precursor: Preparation 2

[0272] Example 8 [ka] Average LAR: 4.9; average total number of α-Gal units: 14.7 % Monomer [SEC]: 80.8% (see Figure 3) Precursor: Preparation 2

[0273] (Reduction of cetuximab to obtain an average LAR of 8 and subsequent conversion to a maleimide-containing linker) General Methods for Conjugation of (Examples 9-11) Cetuximab (4.7 mg / mL) was dissolved in 6% 0.5 M Tris-Cl, 0.025 M EDTA (pH 8.5) at approximately pH 7.5. The mixture was incubated with 8 eq of TCEP:mAb for 90 min at room temperature. To achieve this, an aliquot of the reduced sample was combined with a molar excess of the surrogate payload Mal-vc-PAB-MMAE. The DAR was determined by conjugation (using HIC analysis).

[0274] To the reduced sample, either 16 or 32 eq of linker-maleimide was added, and the reaction mixture was The reaction mixture was incubated at room temperature for 60 minutes. After the incubation, the reaction mixture was diluted with N-acetylcysteine. The sample was purified on G25 resin into PBS and the remaining of unbound linker was subjected to membrane dialysis using a Vivaspin6 30 kDa PES device for 10 dialysis volumes. It was removed by filtration.

[0275] Example 9 [ka] Average LAR: 8; Average total number of α-Gal units: 8 SEC analysis: Rt = 15.3 min, 98.3% monomer content Precursor: Preparation 1

[0276] Example 10 [ka] Average LAR: 8; Average total number of α-Gal units: 8 SEC analysis: Rt = 14.8 min, 98.5% monomer content Precursor: Preparation 20

[0277] Example 11 [ka] Average LAR: 8; Average total number of α-Gal units: 8 SEC analysis: Rt = 14.1 min, 97.0% monomer content Precursor: Preparation 21

[0278] Direct lysine conjugates to N-hydroxysuccinimide-containing linkers of cetuximab General Method for Induction (Examples 12-16) Cetuximab (20 mg) was loaded onto Protein A resin (GE Healthcare, HiTrap MabSelect Sure, 1 m L) and the column was washed with a solution of 50 mM KPi, 50 mM NaCl, and 2 mM EDTA at pH 8. The antibody was eluted with 100 mM citrate buffer, pH 3, and concentrated to approximately 6 mg / mL. The buffer was placed in conjugation buffer (50 mM NaPi, 150 mM NaCl, 2 mM EDTA, pH = 8). The solution was analyzed by SEC to confirm that the serine was in a solution suitable for lysine conjugation. Tuximab was obtained (18.2 mg, 91% yield, 6.4 mg / mL, 100% monomer content).

[0279] The above cetuximab solution was diluted with 6% v / v DMF co-solvent for 5, 10, 15, 20 or 38 min. The reaction was incubated with two molar equivalents of Preparation 22 for 2 hours at 30°C. The reaction was diluted with glycine up to 1 mM The conjugate was quenched by the addition of HCl, buffer exchanged into PBS, and diafiltered. The excess linker was removed by

[0280] Example 12 [ka] Average LAR: 2; Average total number of α-Gal units: 2 SEC analysis: Rt=15.0 min. Monomer content not determined. Precursor: Preparation 22 (5 eq)

[0281] Example 13 [ka] Average LAR: 5; Average total number of α-Gal units: 5 SEC analysis: Rt=14.9 min. Monomer content not determined. Precursor: Preparation 22 (10 eq)

[0282] Example 14 [ka] Average LAR: 8; Average total number of α-Gal units: 8 SEC analysis: Rt=14.8 min. Monomer content not determined. Precursor: Preparation 22 (15 eq)

[0283] Example 15 [ka] Average LAR: 10; average total number of α-Gal units: 10 SEC analysis: Rt=14.7 min. Monomer content not determined. Precursor: Preparation 22 (20 eq)

[0284] Example 16 [ka] Average LAR: 15; average total number of α-Gal units: 15 SEC analysis: Rt=14.2 min, 94.3% monomer content Precursor: Preparation 22 (38 eq)

[0285] Example 17 [ka] Example 17 was treated with 60 eq of Preparation 22 (with 10% v / v DMF) for 2 hours, followed by a further 40 eq The solution was prepared according to general method 3 using Preparation 22 (with 4% v / v DMF) for 2 hours. Average LAR: 20; average total number of α-Gal units: 20 SEC analysis: Rt = 13.9 min, 98.4% monomer content Precursor: Preparation 22 (Fab fragment conjugate (cetuximab))

[0286] (Digestion of cetuximab to cetuximab-Fab) Cetuximab (60 mg, 4.7 mg / mL) was dissolved in digestion buffer (20 mM NaPi, 20 mM cysteine, 10 mM The solution was buffer exchanged into 20 mg / mL immobilized papain (3 mL) and concentrated to 3 mL. Thermo Fisher #20341, Loading: 250 μg / mL resin, Activity: 16-40 BAEE / mg papaya The lysate was equilibrated with digestion buffer and incubated with the concentrated cetuximab at 37°C for 15 hours. The digestion products were collected by filtration and eluted through a Protein A column. The unbound Fab fragments were passed through the column and the flow-through fraction was collected. , 50 mM NaPi, 150 mM NaCl, and 2 mM E by Vivaspin centrifugation (10 kDa MWCO filter). Discontinuous diafiltration into DTA, pH = 8 was performed. The final concentration achieved was 5.6 mg / mL (Figure 1). 16).

[0287] (Reduction of cetuximab-Fab to give LAR=2 and subsequent coupling to a maleimide-containing linker) General Method for Conjugation (Examples 18 and 19) 5.6 mg in conjugation buffer (50 mM NaPi, 150 mM NaCl, and 2 mM EDTA, pH=8) / mL of cetuximab-Fab was reduced by the addition of 5 molar equivalents of TCEP (10 mM in water) for 90 minutes at room temperature. A solution of Preparation 20 (5 molar equivalents) or Preparation 21 (7.5 molar equivalents) was added and the reaction was allowed to stand at room temperature. Incubated for 1 hour. The conjugate was buffer exchanged using PBS and dialyzed. Filtration gave the desired material, which was analyzed by SEC and SDS-PAGE. .

[0288] Example 18 [ka] Average LAR: 2; Average total number of α-Gal units: 2 SEC analysis: Rt=18.2 min, 94.7% monomer content Precursor: Preparation 20

[0289] Example 19 [ka] Average LAR: 2; average total number of α-Gal units: 6 SEC analysis: Rt=17.4 min, 91.7% monomer content Precursor: Preparation 21

[0290] Lysine conjugation to NHS-containing linkers of cetuximab-Fab yielded an average LAR of 7–14. General Method for Injection (Examples 20-24) 5.6 mg in conjugation buffer (50 mM NaPi, 150 mM NaCl, and 2 mM EDTA, pH=8) 1 mL of cetuximab-Fab was diluted with 9% v / v DMF co-solvent in Preparation 22 (15, 30, and 40 equivalents) and The reaction mixture was incubated with Preparation 23 (20 equivalents) at 30°C for 2 hours. The reaction mixture was diluted with glycerol to a concentration of 1 mM. The conjugate was quenched by the addition of HCl, and the conjugate was buffer exchanged into PBS and diafiltered. Excess linker was removed.

[0291] Example 20 [ka] Average LAR: 7; Average total number of α-Gal units: 7 SEC analysis: Rt = 18.0 min, 99% monomer content Precursor: Preparation 22 (15 eq)

[0292] Example 21 [ka] Average LAR: 5; average total number of α-Gal units: 15 SEC analysis: Rt=17.4 min, 99% monomer content Precursor: Preparation 23 (20 eq)

[0293] Example 22 [ka] Average LAR: 11; average total number of α-Gal units: 11 SEC analysis: Rt=17.3 min, 96.9% monomer content Precursor: Preparation 22 (30 eq)

[0294] Example 23 [ka] Average LAR: 14; average total number of α-Gal units: 14 SEC analysis: Rt=17.1 min, 95.7% monomer content Precursor: Preparation 22 (40 eq)

[0295] Example 24 [ka] Example 24 was treated with 60 equivalents of Preparation 22 (with 20% v / v DMF) for 2 hours, followed by an additional 40 e Prepared according to general method 4 using preparation 22 (with 4% v / v DMF) for 2 hours. Average LAR: 17; average total number of α-Gal units: 17 SEC analysis: Rt = 15.9 min, 90.2% monomer content Precursor: Preparation 22 (60 + 40 eq)

[0296] (Antibody conjugate (rituximab)) Sodium citrate anhydrous (7.35 mg / mL), sodium chloride (9 mg / mL), and water were added to the solution. Rituximab (Roche-Rituxan, Lot No: B61) formulated in resolvate 80 (0.7 mg / mL) 05B92UI).

[0297] Direct lysine conjugates to N-hydroxysuccinimide-containing linkers of rituximab General Method for Induction (Example 25) Rituximab was dissolved in 500 mM phosphate buffer (50 mM NaPi, 150 mM NaCl, 2 mM EDTA, pH = 8). The pH was adjusted to pH 7.9 using

[0298] The above solution of rituximab was diluted with 40 molar equivalents of Preparations 22 and 30 along with 10% v / v DMF co-solvent. C for 2 hours, followed by 40 molar equivalents of Preparation 22 (with 4% v / v DMF) The reaction was quenched by the addition of glycine to 1 mM and the concentration was The conjugate was buffer exchanged into PBS and diafiltered to remove excess linker.

[0299] Example 25 [ka] Average LAR: 20; average total number of α-Gal units: 20 SEC analysis: Rt = 14.4 min, 98.5% monomer content Precursor: Preparation 22

[0300] (Fab fragment conjugate (rituximab))

[0301] (Digestion of rituximab to rituximab-Fab) Rituximab (60 mg, 4.7 mg / mL) was dissolved in digestion buffer (20 mM NaPi, 20 mM cysteine, 10 mM The buffer was exchanged into 1000 M EDTA, pH = 7) and concentrated to 20 mg / mL. Immobilized papain (w / w 1 / 160, Thermo Fisher #20341, Loading: 250 μg / mL resin, Activity: 16-40 BAEE / mg papain) The resulting solution was equilibrated in digestion buffer and incubated with concentrated rituximab at 37°C for 5-18 hours. The digestion products were collected by filtration and eluted through a Protein A column. The unbound Fab fragments were passed through the column and the flow-through fraction was collected. Vivaspin centrifugation (10 kDa MWCO filter) into 50 mM NaPi, 150 mM NaCl, and 2 mM EDTA, pH = 8. Discontinuous diafiltration was performed using a diafiltration filter (diafiltration filter). The final concentration achieved was 11.7 mg / mL. The above solution of rituximab-Fab was diluted with 40 molar equivalents of Preparation 22 with 25% v / v DMF co-solvent. and 40 molar equivalents of Preparation 22 (9% v / v DMF) for 2 hours at 30°C. A second addition was made. The reaction was quenched by the addition of glycine to 1 mM. The conjugate was buffer exchanged into PBS and diafiltered to remove excess linker.

[0302] Example 26 [ka] Average LAR: 14; average total number of α-Gal units: 14 SEC analysis: Rt=16.9 min, 87.5% monomer content Precursor: Preparation 22

[0303] (Flow cytometry assay using α-galactosyl IgM antibody) Flow cytometry was used to identify L(as cetuximab) receptors on human cell lines. ) and F (as a carbohydrate molecule capable of binding to human anti-α-galactosyl antibodies It is well known that A431 cells significantly overexpress EGFR receptors. To achieve this, the cells were used to capture EGFR-binding mAb (cetuximab). Binding of α-galactosyl IgM antibody to the compound using litrene (PE)-labeled anti-human IgM antibody was detected.

[0304] A431 cells (ATCC CRL-1555) were collected and diluted in phosphate-buffered saline (PBS) (Sigma D8662) + 0.1% BS 5 × 10 in A (bovine serum albumin - Sigma A2153) 6 The cells were then resuspended at 5 x 10 cells / mL. 5 Cells were incubated in a 200°C chamber with various concentrations of compounds or buffer alone as indicated below. The mixture was incubated at room temperature with shaking at 450 rpm for 1 hour.

[0305] The cells were washed with 2 x 200 μL PBS + 0.1% BSA and then resuspended in 50 μL of 32 μg / mL BSA solution in PBS + 0.1% BSA. L of anti-α-galactosyl IgM antibody (Absolute Antibody Ab00532-15.0) was added and incubated at 4°C for 1 hour. The cells were then washed twice with 200 μL PBS + 0.1% BSA, and then 100 μL The cells were treated with a 1:40 dilution of anti-human IgM-PE (Biolegend 314508) in 2 x 200 μL PBS for 1 hour at 4°C. After a final wash of +0.1% BSA, cells were resuspended in 200 μL PBS + 0.1% BSA and analyzed by flow cytometry. The data from all samples was analyzed using a Beckman Coulter FC500 meter. The data were analyzed using a software package (Beckman Coulter).

[0306] FIG. 4 shows the results of Example 1 (FIG. 4A), Example 2 (FIG. 4B), Example 3 (FIG. 4C), Example 4 (FIG. 4D), and Example 5 (FIG. 4 Example 6 (FIG. 4F), Example 7 (FIG. 4G), and Example 8 (FIG. 4H) were used at 10 nM. The change in fluorescence intensity (PE) indicates the capture of tosyl IgM antibody to the cell surface. This occurs due to a combination event.

[0307] (Flow cytometry assay using α-galactosyl IgG antibody) L (as cetuximab) to receptors on human cell lines using flow cytometry and F (as a carbohydrate molecule capable of binding to human anti-α-galactosyl antibodies). It is well known that A431 cells significantly overexpress EGFR receptors. To achieve this, the cells were used to capture EGFR-binding mAb (cetuximab). Compound binding was detected using a littorin (PE)-labeled α-galactosyl IgG antibody.

[0308] A431 cells (ATCC CRL-1555) were harvested and resuspended in phosphate-buffered saline (PBS) (Sigma D8662) + 0.1% BSA ( 5 × 10 in bovine serum albumin (Sigma A2153) 6 The cells were then resuspended at 5 x 10 cells / mL. 5 Thin The cells were incubated with 10 nM compound, buffer alone, or 10 nM cetuximab while shaking at 450 rpm. The cells were incubated at room temperature for 1 hour. The cells were washed twice with 200 μL PBS + 0.1% BSA, and then Add 50 μL of PE-labeled anti-α-galactosyl IgG antibody at 575 μg / mL in PBS + 0.1% BSA and incubate at 4 °C for 1 Incubated for 1 hour. Anti-α-galactosyl IgG antibody (Absolute Antibody Ab00532.10.0) Custom labeled with PE by Cambridge Research Biochemicals. 2 x 200 μL After a final wash of PBS + 0.1% BSA, cells were resuspended in 200 μL PBS + 0.1% BSA and The data from all samples were evaluated on a Kaluz Fluorescence Intensity Meter (FC500 Beckman Coulter). The data were analyzed in a software package (Beckman Coulter).

[0309] FIG. 5 shows the results of Example 1 (FIG. 5A), Example 2 (FIG. 5B), Example 3 (FIG. 5C), and Example 4 (FIG. 5D) compared to buffer alone. and Example 4 (FIG. 5D) showing the capture of anti-α-galactosyl IgG antibody to the cell surface using 10 nM. A change in fluorescence intensity (PE) occurs due to the binding event at each end of the molecule.

[0310] FIG. 6 shows the results of Example 5 (FIG. 6A), Example 6 (FIG. 6B), and Example 7 (FIG. 6C) compared to 10 nM cetuximab. and Example 8 (FIG. 6D) showing the capture of anti-α-galactosyl IgG antibody to the cell surface using 10 nM. A change in fluorescence intensity (PE) occurs due to the binding event at each end of the molecule.

[0311] Flow cytometry assay using hIVIG-derived α-galactosyl IgG antibody and A431 cells Say) Flow cytometry was used to identify the L(Fab fragment) fragments of EGFR expressed on a human cell line (A431). as a fragment) and F (carbohydrate moiety capable of binding to human anti-α-galactosyl antibody) The binding of the IgG molecule was revealed using a secondary phycoerythrin (PE)-labeled anti-human IgG antibody. , the binding of α-galactosyl IgG antibody to the compound was detected.

[0312] A431 cells (ATCC CRL-1555) were harvested and resuspended in phosphate-buffered saline (PBS) (Sigma D8662) + 0.1% BSA ( 5 × 10 in bovine serum albumin (Sigma A2153) 6 The cells were then resuspended at 5 x 10 cells / mL. 5 Thin Cells were incubated with various compound concentrations up to 1000 nM, buffer alone, or 1000 nM of the conjugate. The mixture was incubated with unmodified Fab fragments for 1 hour at room temperature with shaking at 450 rpm. Cells were washed 2x with 200 μL PBS + 0.1% BSA, followed by 50 μL of hIVIG anti-Ga in PBS + 0.1% BSA. l IgG (70 μg / ml) (custom-purified from human IVIG) was added and incubated for 1 hour at 4°C. The cells were washed 2x with 200 μL PBS + 0.1% BSA, then 100 μL of secondary anti-IgG-PE (clone HP6017, Biolegend 409393) was added. The cells were incubated in the dark at 4°C for 30 minutes. Ta.

[0313] After a final wash of 2 x 200 μL PBS + 0.1% BSA, cells were resuspended in 200 μL PBS + 0.1% BSA. The data from all samples were evaluated on a flow cytometer (FC500 Beckman Coulter). The data was analyzed using the Kaluza software package (version 1.5a, Beckman Coulter). analyzed.

[0314] Figure 7 shows the results of an example where minimal recruitment was observed compared to the unconjugated Fab fragment. 1 shows dose-related, compound-driven mobilization of anti-Gal IgG antibodies from hIVIG 18-23 into A431 cells.

[0315] (Flow cytometry assay using α-galactosyl IgM antibody and A431 cells) The compounds were compared to cetuximab and / or unconjugated Fab fragments to determine the best The assay was performed according to the flow cytometry assay protocol described above at various concentrations from 0.1 to 1000 nM. I tried it.

[0316] Figure 8 shows the results of α-galactosidase B (α-galactosidase B) compared to unconjugated Fab fragments and / or cetuximab. 1 shows dose-related, compound-driven recruitment of ctosyl IgM antibodies to A431 cells.

[0317] (Flow cytometry assay using C3b antibody) Flow cytometry is used to measure the binding of compounds to the target cell lines and complement C3b. The researchers found that A431 cells significantly overexpress EGFR receptors. The cells were used to capture EGFR-binding antibodies or antibody fragments, as this is well known. Using anti-C3b antibody conjugated to erythrin (PE), various concentrations of compounds were After addition, recruitment of C3b molecules to cells from serum was detected.

[0318] A431 cells (ATCC CRL-1555) were harvested and resuspended in phosphate-buffered saline (PBS) (Sigma D8662) + 0.1% BSA ( 5 × 10 in bovine serum albumin (Sigma A2153) 6 The cells were then resuspended at 5 x 10 cells / mL. 5 Thin Cells were incubated with various compound concentrations up to 10,000 ng / ml, buffer alone, or cetuximab Fa b fragment and / or cetuximab at various concentrations, shaking at 450 rpm for 1 h at room temperature The cells were washed twice with 200 μL PBS + 0.1% BSA, then with 100 μL PBS. and 100 μL 20% human serum (HS) (Patricell 23590) or heat-inactivated human serum (HIHS) at 25 μg / ml M86 IgM (Absolute Antibody) was added and incubated at 37°C for 25 minutes.

[0319] The cells were washed 2x with 200 μL PBS + 0.1% BSA, then 100 μL of anti-C3b-PE (3E7 / C3b, B iolegend 846104) was added. The cells were incubated in the dark at 4°C for 30 minutes. After a final wash of 200 μL PBS+0.1% BSA, the cells were resuspended in 200 μL PBS+0.1% BSA and Data from all samples were evaluated on a flow cytometer (FC500 Beckman Coulter). , analyzed in the Kaluza software package (version 1.5a, Beckman Coulter). .

[0320] FIG. 9 shows various concentrations of Example 1 compared to cetuximab Fab fragment and / or cetuximab. The levels of C3b deposition on A431 cells using 3-17, 20, 22, 23, and 24 are shown. When using human serum, all experiments showed <5-fold change over background. was observed (representative HI HS data shown here).

[0321] (Phagocytosis of target cells by macrophages) Using phagocytosis, L (as Fab fragment of cetuximab) was directed to the receptor on the cell line. and F (a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody). The functional effect of binding of EGFR to A431 cells was clarified. Cells were used as target cells. Monocyte-derived macrophages were used as effector cells. Purified hIVIG was used as the source of anti-Gal antibodies. The measured increase in integrated intensity was Such an increase occurs due to phagocytosis of target cells. No significant difference was observed between the two groups (alone or in the presence of unconjugated Fab fragments).

[0322] Effector cells were differentiated in situ in 96-well plates (Corning 3603). Briefly, the leukoreduction system chamber Blood from each healthy donor stored in the National Health Service (ADD) is collected and distributed to the National Health Service (ADD). Peripheral blood mononuclear cells (PBMCs) were purchased from Addenbrooke's Hospital, Cambridge, UK. PBMCs) were cultured in the Lymphoprep™ system according to the manufacturer's instructions (STEMCELL Technologies 07861). Monocytes were isolated using the EasySep™ Human CD14 Positive Selection Kit II (S It was isolated from PBMCs by positive selection using TEMCELL Technologies 17858 and cultured at 100 ng / ml. ImmunoCult™ SF Macrophage Medium ( ) supplemented with recombinant human GM-CSF (Peprotech 300-03) The cells were resuspended in STEMCELL Technologies 10961 and placed in a cell culture incubator (5% CO2, 37°C). After 5 days of differentiation, macrophages were further cultured in a 1000-well plate at 20,000 cells / well in a 1000-well plate at 20°C. For 2 days, 100 ng / ml IFN-γ (Peprotech 300-02) and 1 ng / ml lipopolysaccharide (Invitrogen tlrl-eblp A431 cells (ATCC CRL-1555) were cultured in 10% fetal bovine serum (FBS) and 10% ethanol. Dulbecco's modified Eagle's medium (Gibco® 10500-064) supplemented with serum (Gibco® 10500-064) The target cells were cultured in cell dissociation buffer (Product No. 61965-026) and used as target cells. The cells were collected using Cell Dissociation Buffer (Gibco® 13151014), counted, and diluted to 1.0 x10 6 1 μl of 10 mM pHrodo Green STP ester (Thermo Fisher Scientific P353 69) for 30 min at 37°C. Cells were washed in complete medium, counted, and then incubated with various concentrations of The samples were treated with Example 23, Example 20, or cetuximab-Fab at room temperature for 1 hour with shaking. The cells were then washed in medium-free medium and treated with 70 μg / ml hIVIG (custom-made from human IVIG). Purified) and incubated on ice for 30 minutes, then diluted with Dulbecco's Phosphate Solution (Gibco® 14190-094 ) and incubated for up to 12 hours at 37°C in an IncuCyte® (Sartorius) for effector cell incubation ( The cells were co-cultured with 1000 cells (target:effector ratio 5:1). Images were acquired every 2 hours. Data were collected using Incu Analysis was performed using Cyte® ZOOM software (version 2016A, Sartorius). The graph was created using GraphPad Prism (version 6).

[0323] Figure 10 shows the results of 1 nM of Example 20 and Example 23 compared to unconjugated Fab fragments. Representative compound-mediated phagocytosis in the presence of target cells (EGFR-expressing A431 cells) is shown. The increase in integrated intensity indicates that the target It occurs due to compound-driven phagocytosis of target cells.

[0324] (Flow cytometry assay using α-galactosyl IgM antibody on Raji cells) Flow cytometry was used to measure the binding of L (rituximab or rituximab) to the receptor on human cell lines. ximab Fab), and F (a glycosylated antibody capable of binding to a human anti-α-galactosyl antibody The binding of CD20 receptors to Raji cells was revealed. The cells are used to capture CD20-binding mAb or Fab, as is well known. The binding of α-galactosyl IgM antibody to the compound was measured using PEG-labeled anti-human IgM antibody. Detected.

[0325] Raji cells (ATCC® CCL-86™) were harvested and cultured in phosphate-buffered saline (PBS) (Gibco 5 x 10 in Sigma-Aldrich (trademark) 14190-094) + 0.1% BSA (bovine serum albumin - Sigma A2153) 6 Re in cells / mL After that, 5×10 5 Cells were treated with various concentrations of compounds or Cells were incubated with buffer alone for 1 hour at room temperature with shaking at 450 rpm. The plate was washed with 2 x 200 μL PBS + 0.1% BSA, followed by 50 μL of 32 μg / mL of antibody in PBS + 0.1% BSA. Add α-galactosyl IgM antibody (Absolute Antibody Ab00532-15.0) and incubate at 4°C for 1 hour. The cells were further washed with 2 x 200 μL PBS + 0.1% BSA and then incubated with 100 μL of anti-HIV. The cells were treated with a 1:40 dilution of human IgM-PE (Biolegend 314508) for 1 hour at 4°C. 2 x 200 μL PB After a final wash of PBS+0.1% BSA, cells were resuspended in 200 μL PBS+0.1% BSA and run through a flow cytometer. Data from all samples were analyzed using a FlowJoint (BD FACSVerse™, BD). Graphs were generated using the GraphPad Prism (version 1.0) software package. 6) was used.

[0326] Figure 11 shows the binding of α-galactosyl IgM to Raji cells compared to rituximab and its Fab fragments. 1 shows dose-related compound-driven mobilization of antibodies.

Claims

【Claim 1】 A novel article, method, and manufacturing method substantially described in the specification of this application.

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