Novel Compounds and Their Therapeutic Use

Abstract

The present invention relates to novel compounds capable of linking the immune response to defined therapeutic targets, the use of said compounds in the treatment of cancer and infectious diseases, compositions containing said compounds, processes for their preparation, and novel intermediates used in said processes. [Selected Figure] Figure 1

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to novel compounds having the ability to link an immune response to a defined therapeutic target, the use of such compounds in the treatment of cancer and infectious diseases, compositions containing such compounds, processes for their preparation, and novel intermediates used in such processes.

Background Art

[0002] (Background of the Invention) There is a need to find new ways to mobilize an individual's immune system to fight disease. The human immune system constantly surveys the body for foreign signals in order to identify potentially harmful pathogens or mutated human cells (which can cause cancer growth), and targets them for elimination. There are natural antibodies that can be mobilized against such pathogens or mutated human cells to drive the immune system and eliminate the threat.

[0003] Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. In 2012, cancer occurred in approximately 14.1 million people, causing about 8.2 million deaths, which accounted for 14.6% of total human deaths. The most common cancer types in men are lung cancer, prostate cancer, colorectal cancer, and stomach cancer. The most common types in women are breast cancer, colorectal cancer, lung cancer, and cervical cancer. It is well established that the immune response plays a crucial role in the identification and elimination of cancerous cells. There are drugs that fight cancer by strengthening an individual's immune system to help in the battle against cancer. It is necessary to be able to better target the specific immune response against cancer cells and to generate a wider range of the patient's own tumor-associated antigens. Targeting pre-existing natural antibodies against the patient's own tumor meets this need. It is an urgent task to identify new methods for treating bacterial, viral, and fungal infections. Antimicrobial resistance has become a major global health threat. For example, in the United States, it is estimated that over 2 million people are infected each year with bacteria resistant to a certain class of antibiotics (U.S. Centers for Disease Control and Prevention, 2013).

[0004] A novel approach for treating an infection or cancer is disclosed in WO2005 / 079423, which describes an immune linker comprising two binding sites. The first binding site is capable of binding to an immune response component of an individual. The second binding site is capable of binding to any compound or foreign substance such as an antigen, pathogen, chemical, or an endogenous substance such as an altered cell found in cancer. The resulting action of the immune linker molecule is that the individual's pre-existing immune response is diverted to the target, i.e., cancer cells or a specific pathogen. Examples of the first binding site include compounds or substances that will be recognized as foreign by the individual's immune system and thus trigger an immune response.

[0005] Typical examples of the first binding site include the small molecule hapten dinitrophenyl (DNP), rhamnose, or β-1,6-glucan. Further examples of the first binding site are carbohydrate molecules capable of binding to the human serum antibody anti-α-galactosyl (i.e., galactosyl-α-1,3-galactosyl-β-1,4-N-acetylglucosamine; "anti-Gal").

[0006] Since the function of immunity depends on multivalency, anti-Gal recruitment will depend not only on the concentration of anti-Gal but also on the affinity of the antibody for the target.

[0007] Examples of the second binding site include antibodies or fragments thereof that bind to a specific target molecule. Further examples of the second binding site include established therapeutic antibodies or functional fragments thereof. In this way, the targeted cells or pathogens can be recognized as foreign by the immune system and marked for destruction. Thus, natural antibodies can be recruited against these tumor cells or pathogens and the immune system can be utilized to eliminate the threat.

[0008] WO98 / 34957 describes the stimulation of an immune response using antibodies labeled with α - galactosyl epitopes. The document describes embodiments in which the stimulation of the immune system depends on the incorporation of α - galactosyl epitopes into engineered glycosylation sites within the constant region of the antibody. The production of such appropriately engineered antibodies in cell lines expressing α - 1,3 - galactosyltransferase has been reported to result in the addition of α - galactosyl epitopes. In this case, the number of incorporated epitopes depended on the number of residues engineered for glycosylation. A disadvantage of this approach is the limitation on the number of α - galactosyl residues that can be introduced (limited by the number of site - specific amino acid modifications). A further disadvantage of the approach is that α - 1,3 - galactosyltransferase transports a population of carbohydrate derivatives, which may not be optimal for anti - galactosyl antibody mobilization and further limits the number of immunostimulatory epitopes. Therefore, a linker molecule is needed that allows for optimized loading and presentation of α - galactosyl epitopes to the antibody site to maximize the immune response.

[0009] Accordingly, there is a great need for a linker molecule that contains a spacer group optimized to control the number and position of a first binding site (i.e., a carbohydrate molecule capable of binding to a human anti - α - galactosyl antibody) relative to the position of a second binding site (i.e., an antibody or antigen - binding fragment). Such a linker molecule is designed to attract native antibodies so as to optimize the effectiveness of immunostimulation while minimizing possible side effects and is thus very useful in providing effective anti - cancer therapies and therapies against infectious agents. SUMMARY OF THE INVENTION

[0010] (Summary of the Invention) An immunoconjugate comprising an antibody or an antigen-binding fragment thereof linked via a linker to a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody, wherein the linker comprises at least one phenyl ring capable of presenting one or more carbohydrate epitopes capable of binding to a human anti-α-galactosyl antibody, is provided.

[0011] According to a second aspect of the present invention, there is provided an immunoconjugate which is a compound of formula (A) or a pharmaceutically acceptable salt thereof:

Chemical formula

[0012] According to a further aspect of the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof:

Chemical formula

Brief Description of the Drawings

[0013] (Brief Description of the Drawings)

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Mode for Carrying Out the Invention

[0014] (Detailed Description of the Invention) According to a first aspect of the present invention, there is provided an immunoconjugate comprising an antibody or an antigen-binding fragment thereof linked via a linker to a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody, wherein the linker comprises at least one phenyl ring capable of presenting one or more carbohydrate epitopes capable of binding to a human anti-α-galactosyl antibody.

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

[0016] According to a second aspect of the present invention, there is provided an immunoconjugate which is a compound of formula (A) or a pharmaceutically acceptable salt thereof:

Chemical Formula

[0017] According to a second aspect of the present invention that may be mentioned, there is provided an immunoconjugate which is a compound of formula (A) or a pharmaceutically acceptable salt thereof:

Chemical formula

[0018] According to a further aspect of the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof:

Chemical formula

[0019] According to a further aspect of the invention that may be mentioned, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof:

Chemical formula

[0020] In addition to conjugation to single or multiple sites on a selected antibody or fragment thereof, the present invention enables the ability to present one or more carbohydrate epitopes, and thus retains target binding efficacy while allowing optimal recruitment of native antibodies. The present invention provides that one of ordinary skill in the art can finely tune the optimal number of carbohydrates per selected antibody or fragment thereof for optimal anti-Gal recruitment and retain target efficacy.

[0021] Monoclonal antibodies have greatly improved the outcome of patients with cancer. However, certain patient populations exhibit intrinsic resistance to these therapies, and although good outcomes may be observed, these may not be long-lasting, and acquired resistance to mAb therapy remains a problem, and increased antibody efficacy is desired. Tumors can exhibit or develop mechanisms that result in resistance or decreased response to antibody therapy, such as through increased receptor expression, or changes in signaling pathways, or decreased immune response (Reslan, L., Mabs 2009, 3, 222). For example, patients may exhibit intrinsic resistance to cetuximab resulting from the expression of KRAS mutations that affect EGFR signaling (Lievre, A., J. Clin. Oncol. 2008, 26, 374). In addition, even if a patient initially responds well to cetuximab, most will ultimately acquire resistance (Bianco, R., Endocr. Relat. Cancer 2005, S159; Brand, TM., Cancer Biol. Ther. 2011, 11, 777).

[0022] Further examples of resistance to therapeutic antibodies are seen in cases of non-Hodgkin lymphoma patients treated with rituximab (anti-CD20 monoclonal antibody). Resistance to rituximab is observed in approximately half of treatment-naive patients. Patients who initially respond to rituximab therapy often acquire resistance. The mechanisms of resistance are complex, and strategies to overcome resistance have shown limited success in patients (Best Pract. Res. Clin. Haematol. 2011, 203-216), and thus, there is still a pressing need to improve the activity of therapeutic antibodies to increase and prolong patient responses.

[0023] Many approaches have been incorporated to improve the efficacy of therapeutic antibodies, including antibody-drug conjugates (ADCs), antibody-toxin conjugates (immunotoxins), and engineered antibodies with enhanced effector mechanisms, such as increased antibody-dependent cell cytotoxicity (ADCC). Despite these efforts, few therapies have achieved clinical success, and side effects such as toxicity remain an important issue (Beck, A.: Nat. Rev. Drug. Discov. 2017). Therefore, new strategies are needed to modify mAbs to enhance efficacy and improve patient outcomes.

[0024] The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) is a unique glycoprotein that is naturally produced on glycolipids and glycoproteins and presents multiple epitopes on branched oligosaccharides (J. Immunology (2007), 178 (7), 4676-87). This α-Gal epitope is synthesized, for example, by α1,3-GT, a transferase that is a well-known enzyme that catalyzes the synthesis of Galα-1,3-Gal at multiple glycosylation sites (WO98 / 34957). Although this method is efficient, this synthesis relies on existing or engineered glycosylation sites and only allows for one α-Gal unit per conjugation site.

[0025] Thus, there is an interesting need for a modular approach suitable for the presentation of any one or more of the α-Gal trisaccharide units that optimally utilize the innate immune system.

[0026] The compounds of the present invention include a linker molecule optimized to control and present the number and position of F groups (i.e., carbohydrate molecules capable of binding to human anti-α-galactosyl antibodies) relative to the position of the binding site L (i.e., an antibody or a fragment thereof). For example, a rigid cyclic group has the advantage of providing a scaffold for the optimal placement of one or more F groups relative to L. It will be understood that the exact number and orientation of the F groups relative to L will vary depending on the nature of the L group. Furthermore, the presence of a cyclic group containing a single phenyl ring, biphenyl ring, or triphenyl ring provides the significant advantage of presenting multiple F groups (i.e., carbohydrate molecules capable of binding to human anti-α-galactosyl antibodies) to enhance the immune response arising from the host. The chemical presentation of multiple binding groups has been known in the art heretofore, which is in contrast to the present invention which uses any one of a single six-membered ring system (i.e., phenyl), two six-membered ring systems (biphenyl) linked by a bond, or three six-membered ring systems (triphenyl) linked by two bonds, and is achieved using one or more amino acid groups (see, for example, WO2014 / 178878) or branched linker groups (see, for example, US2014 / 0112975). The technical effect of this difference is that the compounds of the present invention can be prepared more readily than linkers previously known in the art and, advantageously, can avoid the presence of chiral centers and are more resistant to protease degradation. The synthesis of the compounds of the present invention also offers the advantage of not using resins and thus being suitable for scale-up for large-scale pharmaceutical manufacture. Thus, the compounds of the present invention not only are simply therapeutically effective but also offer the advantages of enhancing the immune response from the host and an easy and efficient synthesis in high yield that can be scaled up and down. In addition, the linker of the present invention is not labile and thus typically does not contain a "cleavable linker" component as required by many compounds previously known in the art (see, for example, US8,828,956).Furthermore, the linker of the present invention enables one of ordinary skill in the art to select a particular left- and right-handed combination of groups, along with ease and efficiency of synthesis, to enable an optimal number of F groups per antibody or antibody fragment conjugation site.

[0027] Monoclonal antibody fragments such as Fab, Fab’, Fab’2, Fab2, Fab3, F(ab)2, Fv, scFv, diabody, triabody, tetrabody, nanobody, etc. are also known in the field of oncology. Several advantages over full-length mAbs have been reported, including increased tumor permeability resulting from their smaller size, easier manufacture (they can be expressed in Escherichia coli (E. coli) or yeast, resulting in increased convenience and more efficient scale-up), and reduced immunogenicity. However, fragments such as scFvs or Fab fragments may have reduced efficacy when compared to full-length mAbs. For example, the lack of an Fc domain on a Fab fragment often eliminates the potential for efficacy driven by ADCC, which is a component of the anti-tumor response (Nelson, A. L., mAbs 2009, 2, 77).

[0028] Thus, the benefits of antibody fragments are often offset by the loss of functions associated with them. The use of the phenyl-containing linker of the present invention to provide a new construct conjugated with an optimal number of α-Gal sites provides a new approach to enhancing the anti-tumor action of antibody fragments.

[0029] For both mAbs and their fragments, controlled site-specific conjugation, for example by incorporation of additional cysteine residues, is well known in the art. Such an approach offers several advantages, in particular by enabling derivatization of the mAb or fragment without disrupting the target-binding epitope and by allowing the loading to be controlled, i.e., a certain stoichiometry is achieved (Shen, B. Q., Nat. Biotechnol. 2012, 30, 184). One limitation of site-specific conjugation is that low loading of the conjugated site can result, which may limit efficacy. The use of the phenyl-containing linker of the present invention that allows presentation of multiple α-Gal sites per conjugation site provides a way to achieve a high α-Gal / antibody ratio (high loading), even when the number of conjugation sites is low.

[0030] (Definition of linker) In one embodiment, S1 is: - One to five (e.g., two, three, or five) of the -CH2- groups may be optionally substituted by one or more groups selected from -S-, =N(H)-, -C(=O)-, -NHC(O)-, cyclohexyl, or pyrrolidin-2,5-dione, -(CH2) a - (e.g., -(CH2)2-NHCO-cyclohexyl-CH2-3-pyrrolidin-2,5-dione-, -(CH2)2-NHCO-cyclohexyl-CH2-3-pyrrolidin-2,5-dione-S-(CH2)3-C(=NH)-, or -(CH2)2-NHCO-(CH2)3-CO, etc.); or - One to five (e.g., two) of the -CH2- groups may be optionally substituted by one or more groups selected from -NHC(O)- or pyrrolidin-2,5-dione, -(CH2) b -(CH2-CH2-O) c -(CH2) d - (e.g., -(CH2)2-NHCO-(CH2CH2O)4-(CH2)2-3-pyrrolidin-2,5-dione-, etc.) represents a spacer selected from

[0031] In a further embodiment, S1 is: - one or more of 1 to 10 of the -CH2- groups may be optionally substituted by one or more groups selected from -O-, -S-, =N(H)-, -C(O)NH-, -NHC(O)-, cyclohexyl, or pyrrolidin-2,5-dione, -(CH2) a - (e.g., -(CH2)2-NHCO-cyclohexyl-CH2-pyrrolidin-2,5-dione-, or -(CH2)2-NHCO-cyclohexyl-CH2-pyrrolidin-2,5-dione-S-(CH2)3-C(=NH)-, etc.) represents a spacer selected from

[0032] It will be understood that a, b, c, d, e, f, g, and h are selected to maintain a suitable linker length between groups F and L. Examples of suitable linker lengths between F and L are lengths in the range of about 5 Å to about 50 Å or more, 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 to about 25 Å, about 11 Å to about 20 Å, about 12 Å to about 15 Å. Thus, in one embodiment, a, b, c, d, e, f, g, and h represent a total integer not exceeding 45, such as 5 to 45, such as 7 to 42, such as 30 or less, such as 5 to 30, such as 7 to 29.

[0033] In one embodiment, a represents an integer selected from 1 to 30. In a further embodiment, a represents an integer selected from 2 to 30. In a further embodiment, a represents an integer selected from 2, 4, 6, 9, 11, 18, or 30. In a further embodiment, a represents 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 a further embodiment, a represents an integer selected from 6 to 11. In a further embodiment, a represents an integer selected from 6, 7, or 11. In yet a further embodiment, a represents an integer selected from 6. In an alternative embodiment, a represents an integer selected from 7. In an alternative embodiment, a represents an integer selected from 11.

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

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

[0036] In one embodiment, d represents an integer selected from 1 to 15. In a further embodiment, d represents an integer selected from 2 to 13. In a further embodiment, d represents an integer selected from 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-. In a further embodiment, Y1 represents -C(O)NH-.

[0038] In one embodiment, S2 is -(CH2)- wherein one or two of the -CH2- groups are optionally substituted by one or two groups selected from -N(H)-, -C(O)NH- and -NHC(O)- e -(e.g., -(CH2)3-NHCO-CH2-, -(CH2)3-, -(CH2)3-NHCO-(CH2)4-CONH-CH2-, -(CH2)3-NH-CH2- or -(CH2)3-NHCO-(CH2)3-NHCO-CH2- etc.); or -(CH2)- wherein one to three of the -CH2- groups are optionally substituted by one to three -NHC(O)- groups f -(CH2-CH2-O) g -(CH2) h -(e.g., -(CH2)3-NHCO-(CH2CH2O)4-(CH2)2-NHCO-CH2-, -(CH2)3-NHCO-(CH2CH2O) 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: -(CH2)- wherein one or two of the -CH2- groups are optionally substituted by -C(O)NH- or -NHC(O)- groups e -(e.g., -(CH2)3-NHCO-CH2-, -(CH2)3-NHCO-, -(CH2)3-, -(CH2)3-NHCO-(CH2)4-CONH-CH2- or -(CH2)3-NH-CH2- etc.); or -(CH2)- wherein one or two of the -CH2- groups are optionally substituted by -C(O)NH- or -NHC(O)- groups 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 yet another embodiment, S2 is: -(CH2)- wherein one or two of the -CH2- groups are optionally substituted by one or two -NHC(O)- groups e -(e.g., -(CH2)3-NHCO-CH2- or -(CH2)3-NHCO-(CH2)3-NHCO-CH2-, etc.); or -(CH2)- wherein one to three of the -CH2- groups are optionally substituted by one to three -NHC(O)- groups f -(CH2-CH2-O) g -(CH2) h -(e.g., -(CH2)3-NHCO-(CH2CH2O)4-(CH2)2-NHCO-CH2-, -(CH2)3-NHCO-(CH2CH2O) 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 another embodiment, S2 is: -(CH2)- wherein one or two of the -CH2- groups are optionally substituted by one or two groups selected from -N(H)-, -C(O)NH-, and -NHC(O)- e -(e.g., -(CH2)3-NHCO-CH2-, etc.); or -(CH2)- wherein one to three of the -CH2- groups are optionally substituted by one to three -NHC(O)- groups 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 yet a further embodiment, S2 is: one or two of the -CH2- groups, for example, one is optionally substituted by one or two, for example, one group selected from -N(H)-, -C(O)NH-, and -NHC(O)-, -(CH2) e -(for example, -(CH2)3-NHCO-CH2- etc.) represents a spacer selected from

[0043] In yet a further embodiment, S2 is: one of the -CH2- groups is optionally substituted by an -NHC(O)- group, -(CH2) e -(for example, -(CH2)3-NHCO-CH2- etc.); or two of the -CH2- groups are optionally substituted by -NHC(O)- groups, -(CH2) f -(CH2-CH2-O) g -(CH2) h -(for example, -(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. In a further embodiment, e represents an integer selected from 3 to 10. In a further embodiment, e represents an integer selected from 3, 5, 9, or 10. 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 a further embodiment, e represents an integer selected from 4 to 10. In yet a further embodiment, e represents an integer selected from 4, 5, or 10. In yet another further embodiment, e represents an integer selected from 5.

[0045] In one embodiment, f represents an integer selected from 1 to 8. In a further embodiment, f represents an integer selected from 2 to 8. In a further embodiment, f represents an integer selected from 2 to 6. In yet a further embodiment, f represents an integer selected from 4 to 8. In yet a further embodiment, f represents an integer selected from 4 or 8. In still a further 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 4 or 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. In yet a further embodiment, 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)-. In a further embodiment, Y2 represents a bond or -O-. In yet a further embodiment, Y2 represents -O-.

[0049] In one embodiment, m represents an integer selected from 1 to 4. In a further embodiment, m represents an integer selected from 1 to 3. In a further embodiment, m represents an integer selected from 1 or 3. In yet a further embodiment, m represents an integer selected from 2 or 3. In yet a further embodiment, m represents an integer selected from 1 or 2. In yet a further embodiment, m represents an integer selected from 1. In yet a further embodiment, m represents an integer selected from 2. In yet a further embodiment, m represents an integer selected from 3. In yet a further embodiment, 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 1 to 20. In a further embodiment, z represents an integer selected from 2 to 20 (for example, 2, 4.9, 5, 7, 8, 10, 11, 14, 15, 17, or 20). In a further embodiment, z represents an integer selected from 1 to 8. In a further embodiment, z represents an integer selected from 1 to 5. In yet a further embodiment, z represents an integer selected from 2 to 5. In yet a further embodiment, z represents an integer selected from 2 or 5. In yet a further embodiment, z represents an integer selected from 2. In yet a further embodiment, z represents an integer selected from 5.

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

[0052] According to a further aspect of the present invention, a compound of formula (I) a or a pharmaceutically acceptable salt thereof is provided:

Chemical formula

[0053] According to a further aspect of the invention that may be mentioned, a compound of formula (I) a or a pharmaceutically acceptable salt thereof is provided:

Chemical formula

[0054] In a further embodiment, the present invention provides a compound of formula (I) comprising a compound of Examples 1 to 26 or a pharmaceutically acceptable salt thereof.

[0055] In a further embodiment, the present invention provides a compound of formula (I) which is the free base of a compound of Examples 1 to 26.

[0056] In a further embodiment, the present invention provides a compound of formula (I) comprising a compound of Examples 1-8 or a pharmaceutically acceptable salt thereof.

[0057] In a further embodiment, the present invention provides a compound of formula (I) which is the free base of the compound of Examples 1-8.

[0058] (αGal) As used herein, reference to a "carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody" includes a sugar (i.e., carbohydrate) moiety that binds to an immune response component of the human (i.e., an anti-α-galactosyl antibody) and is capable of consequently inducing an immune response in a human. In one embodiment, the anti-α-galactosyl antibody is an anti-α-galactosyl IgG antibody or an anti-α-galactosyl IgM antibody. Examples of such carbohydrate molecules are α-galactosyl compounds and their modified derivatives. Further examples of suitable carbohydrate molecules are the α-gal epitopes listed in US2012 / 0003251, which are suitable for use in the selective targeting and killing of tumor cells, and these epitopes are incorporated herein by reference. In one embodiment, F is selected from galactosyl-α-1,3-galactosyl-β-1,4-N-acetylglucosamine, α1-3 galactobiose, α1-3-β1-4 galactotriose or galilipentasaccharide.

[0059] In one particular embodiment, F has the structure shown in one of the following formulas:

Chemical formula

[0060] In one particular embodiment, F has the structure shown in the following formula:

Chemical formula

[0061] (Antibodies and their antigen-binding fragments) References herein to the term "antibody(ies)" refer to a molecule or an active fragment of a molecule that binds to a known antigen, particularly, an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule containing a binding site that immunospecifically binds to an antigen. The immunoglobulins according to the present invention can be of any class (IgG, IgM, IgD, IgE, IgA, and IgY), subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or isotype (e.g., IgG in IgG1, IgG2, IgG3, and IgG4, or IgA in IgA1 and IgA2) of an immunoglobulin molecule.

[0062] Within the scope of the present invention, the term "antibody(ies)" includes monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-chain antibodies, bispecific antibodies, human antibodies, and humanized antibodies, as well as active fragments thereof. Examples of active fragments of molecules that bind to a known antigen include Fab, F(ab′)2, scFv, and Fv fragments, including products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the antibodies and fragments mentioned above.

[0063] As used herein, the term "monoclonal antibody" refers to an antibody that is mass-produced from a single clone in the laboratory and recognizes only one antigen. Monoclonal antibodies are typically produced by fusing antibody-producing B cells, which are usually short-lived, to rapidly proliferating cells such as cancer cells (sometimes referred to as "immortal" cells). The resulting hybrid cells, or hybridomas, proliferate rapidly and give rise to clones that produce large amounts of antibody. For the purposes of the present invention, the term "monoclonal antibody" is also understood to include antibodies produced by a mother clone that has not yet achieved complete monoclonality.

[0064] As used herein, the term "chimeric antibody" generally refers to a monoclonal antibody prepared by recombinant DNA techniques, comprising a variable region, i.e., a binding region, derived from a mouse and at least a part of a constant region obtained from a different origin or species. A chimeric antibody comprising a mouse variable region and a human constant region is an exemplary embodiment. Such a mouse / human chimeric antibody is the product of an expressed immunoglobulin gene comprising a DNA segment encoding a mouse immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Another form of "chimeric antibody" encompassed by the present disclosure is one in which the class or subclass has been modified or changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "class switch antibodies". Methods for producing chimeric antibodies include conventional recombinant DNA techniques and gene transfection techniques well known in the art currently. See, for example, Morrison, S. L. et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Patent No. 5,202,238 and U.S. Patent No. 5,204,244.

[0065] As used herein, the terms "humanized antibody" or "humanized version of an antibody" refer to an antibody that has been modified such that the framework or "complementary determining regions" (CDRs) contain CDRs of an immunoglobulin of a different specificity compared to those of the parental immunoglobulin. In one exemplary embodiment, the CDRs of VH and VL are grafted into the framework regions of a human antibody to prepare a "humanized antibody". See, for example, Riechmann, L. et al., Nature 332 (1988) 323-327; and Neuberger, M. S. et al., Nature 314 (1985) 268-270. The variable framework regions of the heavy and light chains can be obtained from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies. The human heavy and light chain variable framework regions are listed, for example, in Lefranc, M.-P., Current Protocols in Immunology (2000)-Appendix 1P A.1P.1-A.1P.37 and are available, for example, via IMGT, the international ImMunoGeneTics information System (registered trademark) (http: / / imgt.cines.fr) or http: / / vbase.mrc-cpe.cam.ac.uk. Optionally, the framework regions can be modified by further mutations. Exemplary CDRs correspond to those that represent sequences that recognize the antigens described above for chimeric antibodies. In one embodiment, such humanized versions are chimerized with human constant regions. The term "humanized antibody" as used herein also includes such antibodies that are modified in the constant region, particularly with respect to C1q binding and / or FcR binding, by, for example, "class switching", i.e., changes or mutations in the Fc portion (e.g., from IgG1 to IgG4 and / or IgG1 / IgG4 mutations), to give rise to the properties according to the present disclosure.

[0066] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions obtained from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, M. A. and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing the entire repertoire of human antibodies or selected ones thereof upon immunization in the absence of endogenous immunoglobulin production. Introduction of the human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen exposure (see, e.g., Jakobovits, A. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A. et al., Nature 362 (1993) 255-258; Brueggemann, M. D. 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). The techniques of Cole, A. et al. and Boerner, P. et al. are also available for the preparation of human monoclonal antibodies (Cole, A. et al., Monoclonal Antibodies and Cancer Therapy, Liss, A. R. (1985) p. 77; and Boerner, P. et al., J. Immunol. 147 (1991) 86-95).As already mentioned, according to the present disclosure, the term "human antibody" as used herein also includes, for example, such antibodies that are modified in the constant region by, for example, "class switch", i.e., changes or mutations in the Fc portion (e.g., from IgG1 to IgG4 and / or IgG1 / IgG4 mutations), such that the properties according to the present disclosure occur with respect to, for example, C1q binding and / or FcR binding.

[0067] As used herein, the term "single-chain antibody" refers to a single-chain Fv molecule (scFv) (Bird et al., 1988, Science 242: 423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 5879-5883) in which the VH domain and the VL domain are linked by a peptide linker that enables the two domains to cooperate to form an antigen-binding site, or a bispecific single-chain Fv (WO03 / 11161).

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

[0069] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, e.g., optionally, its variable domain, or at least its antigen-binding site. Examples of antibody fragments include diabodies formed from antibody fragments, single-chain antibody molecules, and multispecific antibodies. ScFv antibodies are described, for example, in Huston, J. S., Methods in Enzymol. 203 (1991) 46-88. Antibody fragments can be derived from the antibodies of the present invention by several known techniques. For example, a purified monoclonal antibody can be cleaved with an enzyme such as pepsin and then subjected to HPLC gel filtration. Thereafter, appropriate fractions containing the Fab fragment are collected and concentrated by, for example, membrane filtration. For further explanation of common techniques for the isolation of active fragments of antibodies, see, for example, Khaw, B. A. et al., J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al., Methods Enzymology, 121:663-69, Academic Press, 1986.

[0070] As used herein, the terms "specific" and "specifically" are used interchangeably to indicate that other biomolecules do not significantly bind to an antibody that specifically binds to a target biomolecule. In certain embodiments, the level of binding to biomolecules other than the peptide containing the epitope within the peptide results in a binding affinity that is negligible (e.g., undetectable) by ELISA or affinity determination.

[0071] "Negligible binding" means at least about 85%, specifically at least about 90%, more specifically at least about 95%, even more specifically at least about 98%, but especially at least about 99% and up to 100% less binding than binding to the peptide containing the epitope within the peptide.

[0072] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen such as a protein) to which an antigen-binding molecule (e.g., an antibody or antibody fragment) binds. An epitope can be formed from both contiguous or adjacent non-contiguous residues (e.g., amino acid residues) of the target molecule. An epitope formed from contiguous residues (e.g., amino acid residues) is usually also referred to as a linear epitope. An epitope typically contains at least 5 and up to about 12 residues, often 6 to 10 residues (e.g., amino acid residues).

[0073] As used herein, the term "CDR" refers to the hypervariable region of an antibody. The terms "hypervariable region", "HVR", or "HV", as used herein, refer to regions of an antibody variable domain where the sequences are hypervariable and / or form structurally defined loops. Generally, an antibody contains six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Descriptions of several hypervariable regions are used and are included herein. The Kabat complementarity determining regions are based on sequence variability and are the most commonly used (Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The letters "HC" and "LC" before the term "CDR" refer to the CDRs of the heavy chain and light chain, respectively.

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

[0075] To determine the percent (%) identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, 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% of the length of the reference sequence. Thereafter, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, "identity" of an amino acid or nucleic acid is equivalent to "homology" of an amino acid or nucleic acid). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0076] The comparison of sequences between two arrays and the determination of percent identity can be achieved using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined using either the BLOSUM 62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6, using the algorithm of Needleman et al. (1970) J. Mol. Biol. 48:444-453 incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com). In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6, using the GAP program of the GCG software package (available at http: / / www.gcg.com). A particularly preferred set of parameters (and those that practitioners should use when unsure which parameters to apply to determine whether a molecule is within the scope of the sequence identity or homology limitations of the present invention) is the BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0077] Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17 incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

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

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

[0080] In one embodiment, the antigen-binding fragment thereof is an antigen-binding fragment (Fab) or a single-chain variable fragment (scFv). In a further embodiment, the fragment is selected from the group consisting of Fab, Fab′, F(ab)2, F(ab′)2, and scFv. It will be recognized that X1 can represent any suitable antibody or antigen-binding fragment attachment site and that the choice of such group will depend on the amino acid residue selected as the attachment point within the antibody.

[0081] In one embodiment, X1 represents -S- or -N(H)-. In this embodiment, L is conjugated to the compound of formula (I) by one of the following three structures:

Chemical formula

[0082] It will be appreciated that the antibody or antigen-binding fragment of the present invention will be configured to bind to a therapeutic target that is either a cancer cell or a specific pathogen.

[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 specifically binds to a tumor-associated antigen whose cell surface expression on tumor cells is different from its expression on healthy cells.

[0084] In a preferred embodiment, the antibody or antigen-binding fragment binds to a target on cancer cells or pathogens. Preferred targets include: EGFR, HER2, HER3, CD22, EpCAM, PSMA, PSCA, FLT-3, CD30, CD20, CD33, CD23, CD2, CD37, CD25, CD73, CD47, LGR-5, CD80, CD86, CD70, CD74, CD40, CD19, CD79b, CA-125, c-met, CXCR4, DR5, PD-1, PD1L, LeY, MUC1, MUC2, MUC3, MUC4, MUC5ac, MIP-1A, MIP-1B, KIT, TRAIL receptors (R1 and R2), CXCR4, CEACAM, IGF-1R, carbonic anhydrase IX, PDGFRa, CD137, CD276, mesothelin, VEGFR, P-cadherin, CD56, bacterial Psl, bacterial lipopolysaccharide, the galactan-III epitope of bacterial LPS, bacterial PcrV, RSV F protein.

[0085] (Anti-EGFR antibody) In a further embodiment, the antibody or antigen-binding fragment is an epidermal growth factor receptor (EGFR)-binding epitope. EGFR is well known to be overexpressed in several types of human cancers. High expression on certain cancer cells makes EGFR an attractive target for new therapies. In one embodiment, this EGFR-binding antibody or antigen-binding fragment is an epitope that binds to any of the EGFR subfamily selected from EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). Examples of suitable EGFR-binding antibodies include, but are not limited to, cetuximab, nimotuzumab, matuzumab, zalutumumab, and panitumumab.

Table 1

[0086] In a further embodiment, the EGFR antibody is cetuximab, a hybrid mouse / human chimeric antibody comprising both the heavy and light chain sequences disclosed in (a) the literature of Li et al.: (2005) Cancer Cell 7, 301-11; (b) the literature of Dubois et al.: (2008) Anal. Chem. 80, 1737-45; and (c) the IMGT database available at www.imgt.org; www.drugbank.ca, or WO2016 / 196682.

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

[0088] In a further embodiment, a fragment of the EGFR-binding antibody may be selected as an example of L. Typical examples include cetuximab Fab (the literature of Li, S. et al.: (2005) Cancer Cell 7, 301-311); cetuximab scFv (US7,060,808); panitumumab Fab (the literature of Sickmier E. A. et al.: (2016) PLoSOne 11, 9, e0163366); panitumumab scFv (US6,235,883), and D2C7 scFv (US 2013 / 0022598; Clin Cancer Res 2013, 19(17), 4717-4727), but are not limited thereto.

[0089] (anti-CD20 antibody) In a further embodiment, the antibody or antigen-binding fragment is a CD20-binding epitope. It is well known that the removal of CD20-expressing cells (e.g., B cells) has therapeutic benefits in the treatment of blood cancers such as leukemia and lymphoma. Examples of suitable CD20-binding antibodies include, but are not limited to, rituximab, ofatumumab, obinutuzumab, and ocrelizumab.

Table 2

[0090] In a further embodiment, the CD20 antibody is rituximab, a hybrid mouse / human chimeric antibody that includes both the heavy and light chain sequences disclosed in (a) US 5,736,137, (b) Wang, B. et al. (2013) Analyst 138, 3058-3065, and (c) the IMGT database available at www.imgt.org; www.drugbank.ca.

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

[0092] In a further embodiment, a fragment of the CD20-binding antibody may be selected as an example of L. Typical examples include, but are not limited to, rituximab Fab (Du, J. et al. (2007) J. Biological Chem. 282, 15073-15080).

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

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

[0095] (Conjugate) Any antibody or fragment thereof disclosed herein can be conjugated to a cyclic spacer linker described herein having one or more α-Gal. The present invention relates to the ability to select the optimal number of α-Gal units per conjugation site per linker while retaining the effectiveness of the antibody or fragment thereof. The type of conjugation can be selected by one of ordinary skill in the art to allow for the highest yield and purity of the isolated material based on the functionality of the ends of the linker and the surface reactivity or selected reactive sites of the antibody or fragment thereof. In one embodiment, conjugation is readily accomplished by the addition of a thiol to a maleimide site. Alternatively, conjugation can be readily accomplished by amide bond formation between an amino group and an activated ester such as an NHS ester.

[0096] It should be understood that when the conjugate contains several reactive sites leading to multiple conjugation reactions, the above ranges may refer to the actual or average number of linker molecules per antibody or fragment thereof.

[0097] As used herein, the term "LAR" (Linker:Antibody Ratio) refers to the actual or average number of linker molecules successfully conjugated to an antibody or fragment thereof. LAR is equivalent to an integer value defined herein as "z". Various methods for determining linker loading are known in the art. In one embodiment, LAR is determined by the reaction of Mal-vc-PAB-MMAE as an alternative payload of the linker and analyzed using HIC.

[0098] (Properties of the conjugate) In any of the various embodiments described herein, the antibody or fragment thereof will be able to retain its binding efficacy to its target while allowing for optimal mobilization of anti-Gal. In one embodiment, cetuximab retains its ability to bind to EGFR while conjugated to up to an average of 5, for example up to 20, particularly up to 30 linker molecules.

[0099] In one embodiment, the conjugate of the present invention has the following properties: a) Conjugation of the linker to the antibody or fragment thereof should not significantly alter the binding efficacy to the target compared to the unconjugated counterpart; b) Conjugation of the linker containing α-Gal can allow for high levels of LAR without concomitant high levels of aggregation due to the hydrophilic nature of the oligosaccharide; c) Addition of the linker containing α-Gal can allow the antibody or fragment thereof to be more stable; d) Preference for heavy or light chain conjugation depending on the selected linker and / or conjugation method; e) Conjugation of one selected linker containing a high multiplicity of α-Gal units can exhibit optimal properties compared to several conjugation sites using α-Gal of equal loading; f) Conjugation of the linker to the antibody or fragment thereof can lead to an enhanced pharmacokinetic profile may indicate one or more of them.

[0100] (Salts and their derivatives) References to the compounds of formula (I) and subgroups thereof also include, for example, their ionic forms, salts, solvates, isomers (including geometric and stereochemical isomers), tautomers, N-oxides, esters, isotopes and protected forms, as described hereinafter; preferably, their salts or tautomers or isomers or N-oxides or solvates; and more preferably, their salts or tautomers or N-oxides or solvates, even more preferably, their salts or tautomers or solvates. Hereinafter, compounds as defined in any aspect of the present invention, as well as their ionic forms, salts, solvates, isomers (including geometric and stereochemical isomers), tautomers, N-oxides, esters, isotopes and protected forms (excluding intermediate compounds during chemical processes) are referred to as "compounds of the present invention".

[0101] The compounds of formula (I) can exist in the form of salts, for example, acid addition salts, or in certain cases salts with organic and inorganic bases such as carboxylates, sulfonates and phosphates. All such salts are within the scope of the present invention, and references to the compounds of formula (I) include the salt forms of these compounds. In one embodiment, the compounds of formula (I) exist as phosphates.

[0102] The salts of the present invention can be synthesized from the parent compounds containing basic moieties by conventional chemical methods, such as those described in "Pharmaceutical Salts: Properties, Selection, and Use", P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reaction of the basic forms of these compounds with a suitable base or acid in water or an organic solvent, or a mixture of the two; generally, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is used.

[0103] Acid addition salts (mono- or double salts) may be formed with a variety of both inorganic and organic acids. Examples of acid addition salts are acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+) camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic 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), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-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, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic 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, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid, and single or double salts formed with acids selected from the group consisting of acylated amino acids and cation exchange resins.

[0104] One specific group of salts consists of salts formed from 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, naphthalenesulfonic acid, valeric acid, acetic acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactobionic acid. One specific salt is the hydrochloride. Another specific salt is the hydrogen sulfate, also known as the hemisulfate.

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

[0106] When the compounds of formula (I) contain amine functional groups, these can form quaternary ammonium salts by reaction with alkylating agents, for example, according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of formula (I).

[0107] The compounds of the present invention may exist as mono- or poly-salts depending on the pKa of the original acid from which the salts are formed.

[0108] The salt forms of the compounds of the present invention are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are described in Berge et al., 1977, "Pharmaceutically Acceptable Salts", J. Pharm. Sci., Vol. 66, pp. 1-19. However, salts that are not pharmaceutically acceptable can also be prepared as intermediate forms that can subsequently be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salt forms, which may be useful, for example, in the purification or isolation of the compounds of the present invention, also form part of the present invention.

[0109] Those skilled in the art of organic chemistry will understand that many organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are known as "solvates". For example, complexes with water are known as "hydrates". Pharmaceutically acceptable solvates of the compounds of the present invention are within the scope of the present invention.

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

[0111] When a compound contains several amine functional groups, one or more nitrogen atoms may be oxidized to form N-oxides. Specific examples of N-oxides are N-oxides of the nitrogen atoms of tertiary amines or nitrogen-containing heterocycles.

[0112] N-oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid), see for example, "Advanced Organic Chemistry by Jerry March", 4th Edition, pages of Wiley Interscience. More specifically, N-oxides can be generated by the procedure of L. W. Deady (Syn. Comm. 1977, 7, 509 - 514), where the amine compound is reacted with m-chloroperoxybenzoic acid (mCPBA) in an inert solvent such as dichloromethane.

[0113] Certain protected derivatives of the compounds of formula (I) that can be generated prior to the final deprotection step are themselves not pharmaceutically active but, in certain cases, can be administered orally or parenterally and then metabolized in vivo to form the compounds of the present invention that are pharmacologically active, as will be understood by those skilled in the art. Accordingly, such derivatives are described as "prodrugs". All such prodrugs of the compounds of the present invention are included within the scope of the present invention. Examples of prodrug functionalities suitable for the compounds of the present invention are described in Drugs of Today, Volume 19, Number 9, 1983, pages 499-538, and "Topics in Chemistry", Chapter 31, pages 306-316, and in "Design of Produrugs" by H. Bundgaard, Elsevier, 1985, Chapter 1 (the disclosures described therein are incorporated herein by reference). For example, as described by H. Bundgaard in "Design of Produrugs" (the disclosure described therein is incorporated herein by reference), certain sites known to those skilled in the art as "pro-moieties" may be placed on suitable functional groups when such functional groups are present within the compounds of the present invention, as will be further understood by those skilled in the art.

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

[0115] The compounds of formula (I) may exist in numerous different geometric isomeric and tautomeric forms, and reference to the compounds of formula (I) includes all such forms. To avoid uncertainty, if a compound can exist as one of several geometric isomers or tautomers and only one is specifically described or shown, nevertheless all others are encompassed by formula (I).

[0116] The present invention includes isotopically labeled compounds of the invention that are acceptable as pharmaceuticals, i.e., compounds of formula (I) in which one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the atomic mass or mass number normally found in nature.

[0117] Examples of isotopes suitable for inclusion in the compounds of the present invention are isotopes of hydrogen such as 2 H(D) and 3 H(T), isotopes of carbon such as 11 C, 13 C and 14 C, isotopes of fluorine such as 18 F, isotopes of nitrogen such as 13 N and 15 N, isotopes of oxygen such as 15 O, 17 O and 18 O, and the like.

[0118] Certain isotopically labeled compounds of formula (I), such as those incorporating radioisotopes, are useful in tissue distribution studies of drugs and / or substrates. The compounds of formula (I) can also have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between the labeled compounds and other molecules, peptides, proteins, enzymes or receptors. The methods of detection or identification can use compounds labeled with labeling substances such as radioisotopes, enzymes, fluorescent substances, chemiluminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin and luciferase). The radioisotopes tritium, i.e., 3 H(T), and carbon-14, i.e., 14 C are particularly useful for this purpose in view of their ease of incorporation and rapid means of detection.

[0119] Deuterium, i.e., 2Substitution with heavier isotopes, such as H(D), may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or decreased required dose, and may thus be preferred in some situations.

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

[0121] Isotope-labelled compounds of formula (I) can generally be prepared by processes analogous to those described in the accompanying examples and preparations, either by conventional techniques known to those skilled in the art or by using preferably isotope-labelled reagents in place of the unlabelled reagents used previously.

[0122] (Process for preparing the compounds of formula (I)) In this section, unless the context otherwise indicates, as in all other sections of this application, reference to formula (I) also includes all other subgroups and their examples as defined herein.

[0123] The compounds according to the invention described herein may be prepared in a stepwise synthetic sequence as illustrated in the following "Processes and Schemes". These syntheses involve the preparation of various central structures, which can then allow for the selection of the branching and length of the linker connecting the two binding sites. Compounds of formula (I) can be prepared according to synthetic methods well known to those skilled in the art. For example, those skilled in the art will understand that the chemical steps and the choice of protecting groups may be controlled in any order for the synthesis to be successful.

[0124] In one embodiment, the compound of formula (A) is a linker (S) comprising one or more linker components Bmay include. Exemplary linker components include, but are not limited to, 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (vc), alanine-phenylalanine (ala-phe), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), and 4-(n-maleimidomethyl) cyclohexane 1-carboxylate (SMCC).

[0125] In certain embodiments, the compound of formula (A) may include a linker (S B ) that is capable of reacting with a free thiol on the antibody to form a covalent bond. The compounds of the present invention expressly contemplate, but are not limited to, antibody conjugates prepared using the following linker reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB. Other functional groups other than pyrrolidine-2,5-dione (maleimide) that are reactive with thiol groups on the antibody include, but are not limited to, iodoacetamide, bromocetamide, vinylpyridine disulfide, pyridyl disulfide, isocyanate, isothiocyanate, activated ester, halogenated sulfonyl, and acid chloride.

[0126] In certain embodiments, the linker has a functional group capable of reacting with an electrophilic group on the antibody. Exemplary electrophilic groups include, but are not limited to, aldehyde groups, ketone groups, and carbonyl groups. Further, the heteroatom of the reactive functional group of the linker can react with an electrophilic group on the antibody. Typical examples include, but are not limited to, hydrazine, oxime, amino, hydrazide, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide.

[0127] In one embodiment, the compound of formula (A) can include a linker (S B ) that is capable of reacting with a free amine on the antibody to form a covalent bond. The compounds of the invention expressly contemplate, but are not limited to, antibody conjugates prepared using carboxylic acid activating agents such as N-hydroxysuccinimide (NHS), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP).

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

[0129] (a) reacting the antibody or antigen-binding fragment with a compound of formula (III) having at least one reactive thiol group with a compound of formula (II) terminated with maleimide to prepare a compound of formula (IA) wherein X1 represents -S-:

Chemical formula

[0130] (b) reacting the antibody or antigen-binding fragment with a compound of formula (IIIA) having at least one reactive thiol group with a compound of formula (II) terminated with maleimide to prepare a compound of formula (IC) wherein X1 represents -NH2 and S1 contains -S-CH2-CH2-CH2-C(=NH)-:

Chemical formula

[0131] (c) preparing a compound of formula (IB) wherein X1 represents -NH2 by reacting a compound of formula (IIB) terminated with an N-hydroxysuccinimide group with a compound of formula (IIIB) wherein the antibody or antigen-binding fragment has at least one reactive amino group:

Chemical formula

[0132] (d) the interconversion of a compound of formula (I) or a protected derivative thereof to a further compound of formula (I) or a protected derivative thereof.

[0133] Processes (a) and (b) generally include a thiol-maleimide reaction; a Michael addition reaction of a reactive thiol group with an α,β-unsaturated ketone such as maleimide.

[0134] Preferred conditions include incubation of a linker-maleimide intermediate with an antibody or fragment thereof having a reactive thiol in a suitable buffer as described herein at room temperature. A typical linker:antibody ratio (LAR) depends on the number of free thiol groups present on the antibody or fragment thereof, but is typically in the range of 2 - 8.

[0135] Process (c) generally includes an amide bond formation reaction in the presence of an activated ester. Typical conditions include incubation of a linker-NHS ester with an antibody or fragment thereof having a reactive amino in a suitable buffer at room temperature. A typical linker:antibody ratio (LAR) depends on the number of free amino groups present on the antibody or fragment thereof, but is typically in the range of 2 - 20.

[0136] Process (d) typically includes interconversion procedures known to those skilled in the art. For example, in the compound of formula (I), the first substituent may 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 are known to those skilled in the art with respect to the conversion of precursor compounds to the compounds of formula (I) and are described in "March's Advanced Organic Chemistry", 4th Edition, John Wiley & Sons, 1992. For example, metal-catalyzed functionalizations such as using organotin reagents (Stille reaction), Grignard reagents, and reactions with nitrogen nucleophiles are described in "Palladium Reagents and Catalysts" [Jiro Tsuji, Wiley, ISBN 0-470-85032-9], and "Handbook of Organo Palladium Chemistry for Organic Synthesis" [Volume 1, edited by Ei-ichi Negishi, Wiley, ISBN 0-471-31506-0].

[0137] If suitable, the reactions described previously in processes (a), (b), and (c) are followed or preceded by one or more reactions known to those skilled in the art and are carried out in a suitable order to achieve the necessary substitutions defined previously to provide other compounds of formula (I). Non-limiting examples of such reactions, the conditions for which can be found in the literature, include the following: 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 group, Saponification / hydrolysis of ester group, Amidation or transesterification of ester group, Esterification or amidation of carboxyl group, Halogen exchange, Nucleophilic substitution by amine, thiol or alcohol, Reductive amination, Oxime formation of carbonyl group and hydroxylamine group, S-oxidation, N-oxidation, Chlorination.

[0138] The compound of formula (III) may have at least one reactive thiol group available for the reaction. The generation of the reactive thiol group may be achieved by the reduction of the antibody or antigen-binding fragment using TCEP.

[0139] Preferred conditions include either 1.1eq of TCEP:Ab, 4.2eq of TCEP:Ab, or 8eq of TCEP:Ab.

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

[0141] Preferred conditions include 12.2eq of 2-iminothiolane:Ab for low LAR and 30.5eq of 2-iminothiolane:Ab for high LAR.

[0142] The compounds of formula (II) and (IIB) may be prepared from the compounds of formula (V) and (VI) by subsequent reaction with N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or di(N-succinimidyl)glutarate (DSG) by the method described in Scheme 1:

Chemical formula

[0143] The compound of formula (II) may be prepared from the compound of formula (IV) by the interconversion of the terminal amino group to a reactive maleimide group. Preferred conditions include the reaction of the terminal amino group with N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) in DMSO at room temperature. Alternatively, the compound of formula (IIB) may be prepared from the compound of formula (IV) by the interconversion of the terminal amino group to a reactive NHS group. Preferred conditions include the reaction with the cross-linking agent di-(N-succinimidyl)glutarate in a suitable anhydrous organic solvent such as DMF and DMSO or a combination thereof.

[0144] The compound of formula (IV) may be prepared from the compounds of formula (V) and (VI) by process steps (iii) and (iv), as well as an amide bond formation step followed by an appropriate deprotection step. A typical amide bond formation step involves the activation of a carboxylic acid using a reagent containing phosphate, a triazine-based reagent, or a reagent containing carbodiimide in an organic solvent in the presence of an organic base. Preferred conditions include HATU ((1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) with either triethylamine or diisopropylethylamine in DMF or a mixture of DMF and DMSO. When PG contains monomethoxytrityl, the deprotection reaction is mediated by an acid. Preferred conditions include 0.2M aqueous HCl at room temperature.

[0145] Alternatively, the compound of formula (II) may be prepared from the compound of formula (VA) by the method described in Scheme 1A involving the subsequent reaction with N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC):

Chemical formula

[0146] The compound of formula (II) may be prepared from the compounds of formulas (VA) and (VI) by process steps (iii) and (iv) as already described in Scheme 1. The interconversion of the terminal amino group to a reactive maleimide group can be achieved using SMCC as described for process step (ii) in Scheme 1.

[0147] The compounds of formulas (V) and (VA) (wherein S2 is terminated with -NHCO-CH2-) may be prepared from the compounds of formulas (VIII), (IX), and (IXA) by the method described in Scheme 2:

Chemical formula

[0148] The compounds of formulas (V), (IXA), and (VA) may be prepared from the compound of formula (IX) by process steps (iii) and (iv), an amide bond formation step followed by an appropriate deprotection reaction. When PG contains benzyl, the deprotection reaction is mediated by catalytic hydrogenation. Preferred conditions include 10% Pd / C in MeOH / EtOH or water or any combination thereof under a hydrogen atmosphere (15 - 70 psi (103 - 483 kPa)). Alternatively, the deprotection may be mediated by a phase transfer reaction. Preferred conditions include TEA and water at room temperature for 16 hours.

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

[0150] The compound of formula (IX) may be prepared from the compounds of formula (XI) and formula (XII) by the method described in Scheme 3.

Chemical formula

[0151] The compound of formula (IX) may be prepared from the compounds of formula (XI) and formula (XII) by the 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) may be prepared by utilization of the Suzuki reaction for constructing the bi / tri-phenyl unit. Preferred conditions include tetrakistriphenylphosphine palladium(0) or [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium(II) dichloromethane complex with sodium carbonate, potassium acetate, or sodium bicarbonate in dioxane and water at 100 - 110 °C. When a suitable required protecting group such as TBS is utilized, such a protecting group may be deprotected using fluoride-mediated deprotection. Preferred conditions include TBAF in THF at room temperature.

[0153] Alternatively, when Cy is bi / tri-phenyl, the compound of formula (XI) may be directly prepared by utilization of the Suzuki reaction for constructing the bi / tri-phenyl unit using the conditions as described above and herein.

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

[0155] It will be understood that the specific intermediates described herein represent novel compounds not previously known in the art. Accordingly, in accordance with a further aspect of the invention, intermediate compounds selected from the compounds of formula (II), (IIB), (V), (VA), (IX), or (XI) as previously defined are provided.

[0156] Those skilled in the art will understand that they can select a suitable combination of the above-described steps to yield the highest yields with respect to the "Examples and Preparations" described herein.

[0157] (Pharmaceutical Composition) The compound of formula (I) can be administered alone, but on the other hand, it preferably exists as a pharmaceutical composition (e.g., a formulation).

[0158] Accordingly, in a further aspect, the present invention provides a pharmaceutical composition and, as described herein, a method for manufacturing a pharmaceutical composition containing (e.g., mixing) at least one of the compounds of the present invention together with one or more pharmaceutically acceptable excipients and optionally other therapeutic or prophylactic agents. It will be appreciated that when the pharmaceutical composition contains one or more additional therapeutic agents, the agent may include a further compound of formula (I).

[0159] Such pharmaceutically acceptable excipient(s) can be selected, for example, from carriers (e.g., 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, thickening agents, flavoring agents, sweeteners, taste masking agents, stabilizers, or any other excipient commonly used in pharmaceutical compositions. Examples of excipients for various types of pharmaceutical compositions are shown in more detail below.

[0160] As used herein, the term "pharmaceutically acceptable" relates to compounds, substances, compositions, and / or dosage forms that, within the scope of sound medical judgment, are commensurate with a reasonable benefit / risk ratio and are free of excessive toxicity (i.e., generally recognized as safe (GRAS)), and are suitable for use in contact with the tissues of a subject (e.g., a human) without irritation, allergic reaction, or other problems or complications. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other constituents of the formulation.

[0161] The pharmaceutical composition containing the compound of the present invention can be formulated according to known techniques. See, for example, "Remington’s Pharmaceutical Sciences", Mack Publishing Company, Easton, PA, USA.

[0162] The pharmaceutical composition can be in any shape suitable for parenteral, intranasal, intratracheal, sublingual, intraocular, intra-aural, rectal, vaginal, or transdermal administration. If the composition is intended for parenteral administration, it can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration, or for direct delivery to a target organ or tissue by injection, infusion, or other delivery means. This delivery can be by bolus injection, short-term infusion, or long-term infusion, and can be via passive delivery or through the use of a suitable infusion pump or syringe driver.

[0163] Pharmaceutical formulations adapted for parenteral administration include, among other things, aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, co-solvents, surfactants, organic solvent mixtures, cyclodextrin complexing agents, emulsifiers (for the formation and stabilization of emulsion formulations), liposome components for the formation of liposomes, gelling polymers for the formation of polymeric gels, lyoprotectants, and combinations of these agents, in order to stabilize the active ingredient in dissolved form and to make the formulation isotonic with the blood of the intended recipient. Pharmaceutical formulations for parenteral administration can also take the form of aqueous and non-aqueous sterile suspensions, which may contain suspending and thickening agents (R.G. Strickly, "Solubilizing Excipients in oral and injectable formulations", Pharmaceutical Research, Vol 21(2) 2004, p 201-230).

[0164] The formulation may be presented in unit-dose or multi-dose containers, such as sealed ampoules, vials, and pre-filled syringes, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use.

[0165] This pharmaceutical preparation can be prepared by lyophilizing the compound of the present invention. Lyophilization refers to the procedure of freeze-drying the composition. Therefore, freeze-drying and lyophilization are used synonymously herein.

[0166] Injectable solutions and suspensions, when needed, may be prepared from sterilized powders, granules and tablets.

[0167] The pharmaceutical composition of the present invention for parenteral injection may also contain a pharmaceutically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion immediately before use.

[0168] Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as sunflower oil, safflower oil, corn oil, or olive oil), and organic esters for injection such as ethyl oleate. For example, by using a thickening or coating substance such as lecithin, by maintaining the particle size required in the case of a dispersant, and by using a surfactant, appropriate fluidity can be maintained.

[0169] The composition of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It is also desirable to contain agents for adjusting the tonicity, such as saccharides, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by containing agents for delaying absorption such as aluminum monostearate and gelatin.

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

[0171] In another preferred embodiment, the pharmaceutical composition is in a form suitable for subcutaneous (s.c.) 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 which aids their absorption. In addition, the nanoparticles may have the potential to penetrate directly into cells. Nanoparticle drug delivery systems are described in "Nanoparticle Technology for Drug Delivery", edited by Ram B Gupta and Uday B. Kompella, Informa Healthcare, ISBN 9781574448573, 13th Edition, published in March 2006. Nanoparticles for drug delivery are also described in J. Control. Release, 2003, 91 (1-2), 167-172, and in the literature by Sinha et al., Mol. Cancer Ther. August 1, (2006) 5, 1909.

[0173] The pharmaceutical composition typically contains approximately 1% (w / w) to approximately 95% (w / w) of the active ingredient and 99% (w / w) to 5% (w / w) of a pharmaceutically acceptable excipient or combination of excipients. Preferably, the composition contains approximately 20% (w / w) to approximately 90% (w / w) of the active ingredient and 80% (w / w) to 10% of a pharmaceutically acceptable excipient or combination of excipients. The pharmaceutical composition contains approximately 1% to approximately 95%, preferably approximately 20% to approximately 90% of the active ingredient. The pharmaceutical composition according to the present invention may be in a unit dosage form, such as, for example, in the form of ampoules, vials, suppositories, pre-filled syringes, dragées, tablets or capsules.

[0174] Excipient(s) acceptable as this medicament can be selected according to the desired physical form of this preparation, and can be selected, for example, from diluents (e.g., solid diluents such as fillers or extenders; and liquid diluents such as solvents and co-solvents), disintegrants, buffers, lubricants, glidants, release control agents (e.g., polymers or waxes that suppress or delay release), binders, granulating agents, pigments, plasticizers, antioxidants, preservatives, flavoring agents, taste masking agents, tonicity modifiers, and coating agents.

[0175] Those skilled in the art will have the expertise to select the suitable amounts of the components for use in the preparation. For example, tablets and capsules typically contain 0-20% (depending on the drug dosage) of disintegrant, 0-5% of lubricant, 0-5% of glidant, and / or 0-99% (w / w) of filler / extender. They also typically contain 0-10% (w / w) of polymer binder, 0-5% (w / w) of antioxidant, and 0-5% (w / w) of pigment. Sustained release tablets will additionally contain 0-99% (w / w) of release control (e.g., delay) polymer (depending on the dosage). The film coating of tablets or capsules typically contains 0-10% (w / w) of polymer, 0-3% (w / w) of pigment, and / or 0-2% (w / w) of plasticizer.

[0176] Parenteral or subcutaneous preparations typically contain 0-20% (w / w) of buffer solution, 0-50% (w / w) of co-solvent, and / or 0-99% (w / w) of water for injection (WFI) (depending on the dosage and when lyophilized). Preparations for intramuscular depot formulations also contain 0-99% (w / w) of oil.

[0177] The compounds of the present invention can also be formulated as solid dispersants. A solid dispersant is a homogeneous and very fine dispersion phase of two or more solids. One type of solid dispersant, a solid solution (molecular dispersion system), is well-known for use in pharmaceutical technology (see Chiou and Riegelman, J. Pharm. Sci., 60, 1281-1300 (1971)) and is useful for increasing the dissolution rate and bioavailability of drugs that are poorly soluble in water.

[0178] The pharmaceutical formulation may be provided to the patient in a "patient pack" that contains the entire treatment process, usually in a blister pack. The patient pack has advantages over conventional prescriptions, where the pharmacist divides the supply of the drug for the patient from the bulk supply, in which case the patient always has access to the enclosed instructions included in the patient pack, which are usually not described in the patient's prescription. The inclusion of the enclosed instructions has been shown to improve patient compliance with the doctor's instructions. An example of a patient pack includes a pre-filled syringe. Such a pre-filled syringe already contains the drug substance. The tip portion of the pre-filled syringe with the needle attached is sealed by a nozzle cap. Before injection, the nozzle cap is removed from the tip portion and the needle is attached there. Next, the plunger rod is pushed into the tip portion to slide the gasket, and as a result, the drug is released.

[0179] Compositions for nasal delivery include ointments, creams, sprays, patches, gels, droplets, and inserts (e.g., intraocular inserts). Such compositions can be formulated according to known methods.

[0180] Examples of formulations for rectal or vaginal administration include pessaries and suppositories, which may be formed, for example, from a shaped moldable or waxy substance containing the active compound. A solution of the active compound may also be used for rectal administration.

[0181] Compositions for administration by inhalation can be in the form of an inhalable powder composition or a liquid or powder spray, and can be administered in a standard form using a powder inhaler or an aerosol metering dispenser. Such devices are well known. For administration by inhalation, the powdered formulation typically contains the active compound together with an inert solid powdered diluent such as lactose.

[0182] The compounds of the present invention are generally presented in unit dosage forms and thus typically contain an amount of the compound sufficient to provide the desired level of biological activity. For example, the formulation may contain from 1 ng to 2 g of the active ingredient, for example from 1 ng to 2 mg of the active ingredient. Within these ranges, certain sub-ranges of the compounds are from 0.1 mg to 2 g of the active ingredient (more generally from 10 mg to 1 g, for example from 50 mg to 500 mg), or from 1 μg to 20 mg (for example, from 1 μg to 10 mg, for example from 0.1 mg to 2 mg of the active ingredient).

[0183] This active compound will be administered in an amount sufficient to achieve the desired therapeutic effect to patients (e.g., human or animal patients) in need thereof.

[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.

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

[0186] For example, in embodiments where the antibody or an antigen-binding fragment thereof is an EGFR antibody (e.g., cetuximab or nimotuzumab) or a fragment thereof, the compound of formula (I) is for use in the treatment of cancer.

[0187] Accordingly, in a further aspect of the invention, there is provided a compound of formula (I) as defined herein, wherein the antibody or antigen-binding fragment thereof is an EGFR antibody (e.g., cetuximab or nimotuzumab) or a fragment thereof for use in the treatment of cancer.

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

[0189] Furthermore, in embodiments where the antibody or antigen-binding fragment thereof is a pathogen-specific antibody or a fragment thereof, the compound of formula (I) is for use in the treatment of bacterial infections.

[0190] Accordingly, in a further aspect of the invention, there is provided a compound of formula (I) as defined herein, wherein the antibody or antigen-binding fragment thereof is a pathogen-specific antibody or a fragment thereof for use in the treatment of bacterial infections.

[0191] In a further aspect of the invention, there is provided a method of treating bacterial infections comprising administering to an individual in need thereof a compound of formula (I) as defined herein, wherein the antibody or antigen-binding fragment thereof is a pathogen-specific antibody or a fragment thereof.

[0192] The compounds of the invention are generally administered to subjects in need of such administration, such as human or animal patients, preferably humans.

[0193] The compounds of the present invention will typically be administered in amounts that are therapeutically or prophylactically useful and generally non-toxic. However, in certain situations (e.g., in cases of life-threatening diseases), the benefits of administering the compounds of the present invention outweigh any drawbacks of toxic or side effects, and in such cases, it may be desirable to administer the compounds of the present invention in amounts that are associated with some degree of toxicity.

[0194] The compounds of the present invention may be administered over a long period (i.e., chronically) or for only a short period (i.e., acutely) in order to maintain a beneficial therapeutic effect. Alternatively, they may be administered in a continuous manner or in a manner that provides intermittent dosing (e.g., in a pulse-like manner).

[0195] Typical daily amounts of the compounds of the present invention can range from 100 pg to 100 mg / kg body weight, more typically from 5 ng to 25 mg / kg body weight, and more generally from 10 ng to 15 mg per kg of body weight (e.g., 10 ng to 10 mg, and more typically from 1 μg / kg to 20 mg / kg, e.g., 1 μg to 10 mg / kg), but higher or lower dosages may be administered if necessary. The compounds of the present invention can be administered on a daily basis or on an alternating basis, 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, or every 14 days, or every 21 days, or every 28 days. Alternatively, the compounds of the present invention can be administered by infusion multiple times a day.

[0196] The compounds of the present invention may be administered, for example, in a dosage range of 1 to 1500 mg, 2 to 800 mg, or 5 to 500 mg, such as 2 to 200 mg or 10 to 1000 mg. Specific examples of dosages include 10, 20, 50, and 80 mg. The compounds of the present invention may be administered once or more than once daily. The compounds of the present invention can be administered continuously (i.e., taken daily without interruption over the course of a treatment regimen). Alternatively, the compounds of the present invention can be administered intermittently (i.e., taken continuously for a predetermined period such as one week, then interrupted for a period such as one week, then taken continuously for another period such as one week, and so on throughout the course of a treatment regimen). Examples of treatment regimens for intermittent administration include regimens where the administration is for one or more cycles, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cycles, one week on, one week off; or, two weeks on, one week off; or, three weeks on, one week off; or, two weeks on, two weeks off; or, four weeks on, two weeks off; or, one week on, three weeks off cycles.

[0197] In one particular dosing schedule, the patient is given an intravenous infusion of the compound of the present invention for one hour each day for a period of up to 10 days, particularly for up to 5 days per week, and this treatment will be repeated at desired intervals such as 2 to 4 weeks, particularly every 3 weeks.

[0198] More specifically, the patient may be given an intravenous infusion of the compound of the present invention for one hour each day for a period of 5 days, and this treatment may be repeated every 3 weeks.

[0199] In another particular dosing schedule, the patient is given an intravenous infusion lasting from 30 minutes to 1 hour, followed by a maintenance intravenous infusion for a variable period of, for example, 1 to 5 hours, such as 3 hours.

[0200] In a further particular dosing schedule, the patient is given a continuous intravenous infusion for a period of 12 hours to 5 days, particularly a continuous intravenous infusion for 24 hours to 72 hours.

[0201] However, ultimately, the amount of the compound of the invention to be administered and the type of composition used will correspond to the nature or physiological state of the disease being treated and will be at the discretion of the physician.

[0202] It will be appreciated that the compounds of the invention can be used as a single agent or in combination with other therapeutic agents. Combination experiments can be carried out, for example, as described in the literature of Chou TC, Talalay P., "Quantitative analysis of dose - effect relationships: the combined effects of multiple drugs or enzyme inhibitors", Adv Enzyme Regulat, 1984;22: 27-55.

[0203] When the compounds of the invention are administered in combination therapy with 1, 2, 3, 4 or more therapeutic agents (preferably 1 or 2, more preferably 1), these agents can be administered simultaneously or sequentially. In the latter case, two or more agents will be administered in an amount and manner sufficient to ensure that an advantageous or synergistic effect is achieved within a certain period. When administered sequentially, they can be administered at closely spaced intervals (e.g., over a period of 5 - 10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 hours or more apart, or at even longer intervals if necessary), and the exact dosing regimen will be appropriate to the characteristics of the therapeutic agent(s). These doses can be repeated, for example, every 7, 14, 21 or 28 days, and for each course of treatment, can be administered, for example, once, twice or more.

[0204] The preferred methods and order of administration of the components to be combined, as well as each dosage amount and regimen, will be recognized as being dependent on the particular other medical agents being administered, the compounds of the present invention, their routes of administration, the particular tumors being treated, and the particular host being treated. The optimal methods and order of administration, as well as dosage amounts and regimens, can be readily determined by one of ordinary skill in the art using conventional methods and taking into account the information described herein.

[0205] The weight ratio of the compound of the present invention to one or more other therapeutic agent(s) when administered in combination may be determined by one of ordinary skill in the art. Such ratio, as well as the exact dosage and frequency of administration, will depend on the particular compound of the present invention and the other therapeutic agent(s) used, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, diet, time of administration, and general physical condition of the particular patient, the mode of administration, as well as other drugs the individual is taking. Further, it is evident that the effective daily amount may be decreased or increased depending on the response of the subject being treated and / or the evaluation of the physician prescribing the compound of the present invention. The particular weight ratio of the compound of the present invention to another therapeutic agent may be in the range of 1 / 10 to 10 / 1, more specifically 1 / 5 to 5 / 1, and even more specifically 1 / 3 to 3 / 1.

[0206] (Anticancer treatment) Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, for example, cancers of the bladder and urinary tract, breast, gastrointestinal tract (esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar carcinoma, and mesothelioma), head and neck (e.g., cancers of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovary, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal gland, prostate, skin, and appendage carcinomas (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus), tumors of epithelial origin (including various types of adenomas and carcinomas, such as adenocarcinoma, squamous carcinoma, transitional cell carcinoma, and other carcinomas); hematological malignancies and related conditions of the lymphatic system (e.g., B-cell lymphomas such as acute lymphoblastic leukemia [ALL], chronic lymphocytic leukemia [CLL], diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), hematological malignancies and related conditions of the myeloid system (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], eosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia), hematological malignancies (i.e., leukemia, lymphoma), as well as pre-cancerous hematological disorders and borderline malignant tumor disorders; tumors of mesenchymal origin, such as sarcomas of soft tissue, bone, or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi sarcoma, Ewing sarcoma, synovial sarcoma, epitheloid granuloma, gastrointestinal stromal tumor, benign and malignant histiocytic species, and dermatofibrosarcoma protuberans, etc.; tumors of the central nervous system or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma, meningioma, ependymoma, pineal tumor, and schwannoma); endocrine tumors (e.g., pituitary tumor, adrenal tumor, islet cell tumor, parathyroid tumor, carcinoid tumor, and medullary carcinoma of the thyroid);Tumors of the eye and its appendages (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratoma, seminoma, undifferentiated embryonal cell tumor, cystic teratoma, and choriocarcinoma); and pediatric and fetal tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor, and undifferentiated neuroectodermal tumor); or congenital or other syndromes (e.g., xeroderma pigmentosum) that predispose a patient to malignancy, but are not limited thereto;

[0207] In one embodiment, the cancer is a solid tumor. In a further embodiment, the cancer is breast cancer, ovarian cancer, cervical cancer, colorectal cancer, liver cancer, prostate cancer, or lung cancer.

[0208] In one embodiment, the cancer includes a hematological malignancy. In a further embodiment, the hematological malignancy is one of multiple myeloma, non-Hodgkin lymphoma, or chronic lymphocytic leukemia.

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

[0210] The compounds of the present invention may also be administered in combination with non-chemotherapeutic treatments such as radiotherapy, photodynamic therapy, gene therapy; surgery and dietary restrictions.

[0211] For use in combination therapy with another chemotherapeutic agent, the compounds of the present invention and one, two, three, four or more other therapeutic agents can be formulated together, for example, in a dosage form containing two, three, four or more therapeutic agents, i.e., in a single pharmaceutical composition containing all the components. In an alternative embodiment, the individual therapeutic agents may be formulated separately and provided together in the form of a kit, optionally with their instructions for use.

[0212] (Anti-infective therapy) Examples of infectious agents include any pathogen such as bacteria, fungi, parasites, or viruses. Thus, in one embodiment, the disease or disorder mediated and / or caused by an infectious agent is a bacterial infection.

[0213] Examples of such bacterial infections include infections caused by the following bacteria: Staphylococcus species such as Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus (MRSA)); Clostridium species (e.g., Clostridium difficile, Clostridium tetani, and Clostridium botulinum); Enterobacter species, Mycobacterium tuberculosis; Shigella species such as Shigella dysenteriae; Campylobacter species such as Campylobacter jejuni; Enterococcus species such as Enterococcus faecalis; Bacillus anthracis; Yersinia pestis, Bordetella pertussis, Streptococcal species; Salmonella thyphimurim, Salmonella enterica; Chlamydia species, Treponema pallidum, Neisseria gonorrhoeae, Borrelia burgdorferi, Vibrio cholerae, Corynebacterium diphtheriae, Helicobacter pylori; and Gram-negative bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli (including strains resistant to one or more classes of antibiotics, particularly multidrug-resistant (MDR) strains).

[0214] (Vaccine therapy) In a further aspect of the invention, there is provided a vaccine comprising an immunoconjugate as defined herein.

[0215] In a further aspect of the invention, there is provided an adjuvant comprising an immunoconjugate as defined herein.

Examples

[0216] (Example) The present invention is illustrated, but not limited, with reference to the specific embodiments described in the following examples. Compounds are named using an automated nomenclature package such as AutoNom (MDL) or ChemDraw, or are named by the chemical supplier.

[0217] The following synthetic procedures are provided for illustration of the methods used; for a given preparation or step, the precursors used are not necessarily derived from the individual batches synthesized according to the steps in a given description.

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

[0219] (LCMS method A: acidic run for 4.5 minutes)

Table 3

Table 4

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 × 30 mm, 2.7 μm Solvent: A = water; B = 0.1% formic acid in acetonitrile. Column Temperature: 20°C, Injection Volume: 5 μL.

[0221] (LCMS Method D: Acidic Run for 4.5 Minutes)

Table 6

[0222] (NMR) Details of NMR were recorded on either Oxford Instruments AS400.

[0223] (Abbreviations) When the following abbreviations are used, the following meanings apply: AcOH is acetic acid; aq. is aqueous; br is a broad singlet; δ is the chemical shift in ppm; d is a doublet; dd is a doublet of doublets; ddd is a doublet of doublets of doublets; DCM is dichloromethane; DIPEA is diisopropylethylamine; DMF is dimethylformamide; DMSO is dimethyl sulfoxide; DMSO-d6 is a perdeuterated dimethyl sulfoxide NMR solvent; DSG is di-(N-succinimidyl) glutarate; EtOH is ethanol; EtOAc is ethyl acetate; HATU is O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; HPLC is high performance liquid chromatography; IMS is industrial methylated spirit (typically 5% - 10% MeOH in EtOH); μ is micro; m is multiplet; Mal is maleimide; MeCN is acetonitrile; MeOH is methanol; mins is minutes; mL is milliliter; 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 hydrogenation catalyst (water-wetted) of palladium on carbon (typically 5% - 10%); Pd(PPh3)4 is tetrakistriphenylphosphine palladium(0); ppm is parts per million; q is quartet; Rt is retention time; s is singlet; SMCC is succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; t is triplet; 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 referring to α-Gal, apply the following intermediate: 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)amine [Chemical formula] This intermediate can be prepared by the method described in the literature of Bovin et al. (Mendeleev Communications (2002), (4), 143-145).

[0225] Preparations 1 to 19 describe the methods used to prepare intermediates from important linker molecules required for conjugation to the examples, as explained by processes (a) to (d) and Schemes 1, 1A, 2, and 3 as described above.

[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)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-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide)ethyl)-[1,1'-biphenyl]-3-carboxamide [Chemical Structure] To a solution of 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)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 (Preparation 3, 5.0 mg, 2.26 μmol) in DMSO (400 μL) was added a solution of SMCC (2.2 mg, 6.57 μmol) in DMSO (100 μL). The resulting solution was stirred at room temperature for 18 h and then dried under vacuum. 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)cyclohexane-1-carboxamido)ethyl)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-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)acetamide))

Chem.

[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)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

Chem.

[0229] (Step 2) 0.2 M HCl(aq) was added dropwise until pH 3-4, and the solution was stirred at room temperature for 18 hours. The reaction solution was concentrated under vacuum and purified using reverse-phase column chromatography eluting with 5-40% MeCN in water containing 0.1% ammonia, affording the title compound as a colorless solid (12.4 mg, 28%). 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-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)acetamide))

Chem.

[0231] (Step 2) 0.2 M HCl(aq) was added dropwise to pH 3 - 4 and the solution was stirred at room temperature for 18 h. The reaction mixture was concentrated in vacuo and purified by reverse-phase column chromatography eluting with 5 - 40% MeCN in water containing 0.1% ammonia to give the title compound as a colorless solid (47.0 mg, 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

Chem.

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

[0234] (Method B) To 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-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-carboxylate (Preparation 7, 278 mg, 123 μmol) dissolved in water (7 mL) was added TEA (7 mL), and the reaction mixture was vigorously stirred at room temperature for 16 h. The reaction mixture was concentrated under vacuum and purified using reverse-phase column chromatography eluting with 5-40% MeCN / water containing 0.1% NH3 to give the title compound as a colorless solid (224 mg, 83%).

[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)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) [Chemical formula] Benzyl 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)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-carboxylate (Preparation 8, 93.5 mg, 98.7 μmol) was dissolved in MeOH / H2O (1:1 v / v, 5 mL), and Pd / C (10%, 10 mg) was added. The reaction was placed under an atmosphere of hydrogen (50 psi (345 kPa)) and stirred at room temperature for 3 h. The catalyst was removed by filtration through a syringe filter, and the solvent was removed under reduced pressure to give a crude product, which was purified using reverse-phase column chromatography eluting with 5 - 40% MeCN / H2O containing 0.1% NH3 to give 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-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-carboxylate)

Chem.

[0237] (Preparation 8 Benzyl 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)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-carboxylate)

Chemical Structure

[0238] (Preparation 9 2,2',2''-((5'-((benzyloxy)carbonyl)-[1,1'-biphenyl]-3,3',5-triyl)tris(oxy))triacetic acid)

Chemical Structure

Chemical Structure

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

[0240] (Formulation 11 Tri-tert-butyl 2,2',2''-((5'-((benzyloxy)carbonyl)-[1,1'-biphenyl]-3,3',5-triyl)tris(oxy))triacetate) [Chem.] To benzyl 3',5,5'-trihydroxy-[1,1'-biphenyl]-3-carboxylate (Formulation 13, 356 mg, 1.06 mmol) dissolved in DMF (10 mL) were added tert-butyl bromoacetate (625 μL, 4.23 mmol) and potassium carbonate (1.17 g, 8.47 mmol). The resulting suspension was stirred under nitrogen for 16 h 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), 2 M aqueous NaOH (10 mL), dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography eluting with 7 - 60% EtOAc in heptane to give the title compound as a clear colorless gum (618 mg, 86%). LCMS method C: Rt = 4.34 minutes, no mass ions were observed.

Chem.

[0241] (Preparation 12 Benzyl 4'-(2-(tert-butoxy)-2-oxoethoxy)-[1,1'-biphenyl]-3-carboxylate)

Chem.

Chem.

[0242] (Preparation 13 Benzyl 3',5,5'-trihydroxy-[1,1'-biphenyl]-3-carboxylate)

Chem.

Chemical formula

[0243] (Preparation 14 benzyl 4'-hydroxy-[1,1'-biphenyl]-3-carboxylate)

Chemical formula

Chem.

[0244] (Preparation 15 (Benzyl 3-bromo-5-hydroxybenzoate)

Chem.

Chem.

[0245] (Preparation 16 (Benzyl 3',5'-bis((tert-butyldimethylsilyl)oxy)-5-hydroxy-[1,1'-biphenyl]-3-carboxylate)

Chem.

[0246] (Preparation 17 ((5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-phenylene)bis(oxy))bis(tert-butyldimethylsilane))

Chemical formula

Chemical formula

[0247] (Preparation 18 1,3-bis((tert-butyldimethylsilyl)oxy)benzene)

Chemical formula

Chemical formula

[0248] (Preparation 19 N 1 -((4-Methoxyphenyl)diphenylmethyl)ethane-1,2-diamine)

Chem.

Chem.

[0249] (Preparation 20 4'-(2-((2-(3-((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)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)amino)-3-oxopropoxy)ethyl)amino)-2-oxoethoxy)-N-(2-(3-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamide)ethyl)-[1,1'-biphenyl]-3-carboxamide)

Chem.

[0250] (Preparation 21 3,3',3''-((((2,2',2''-((5'-((2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamide)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-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)propanamide))

Chem.

[0251] (Preparation 22 2,5-Dioxopyrrolidin-1-yl 5-((2-(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)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-Carboxamide)Ethyl)Amino)-5-Oxopentanoate [Chemical Structure] 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)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 (Preparation 3, 21.76 mg, 24 mmol) was dissolved in anhydrous DMF:DMSO (1062 μL) in a 1:1 ratio with stirring. Di-(N-Succinimidyl) Glutarate (39.49 mg, 120 mmol) was added as a 300 mM solution in anhydrous DMF:DMSO (400 μL) in a 1:1 ratio. The reaction mixture was stirred at room temperature for 1 hour in a positive pressure nitrogen atmosphere. The reaction mixture was purified using reverse-phase column chromatography (10×250 mm Hichrom ACE 10) eluting with 1 - 50% MeCN in 1% TFA in water. The desired fraction was lyophilized over 24 hours until dry 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-(((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)-3-oxopropoxy)ethyl)amino)-2-oxoethoxy)-[1,1'-biphenyl]-3-carboxamide)ethyl)amino)pentanoate) [Chemical formula] The title compound was prepared by the method described for Preparation 22 using Preparation 25 and directly employed in the conjugation step.

[0253] (Preparation 24 4'-(2-((2-(3-((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)-3-oxopropoxy)ethyl)amino)-2-oxoethoxy)-N-(2-aminoethyl)-[1,1'-biphenyl]-3-carboxamide) [Chemical formula] The title compound was prepared by the method described for Preparation 3 using 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)-[1,1'-biphenyl]-3-carboxylic acid (WO2017060729). 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)-3,4,5-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)propanamide))

Chemical Structure

[0255] (Example) (Materials and methods:) vcMMAE (vcE): ADCB TOX001, 10 mM in DMA TCEP - Biovectra Cat 1300 Lot: 42359 N-acetylcysteine - Sigma A7250 Lot: WXBC3104V Dimethylacetamide - Sigma - Aldrich 271012 Lot: STBF9638V KNE buffer: 50 mM KPI, 50 mM NaCl, 2 mM EDTA, pH 7.5 PBS: Reconstituted using Sigma, P5368 #SLBQ7495, WFI: Sigma W3500 #RNBF6963 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 evaluated by chromatography on a TOSOH TSK gel G3000SWXL 7.8 mm × 30 cm, 5 μm column at 0.5 mL / min in 10% IPA, 0.2 M potassium phosphate, 0.25 M potassium chloride, pH 6.95. Samples were loaded neat and data were collected at 214, 252, and 280 nm. All data reported are at 280 nm.

[0257] (Hydrophobic interaction chromatography (HIC)) HIC was performed at 0.8 mL / min using a 12-minute linear gradient between mobile phase A - 1.5 M (NH4)2SO4, 25 mM NaPi, pH 6.95 ± 0.05 and mobile phase B - 75% 25 mM NaPi, pH 6.95 ± 0.05, 25% IPA on a TOSOH Butyl-NPR 4.6 mm × 3.5 cm, 2.5 μm column. Samples were loaded neat to a maximum injection volume of 10 μL and data were collected at 280, 252, and 214 nm; all data reported are at 214 nm.

[0258] (Polymeric reversed-phase chromatography (PLRP)) The reverse phase was carried out at 0.25 mL / min / 80 °C using a linear gradient over 25 min between 0.1% TFA 25% MeCN and 0.1% TFA 50% MeCN on a Polymer Labs PLRP-S 2.1 mm×50 mm, 5 μm, 1000 Å column. 10 μg was mixed with 5 μL of 0.1 M DTT, made up to 50 μL with 0.5 M Tris / Cl, pH 8.0 and incubated at 37 °C for 15 min to reduce the sample. The reduced sample was diluted 1:1 using 49% acetonitrile, 49% water, 2% formic acid to stop the reduction and stabilize the sample until PLRP analysis. 20 μL of the sample was loaded onto the analytical column. All data were reported at 214 nm.

[0259] Note: For HIC and PLRP, individual linker loadings cannot be separated due to the hydrophilicity of α-Gal. The linker:Ab ratio (LAR) was determined by the first thiol-reactive antibody and a fixed amount of vcE chase of the completed reaction mixture.

[0260] The 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 / 2020) having a molecular weight of 152,000 Da was used for the following conjugation. Calculations were based on Abs at 1.45 cm -1 mg / mL -1 of 0.1% 280 nm, UV analysis at 4.7 mg / mL, and a calibration curve by SEC at 214 nm.

[0263] (Schematic method for reduction of cetuximab and subsequent conjugation to a linker containing maleimide (Examples 1-4)) To cetuximab (4.7 mg / mL), 6% 0.5 M Tris-Cl, 0.025 M EDTA, pH 8.5 was added, and it was incubated with 1.1 eq of TCEP:mAb (for average LAR = 2) or 4.2 TCEP:mAb (for average LAR = 5) for 90 minutes and 120 minutes at room temperature, respectively. To analyze the degree of reduction, an aliquot of the reduced sample was conjugated with a molar excess of the alternative payload Mal-vc-PAB-MMAE to determine the DAR (using HIC analysis). To this reduced sample, 8 eq of linker-maleimide was added, and the reaction mixture was incubated at room temperature for 60 minutes. After incubation, the reaction was quenched for 30 minutes by the addition of N-acetylcysteine (10 mM aqueous solution). The sample was purified in PBS with G25 resin, and the remaining unbound linker was removed by membrane diafiltration with 10 dialysis volumes using a Vivaspin6 device.

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

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

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

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

[0268] (Schematic method for the thiol interconversion of cetuximab and subsequent conjugation to a linker containing maleimide (Examples 5 - 8)) Cetuximab (4.7 mg / mL) was bound to 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, and the mAb was eluted with 4 CV of 0.1 M glycine at pH 3. 20% 1.76 M HEPES at pH 10.8 was added to the elution fraction (pooled by UV280 absorbance) containing the target protein. Then, cetuximab was incubated with 12.2 eq of 2-iminothiolane:mAb (for low LAR samples) or 30.5 eq of 2-iminothiolane:mAb (for high LAR samples) at 23 °C for 120 minutes. An aliquot of the sample was buffer-exchanged with 5 mM His, 50 mM trehalose pH 6.0 and conjugated with a molar excess of the alternative payload Mal-vc-PAB-MMAE to determine the DAR. Prior to coupling, the remaining thiolated mixture was quenched by NAP25 buffer-exchange into 5 mM His, 50 mM trehalose, pH 6.0. The linker-maleimide conjugation was carried out at 23 °C for 120 minutes using 4 eq of linker-maleimide:thiol using a final 5% of DMA. Immediately after incubation, the sample was buffer-exchanged into PBS by G25 resin. The excess linker was removed by 10 DV membrane diafiltration (Vivaspin6). Throughout the experiment, the amount of free thiol can be monitored using Ellman's assay.

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

[0270] (Example 6) [Chemical formula] 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)

Chemical Structure

[0272] (Example 8)

Chemical Structure

[0273] (Schematic method for the reduction of cetuximab and subsequent conjugation to a linker containing maleimide to obtain an average LAR = 8 (Examples 9 - 11)) To cetuximab (4.7 mg / mL), 6% 0.5 M Tris-Cl, 0.025 M EDTA (pH 8.5) was added to about pH = 7.5, and incubated with 8 eq of TCEP:mAb at room temperature for 90 minutes. To analyze the degree of reduction, an aliquot of the reduced sample was conjugated with a molar excess of alternative payload Mal-vc-PAB-MMAE to determine the DAR (using HIC analysis).

[0274] To the reduced sample, either 16 or 32 eq of linker-maleimide was added, and the reaction solution was incubated at room temperature for 60 minutes. After incubation, the reaction solution was quenched for 30 minutes by the addition of N-acetylcysteine (10 mM aqueous solution), the sample was purified in PBS with G25 resin, and the remaining unbound linker was removed by membrane diafiltration with 10 dialysis volumes using a Vivaspin6 30 kDa PES device.

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

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

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

[0278] (Schematic method for direct lysine conjugation of cetuximab to a linker containing N-hydroxysuccinimide (Examples 12 - 16)) Cetuximab (20 mg) was bound to Protein A resin (GE Healthcare, HiTrap MabSelect Sure, 1 mL), 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 at pH = 3 and buffer-exchanged into lysine conjugation buffer (50 mM NaPi, 150 mM NaCl, 2 mM EDTA, pH = 8) with concentration to approximately 6 mg / mL. The solution was analyzed by SEC to obtain cetuximab in a solution suitable for lysine conjugation (18.2 mg, yield 91%, 6.4 mg / mL, 100% monomer content).

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

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

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

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

[0283] (Example 15)

Chemical Structure

[0284] (Example 16)

Chemical Structure

[0285] (Example 17)

Chemical Structure

[0286] (Digestion of Cetuximab to Cetuximab-Fab) Cetuximab (60 mg, 4.7 mg / mL) was buffer-exchanged into digestion buffer (20 mM NaPi, 20 mM cysteine, 10 mM EDTA, pH = 7) and concentrated to 3 mL at 20 mg / mL. Immobilized papain (3 mL, Thermo Fisher #20341, loading: 250 μg / mL resin, activity: 16 - 40 BAEE / mg papain) 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. Unbound Fab fragments were passed through the column and the flow-through fraction was collected. The Fab fragments were subjected to discontinuous diafiltration into 50 mM NaPi, 150 mM NaCl, and 2 mM EDTA, pH = 8 using Vivaspin centrifugation (10 kDa MWCO filter). The final concentration achieved was 5.6 mg / mL (see Figure 16).

[0287] (Schematic method for reduction of cetuximab-Fab to obtain LAR = 2 and subsequent conjugation to a linker containing maleimide (Examples 18 and 19)) 5.6 mg / mL of cetuximab-Fab in conjugation buffer (50 mM NaPi, 150 mM NaCl, and 2 mM EDTA, pH = 8) was reduced at room temperature for 90 minutes by the addition of 5 molar equivalents of TCEP (10 mM in water). A solution of Preparation 20 (5 molar equivalents) or Preparation 21 (7.5 molar equivalents) was added and the reaction mixture was incubated at room temperature for 1 hour. The conjugate was buffer-exchanged using PBS and diafiltered to obtain the desired substance. The conjugate was analyzed by SEC and SDS-PAGE.

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

[0289] (Example 19)

Chemical Structure

[0290] (Schematic method for the conjugation of lysine to a linker containing NHS of cetuximab-Fab to obtain an average LAR of 7 - 14 (Examples 20 - 24)) 5.6 mg / mL of cetuximab-Fab in conjugation buffer (50 mM NaPi, 150 mM NaCl, and 2 mM EDTA, pH = 8) was incubated with 9% v / v DMF co-solvent, Preparation 22 (15, 30, and 40 equivalents) and Preparation 23 (20 equivalents) at 30 °C for 2 hours. The reaction solution was quenched by the addition of glycine to a concentration of 1 mM, and the conjugate was buffer-exchanged into PBS and diafiltered to remove excess linker.

[0291] (Example 20)

Chemical Structure

[0292] (Example 21)

Chemical Structure

[0293] (Example 22)

Chemical Structure

[0294] (Example 23)

Chemical formula

[0295] (Example 24)

Chemical formula

[0296] (Antibody conjugate (rituximab)) Rituximab (Roche - Rituxan, lot No: B6105B92UI) formulated in polysorbate 80 (0.7 mg / mL) with sodium citrate anhydrous (7.35 mg / mL), sodium chloride (9 mg / mL), and water.

[0297] (Schematic method for direct lysine conjugation of rituximab to a linker containing N-hydroxysuccinimide (Example 25)) Rituximab was adjusted to pH = 7.9 using 500 mM phosphate buffer (50 mM NaPi, 150 mM NaCl, 2 mM EDTA, pH = 8).

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

[0299] (Example 25) [Chemical formula] Average LAR: 20; Average total number of α-Gal units: 20 SEC analysis: Rt = 14.4 minutes, 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 buffer-exchanged into digestion buffer (20 mM NaPi, 20 mM cysteine, 10 mM 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) 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. Unbound Fab fragments were passed through the column and the flow-through fractions were collected. Discontinuous diafiltration was performed on the Fab fragments by Vivaspin centrifugation (10 kDa MWCO filter) into 50 mM NaPi, 150 mM NaCl, and 2 mM EDTA, pH = 8. The final concentration achieved was 11.7 mg / mL. The above rituximab-Fab solution was incubated with 25% v / v DMF co-solvent with 40 molar equivalents of Preparation 22 at 30 °C for 2 hours, followed by a second addition of 40 molar equivalents of Preparation 22 (with 9% v / v DMF). The reaction solution was quenched by the addition of glycine to 1 mM, and the conjugate was buffer-exchanged into PBS and diafiltered to remove excess linker.

[0302] (Example 26) [Chemical formula] Average LAR: 14; Average total number of α-Gal units: 14 SEC analysis: Rt = 16.9 minutes, 87.5% monomer content Precursor: Preparation 22

[0303] (Flow cytometry assay using α-galactosyl IgM antibody) Flow cytometry was used to show the binding of L (as cetuximab) and F (as a carbohydrate molecule capable of binding to human anti-α-galactosyl antibodies) to receptors on human cell lines. Since it is well known that A431 cells significantly overexpress the EGFR receptor, these cells were used to capture the EGFR-binding mAb (cetuximab). A secondary phycoerythrin (PE)-labeled anti-human IgM antibody was used to detect the binding of the α-galactosyl IgM antibody to the compound.

[0304] A431 cells (ATCC CRL-1555) were harvested and resuspended in phosphate-buffered saline (PBS) (Sigma D8662) + 0.1% BSA (bovine serum albumin - Sigma A2153) at 5×10 6 cells / mL. Then, 5×10 5 cells were incubated for 1 hour at room temperature with various concentrations of the compound or buffer alone, with shaking at 450 rpm.

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

[0306] Figure 4 shows the capture of anti-α-galactosyl IgM antibody on the cell surface using Examples 1 (Figure 4A), 2 (Figure 4B), 3 (Figure 4C), 4 (Figure 4D), 5 (Figure 4E), 6 (Figure 4F), 7 (Figure 4G), and 8 (Figure 4H) at 10 nM. The change in fluorescence intensity (PE) occurs due to the binding events at each end of the molecule.

[0307] (Flow Cytometry Assay Using α-Galactosyl IgG Antibody) Flow cytometry was used to elucidate the binding of L (as cetuximab) and F (as a carbohydrate molecule capable of binding to human anti-α-galactosyl antibody) to receptors on human cell lines. Since it is well known that A431 cells significantly overexpress the EGFR receptor, these cells were used to capture the EGFR-binding mAb (cetuximab). A phycoerythrin (PE)-labeled α-galactosyl IgG antibody was used to detect the binding of the compound.

[0308] A431 cells (ATCC CRL-1555) were collected and resuspended at 5 × 10 6 cells / mL in phosphate-buffered saline (PBS) (Sigma D8662) + 0.1% BSA (bovine serum albumin - Sigma A2153). Next, 5 × 10 5Cells were incubated at room temperature for 1 hour with 10 nM of the compound, buffer alone, or 10 nM cetuximab, while shaking at 450 rpm. The cells were washed with 2 × 200 μL PBS + 0.1% BSA, and then 50 μL of a 575 μg / mL PE-labeled anti-α-galactosyl IgG antibody in PBS + 0.1% BSA was added and incubated at 4°C for 1 hour. The anti-α-galactosyl IgG antibody (Absolute Antibody Ab00532.10.0) was custom-labeled with PE by Cambridge Research Biochemicals. After a final wash with 2 × 200 μL PBS + 0.1% BSA, the cells were resuspended in 200 μL PBS + 0.1% BSA and evaluated on a flow cytometer (FC500 Beckman Coulter). Data from all samples were analyzed in the Kaluza software package (Beckman Coulter).

[0309] Figure 5 shows the capture of anti-α-galactosyl IgG antibody on the cell surface using Examples 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), and 4 (Figure 5D) at 10 nM, compared to buffer alone. Changes in fluorescence intensity (PE) occur due to binding events at each end of the molecule.

[0310] Figure 6 shows the capture of anti-α-galactosyl IgG antibody on the cell surface using Examples 5 (Figure 6A), 6 (Figure 6B), 7 (Figure 6C), and 8 (Figure 6D) at 10 nM, compared to 10 nM cetuximab. Changes in fluorescence intensity (PE) occur due to binding events at each end of the molecule.

[0311] (Flow cytometry assay using α-galactosyl IgG antibody derived from hIVIG and A431 cells) Flow cytometry was used to reveal the binding of L (as a Fab fragment) and F (as a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody) to EGFR expressed on the human cell line (A431). A secondary phycoerythrin (PE)-labeled anti-human IgG antibody was used to detect the binding of the α-galactosyl IgG antibody to the compound.

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

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

[0314] Figure 7 shows the dose-dependent compound-driven mobilization of anti-Gal IgG antibodies from hIVIG of Examples 18 - 23 to A431 cells compared to unconjugated Fab fragment where minimal mobilization was observed.

[0315] (Flow cytometry assay using α-galactosyl IgM antibody and A431 cells) The compound was tested according to the above flow cytometry assay protocol at various concentrations up to 1000 nM, compared to cetuximab and / or unconjugated Fab fragments.

[0316] Figure 8 shows dose-dependent compound-driven mobilization of α-galactosyl IgM antibody to A431 cells, compared to unconjugated Fab fragments and / or cetuximab.

[0317] (Flow cytometry assay using C3b antibody) Flow cytometry was used to reveal the binding of the compound to the target cell line and the mobilization of the C3b complement component to the cells. Since it is well known that A431 cells significantly overexpress the EGFR receptor, the cells were used to capture the EGFR-binding antibody or antibody fragment. An anti-C3b antibody conjugated to phycoerythrin (PE) was used to detect the mobilization of C3b molecules to serum-derived cells after the addition of various concentrations of the compound.

[0318] A431 cells (ATCC CRL-1555) were collected and resuspended at 5×10 6 cells / mL in phosphate-buffered saline (PBS) (Sigma D8662) + 0.1% BSA (bovine serum albumin - Sigma A2153). Next, 5×10 5 cells were incubated for 1 hour at room temperature with various compound concentrations up to 10000 ng / ml, buffer alone, or various concentrations of cetuximab Fab fragment and / or cetuximab, with shaking at 450 rpm. The cells were washed with 2×200 μL PBS + 0.1% BSA, and then 100 μL PBS and 100 μL 20% human serum (HS) (Patricell 23590) or heat-inactivated human serum (HIHS) were added together with 25 μg / ml M86 IgM (Absolute Antibody) and incubated at 37°C for 25 minutes.

[0319] The cells were washed with 2x200μL PBS+0.1%BSA, then 100μL of anti-C3b-PE (3E7 / C3b, Biolegend 846104) was added. The cells were incubated in the dark at 4°C for 30 minutes. After a final wash of 2x200μL PBS+0.1%BSA, the cells were resuspended in 200μL PBS+0.1%BSA and evaluated on a flow cytometer (FC500 Beckman Coulter). Data from all samples were analyzed in the Kaluza software package (version 1.5a, Beckman Coulter).

[0320] Figure 9 shows the levels of C3b deposition on A431 cells using various concentrations of Examples 13-17, 20, 22, 23, and 24 compared to cetuximab Fab fragments and / or cetuximab. A <5-fold change over background was observed for all examples when heat-inactivated human serum was employed (representative HI HS data shown here).

[0321] (Phagocytosis of target cells by macrophages) Phagocytosis was used to determine the functional effect of binding of L (as a Fab fragment of cetuximab) and F (as a carbohydrate molecule capable of binding human anti-α-galactosyl antibodies) to receptors on cell lines. A431 cells, which are reported to express EGFR, were used as target cells. Monocyte-derived macrophages were used as effector cells. Purified hIVIG was used as a source of anti-Gal antibodies. The increase in the measured integrated intensity occurs due to phagocytosis of the target cells. No such increase was observed under control conditions (cells alone or in the presence of unconjugated Fab fragments).

[0322] Effector cells were differentiated in situ in a 96-well plate (Corning 3603). Briefly, blood from each healthy donor stored in a leukoreduction system chamber was purchased from the National Health Service (Addenbrooke’s Hospital, Cambridge, UK). Peripheral blood mononuclear cells (PBMCs) were isolated using the Lymphoprep™ system according to the manufacturer's instructions (STEMCELL Technologies 07861). Monocytes were isolated from PBMCs by positive selection using the EasySep™ Human CD14 Positive Selection Kit II (STEMCELL Technologies 17858) and resuspended in ImmunoCult™ SF Macrophage Medium (STEMCELL Technologies 10961) supplemented with 100 ng / ml recombinant human GM-CSF (Peprotech 300-03) and plated at 20,000 cells / well in a cell culture incubator (5% CO2, 37°C). Five days after differentiation, macrophages were polarized towards the M1 phenotype using 100 ng / ml IFN-γ (Peprotech 300-02) and 1 ng / ml lipopolysaccharide (Invitrogen tlrl-eblps) for an additional two days. A431 cells (ATCC CRL-1555) were cultured in Dulbecco's modified Eagle's medium (Gibco® 61965-026) supplemented with 10% fetal bovine serum (Gibco® 10500-064) and used as target cells. Target cells were harvested using Cell Dissociation Buffer (Gibco® 13151014), counted, and 1.0 × 10 6Cells were labeled with 1 μl of 10 mM pHrodo Green STP ester (Thermo Fisher Scientific P35369) per cell at 37 °C for 30 minutes. The cells were washed in complete medium, counted, and then treated with various concentrations of Example 23, Example 20, or cetuximab-Fab with shaking at room temperature for 1 hour. The cells were then washed in serum-free medium and incubated with 70 μg / ml hIVIG (custom purified from human IVIG) on ice for 30 minutes, washed in Dulbecco's phosphate solution (Gibco® 14190-094), and co-cultured with effector cells (target: effector ratio 5:1) at 37 °C in IncuCyte® (Sartorius) for up to 12 hours. Images were acquired every 2 hours. Data were analyzed using IncuCyte® ZOOM software (version 2016A, Sartorius). Graphs were created using GraphPad Prism (version 6).

[0323] Figure 10 shows representative compound-mediated phagocytosis in the presence of 1 nM of Example 20 and Example 23 compared to unconjugated Fab fragments. Target cells (EGFR-expressing A431 cells) were phagocytosed by effector cells (macrophages). The increase in integrated intensity results from compound-driven phagocytosis of the target cells.

[0324] (Flow cytometry assay using α-galactosyl IgM antibody in Raji cells) Flow cytometry was used to reveal the binding of L (as rituximab or rituximab Fab) and F (as a glycoprotein molecule capable of binding to human anti-α-galactosyl antibodies) to receptors on human cell lines. Since Raji cells are well known to overexpress the CD20 receptor significantly, these cells were used to capture CD20-binding mAb or Fab. Binding of the α-galactosyl IgM antibody to the compound was detected using a secondary phycoerythrin (PE)-labeled anti-human IgM antibody.

[0325] Raji cells (ATCC® CCL-86™) were collected and resuspended in phosphate-buffered saline (PBS) (Gibco® 14190-094) + 0.1% BSA (bovine serum albumin - Sigma A2153) at 5×10 6 cells / mL. Then, 5×10 5 cells were incubated at room temperature for 1 hour with various concentrations of the compound or buffer alone as shown below, with shaking at 450 rpm. The cells were washed with 2×200 μL of PBS + 0.1% BSA, and then 50 μL of anti-α-galactosyl IgM antibody (Absolute Antibody Ab00532-15.0) at 32 μg / mL in PBS + 0.1% BSA was added and incubated at 4°C for 1 hour. The cells were further washed with 2×200 μL of PBS + 0.1% BSA and then treated with a 1:40 dilution of 100 μL of anti-human IgM-PE (Biolegend 314508) at 4°C for 1 hour. After a final wash with 2×200 μL of PBS + 0.1% BSA, the cells were resuspended in 200 μL of PBS + 0.1% BSA and evaluated using a flow cytometer (BD FACSVerse™, BD). Data from all samples were analyzed using the FlowJo® software package. Graphs were created using GraphPad Prism (version 6).

[0326] Figure 11 shows the dose-dependent compound-driven mobilization of α-galactosyl IgM antibody to Raji cells compared to rituximab and its Fab fragment. This application provides an invention in the following aspects. (Aspect 1) An immunoconjugate comprising an antibody or an antigen-binding fragment thereof linked via a linker to a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody, wherein the linker comprises at least one phenyl ring capable of presenting one or more carbohydrate epitopes capable of binding to a human anti-α-galactosyl antibody. The immunoconjugate is characterized by the above. (Aspect 2) The immunoconjugate according to Aspect 1, wherein the linker is a phenyl, biphenyl, or triphenyl group; or a biphenyl group-containing group. (Aspect 3) The immunoconjugate according to Aspect 1 or Aspect 2, which is a compound of formula (A) or a pharmaceutically acceptable salt thereof: (Chemical Formula 1) TIFF0007695773000087.tif44170 (In the formula, F is a carbohydrate molecule capable of binding to a human anti-α-galactosyl antibody; Cy is phenyl, biphenyl, or triphenyl; S A and S B represent a spacer selected for 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). (Aspect 4) The immunoconjugate according to any one of Aspects 1 to 3, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof: (Chemical Formula 2) TIFF0007695773000088.tif38170 (In the formula, L represents a binding site selected from an antibody or an antigen-binding fragment thereof; S 1 is such that 1 to 10 of the -CH 2 - groups may be optionally substituted by one or more groups selected from -O-, -S-, =N(H)-, -C(=O)-, -C(O)NH-, -NHC(O)-, cyclohexyl, or pyrrolidine-2,5-dione, -(CH 2 ) a - or -(CH 2 ) b -(CH 2 -CH 2 -O) c -(CH 2 ) d - represents a spacer selected from the - groups; 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; S 2 is such that 1 to 3 of the -CH 2 - groups may be optionally substituted by one or more groups selected from -N(H)-, -C(O)NH-, and -NHC(O)-, -(CH 2 ) e - or -(CH 2 ) f -(CH 2 -CH 2 -O) g -(CH 2 ) h - represents a spacer selected from the - groups; 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; X 1 represents an antibody or antigen-binding fragment attachment site; Y 1 and Y 2 are independently a bond, -O-, -S-, -NH-, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -NHSO 2 -, -SO 2 NH-, 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 Cy represents biphenyl or triphenyl, the -Y 1 -S 1 -X 1 -L group may be present on any of the phenyl rings, and one or more of the [F-S 2 -Y 2 ] m - groups may be present on any of the phenyl rings). (Embodiment 5) S 1 is: -CH 2 One to five (e.g., 2, 3, or 5) of the - groups may be optionally substituted with one or more groups selected from -S-, =N(H)-, -C(=O)-, -NHC(O)-, cyclohexyl, or pyrrolidine-2,5-dione, -(CH 2 ) a -(e.g., -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 -3-pyrrolidine-2,5-dione-, -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 -3-pyrrolidine-2,5-dione-S-(CH 2 ) 3 -C(=NH)-, or -(CH 2 ) 2 -NHCO-(CH 2 ) 3 -CO, etc.); or -CH 2 One to five (e.g., 2) of the - groups may be optionally substituted with one or more groups selected from -NHC(O)- or pyrrolidine-2,5-dione, -(CH 2 ) b -(CH 2 -CH 2 -O) c -(CH 2 ) d -(e.g., -(CH 2 ) 2 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -3-pyrrolidine-2,5-dione-, etc.) represents a spacer selected from, the immunoconjugate described in Embodiment 4. (Embodiment 6) X 1 is, represents -S- or -N(H)-, the immunoconjugate described in Embodiment 4 or 5. (Embodiment 7) The immunoconjugate according to any one of Aspects 4 to 6, wherein a represents an integer selected from 1 to 30; or 2 to 30; or 2, 4, 6, 9, 11, 18, or 30; or 6 to 30; or 6, 11, 18, or 30; or 5 to 15; or 6 to 11; or 6, 7, or 11; or 6; or 7; or 11. (Aspect 8) The immunoconjugate according to any one of Aspects 4 to 7, wherein b represents an integer selected from 0 to 3; or 0 or 3; or 1 to 3; or 2 or 3; or 3. (Aspect 9) The immunoconjugate according to any one of Aspects 4 to 8, wherein c represents an integer selected from 1 to 15; or 1 to 12; or 4 to 12; or 4 or 12; or 4. (Aspect 10) The immunoconjugate according to any one of Aspects 4 to 9, wherein d represents an integer selected from 1 to 15; or 2 to 13; or 2, 5, or 13; or 13; or 3. (Aspect 11) Y 1 The immunoconjugate according to any one of Aspects 4 to 10, wherein represents a bond, -C(O)NH-, or -O-; or -C(O)NH-. (Aspect 12) S 2 is: -CH 2 One or two of the - groups are optionally substituted with one or two groups selected from -N(H)-, -C(O)NH-, and -NHC(O)-, -(CH 2 ) e -(for example, -(CH 2 ) 3 -NHCO-CH 2 -, -(CH 2 ) 3 -, -(CH 2 ) 3 -NHCO-(CH 2 ) 4 -CONH-CH 2 -, -(CH 2 ) 3 -NH-CH 2 -, or -(CH 2 ) 3 -NHCO-(CH 2 ) 3 -NHCO-CH 2 - etc.); or -CH 2 One to three of the - groups are optionally substituted with one to three -NHC(O)- groups, -(CH 2 ) f -(CH 2 -CH 2 -O) g -(CH 2 ) h -(for example, -(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2 -, -(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 12 -(CH 2 ) 2 -NHCO-CH 2 -, or -(CH 2 ) 3 -NHCO-(CH 2 ) 3 -NHCO-(CH 2CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2 - etc.) represents a spacer selected from; or S 2 is: -CH 2 One or two of the - groups are optionally substituted with a -C(O)NH- or -NHC(O)- group, -(CH 2 ) e -(for example, -(CH 2 ) 3 -NHCO-CH 2 -, -(CH 2 ) 3 -NHCO-, -(CH 2 ) 3 -, -(CH 2 ) 3 -NHCO-(CH 2 ) 4 -CONH-CH 2 -, or -(CH 2 ) 3 -NH-CH 2 - etc.), or one or two of the -CH 2 - groups are optionally substituted with a -C(O)NH- or -NHC(O)- group, -(CH 2 ) f -(CH 2 -CH 2 -O) g -(CH 2 ) h -(for example, -(CH 2 ) 3 -NHCO-(CH 2 ) 2 -(OCH 2 CH 2 ) 4 -NHCO-CH 2 -, or -(CH 2 ) 4 -NHCO-(CH 2 ) 2 -(OCH 2 CH 2 ) 4 -NHCO-CH 2 - etc.) represents a spacer selected from; or S 2 is: -CH 2 - in which one or two of the -CH- groups are optionally substituted by one or two -NHC(O)- groups, -(CH 2 ) e -(for example, -(CH 2 ) 3 -NHCO-CH 2 -, or -(CH 2) 3 -NHCO-(CH 2 ) 3 -NHCO-CH 2 - etc.); or -CH 2 - in which 1 to 3 of the -CH- groups are optionally substituted by 1 to 3 -NHC(O)- groups, -(CH 2 ) f -(CH 2 -CH 2 -O) g -(CH 2 ) h -(for example, -(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2 -, -(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 12 -(CH 2 ) 2 -NHCO-CH 2 -, or -(CH 2 ) 3 -NHCO-(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2 - etc.) represents a spacer selected from; or S 2 is: -CH 2 - in which one or two of the -CH- groups are optionally substituted by one or two groups selected from -N(H)-, -C(O)NH-, and -NHC(O)-, -(CH 2 ) e -(for example, -(CH 2 ) 3 -NHCO-CH 2 - etc.); or -CH 2 - in which 1 to 3 of the -CH- groups are optionally substituted by 1 to 3 -NHC(O)- groups, -(CH 2 ) f -(CH 2 -CH 2 -O) g -(CH 2 ) h -(for example, -(CH 2 ) 3-NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2 - etc.) represents a spacer selected from; or S 2 is: -CH 2 - in which one or two of the -CH- groups, for example, one is optionally substituted by one or two, for example, one group selected from -N(H)-, -C(O)NH-, and -NHC(O)-, -(CH 2 ) e -(for example, -(CH 2 ) 3 -NHCO-CH 2 - etc.) represents a spacer selected from; or S 2 is: -CH 2 - in which one of the -CH- groups is optionally substituted by an -NHC(O)- group, -(CH 2 ) e -(for example, -(CH 2 ) 3 -NHCO-CH 2 - etc.); or -CH 2 - in which two of the -CH- groups are optionally substituted by -NHC(O)- groups, -(CH 2 ) f -(CH 2 -CH 2 -O) g -(CH 2 ) h -(for example, -(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2 - etc.) represents a spacer selected from any one of aspects 4 to 11, the immunoconjugate described in any one of the preceding paragraphs. (Aspect 13) The immunoconjugate according to any one of aspects 4 to 12, wherein e represents an integer selected from 1 to 10; or 3 to 10; or 3, 5, 9, or 10; or 5 to 9; or 5 or 9; or 4 to 10; or 4, 5, or 10; or 5. (Aspect 14) The immunoconjugate according to any one of aspects 4 to 13, wherein f represents an integer selected from 1 to 8; or 2 to 8; or 2 to 6; or 4 to 8; or 4 or 8; or 4. (Aspect 15) The immunoconjugate according to any one of aspects 4 to 14, wherein g represents an integer selected from 1 to 15; or 4 to 12; or 4 or 12; or 1 to 5; or 1 to 4; or 4. (Aspect 16) The immunoconjugate according to any one of aspects 4 to 15, wherein h represents an integer selected from 1 to 4; or 4. (Aspect 17) Y 2 The immunoconjugate according to any one of aspects 4 to 16, wherein represents a bond, -O-, or -NHC(O)-; or a bond or -O-; or -O-. (Aspect 18) The immunoconjugate according to any one of aspects 4 to 17, wherein m represents an integer selected from 1 to 4; or 1 to 3; 1 or 3; or 2 or 3; or 1 or 2; or 1; or 2; or 3; or 4. (Aspect 19) The immunoconjugate according to any one of aspects 4 to 18, wherein z represents an integer selected from 2 to 20; or 2; or 4.9; or 5; or 7; or 8; or 10; or 11; or 14; or 15; or 17; or 20. (Aspect 20) The immunoconjugate according to any one of aspects 4 to 19, wherein Cy represents phenyl or biphenyl; or biphenyl or triphenyl; or phenyl or triphenyl; or biphenyl. (Aspect 21) Compound of formula (I) a or a pharmaceutically acceptable salt thereof: (Chemical formula 3) TIFF0007695773000089.tif41170 (wherein, L represents a binding site selected from an antibody or an antigen-binding fragment thereof; S 1 is: -CH 2 Two, three, or five of the - groups may be optionally substituted by one or more groups selected from -S-, =N(H)-, -C(=O)-, -NHC(O)-, cyclohexyl, or pyrrolidine-2,5-dione, -(CH 2 ) a -; or -CH 2 Two of the - groups may be optionally substituted by one or more groups selected from -NHC(O)- or pyrrolidine-2,5-dione, -(CH 2 ) b -(CH 2 -CH 2 -O) c -(CH 2 ) 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; S 2 is: -CH 2 -wherein one of the - groups is optionally substituted by an -NHC(O)- group, -(CH 2 ) e -; or -CH 2 -wherein two of the - groups are optionally substituted by an -NHC(O)- group, -(CH 2 ) f -(CH2 -CH 2 -O) g -(CH 2 ) 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; X 1 represents -S- or -N(H)-; Y 1 represents -C(O)NH-; Y 2 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 -Y 1 -S 1 -X 1 -L group may be present on either of the phenyl rings, and one or more of the [F-S 2 -Y 2 ] m - groups may be present on either of the phenyl rings). (Embodiment 22) The immunoconjugate according to any one of Embodiments 1 to 21, wherein the antibody is a polyclonal antibody. (Embodiment 23) The immunoconjugate according to any one of Embodiments 1 to 22, wherein the antibody is a humanized antibody, a human antibody, a mouse antibody, or a chimeric antibody. (Embodiment 24) The immunoconjugate according to any one of Embodiments 1 to 23, wherein the antigen-binding fragment thereof is an antigen-binding fragment (Fab) or a single-chain variable fragment (scFv). (Embodiment 25) The immunoconjugate according to Embodiment 24, wherein the fragment is selected from the group consisting of Fab, Fab′, F(ab)2, F(ab′)2, and scFv. (Embodiment 26) The immunoconjugate according to any one of Embodiments 1 to 25, wherein the antibody or its antigen-binding fragment is an EGFR antibody or its fragment, for example, cetuximab, cetuximab Fab, or nimotuzumab, particularly an EGFR antibody or its fragment having at least 80% sequence identity to SEQ ID NOs: 1 to 4. (Embodiment 27) The immunoconjugate according to any one of Embodiments 1 to 25, wherein the antibody or its antigen-binding fragment is a CD20 antibody or its fragment, for example, rituximab or rituximab Fab. (Embodiment 28) The immunoconjugate according to any one of aspects 1 to 25, wherein the antibody or antigen-binding fragment thereof is selected from pathogen-specific antibodies or fragments thereof. (Aspect 29) The immunoconjugate according to any one of aspects 1 to 28, wherein F is selected from galactosyl-α-1,3-galactosyl-β-1,4-N-acetylglucosamine, α1-3 galactobiose, α1-3-β1-4-galactotriose, or galili pentasaccharide. (Aspect 30) The compound according to aspect 1 or 21, or a pharmaceutically acceptable salt thereof, selected from any one of Examples 1 to 26. (Aspect 31) A pharmaceutical composition comprising the compound according to any one of aspects 1 to 30 or a pharmaceutically acceptable salt thereof. (Aspect 32) The pharmaceutical composition according to aspect 31, further comprising one or more additional therapeutic agents. (Aspect 33) The compound according to any one of aspects 1 to 30 or a pharmaceutically acceptable salt thereof for use in therapy. (Aspect 34) The compound according to aspect 26 or a pharmaceutically acceptable salt thereof for use in the treatment of cancer. (Aspect 35) The compound according to aspect 27 or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, such as blood cancer, particularly leukemia and lymphoma. (Aspect 36) The compound according to aspect 28 or a pharmaceutically acceptable salt thereof for use in the treatment of bacterial infections. (Aspect 37) A process for preparing the compound of formula (I) according to aspect 4, comprising: (a) reacting the antibody or antigen-binding fragment thereof with a compound of formula (III) having at least one reactive thiol group, S 1 with a compound of formula (II) terminated with pyrrolidine-2,5-dione to prepare a compound of formula (IA) wherein X 1 represents -S-: (Chemical formula 4) TIFF0007695773000090.tif47170 (wherein F, S 2 、Y 2 , m, Cy, Y 1 , and S 1 are as defined in aspect 4); or (b) reacting the antibody or antigen-binding fragment thereof with a compound of formula (IIIA) having at least one reactive thiol group, S 1 with a compound of formula (II) terminated with pyrrolidine-2,5-dione to prepare a compound of formula (IC) wherein X 1 represents -NH 2 and S 1 represents -S-CH 2 -CH 2 -CH 2 -C(=NH)-: (Chemical formula 5) TIFF0007695773000091.tif43170 (wherein F, S 2 、Y 2 , m, Cy, Y 1 、S 1 , and L are as defined in aspect 4); or (c) S 1 Reacting a compound of formula (IIB) terminated with an N-hydroxysuccinimide group with a compound of formula (IIIB) in which the antibody or antigen-binding fragment has at least one reactive amino group, whereby X 1 is -NH 2 To prepare a compound of formula (IB) representing: (Chemical formula 6) TIFF0007695773000092.tif46170 (In the formula, F, S 2 、Y 2, m, Cy, Y 1 、S 1 , and L are as defined in Embodiment 4); and / or (d) The interconversion of a compound of formula (I) or a protected derivative thereof to a further compound of formula (I) or a protected derivative thereof The process comprising: (Embodiment 38) A compound of formula (II) or formula (IIB) according to Embodiment 37, or a compound of formula (V), (VA), (IX), or (XI) described in the specification.

Claims

1. An immunoconjugate that is a compound of formula (I) or a pharmaceutically acceptable salt thereof: 【Chemical Formula 1】 (In the formula, L represents a binding site selected from an antibody or an antigen-binding fragment thereof; S 1 is -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 -3-pyrrolidine-2,5-dione-, -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 -3-pyrrolidine-2,5-dione-S-(CH 2 ) 3 -C(=NH)-, -(CH 2 ) 2 -NHCO-(CH 2 ) 3 -CO-, or -(CH 2 ) 2 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -3-pyrrolidine-2,5-dione-; S 2 is -(CH 2 ) 3 -NHCO-CH 2 -, -(CH 2 ) 3 -, -(CH 2 ) 3 -NHCO-(CH 2 ) 4 -CONH-CH 2 -, -(CH 2 ) 3 -NH-CH 2 -, -(CH 2 ) 3 -NHCO-(CH 2 ) 3 -NHCO-CH 2 -, -(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2 -、-(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 12 -(CH 2 ) 2 -NHCO-CH 2 -、 or -(CH 2 ) 3 -NHCO-(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2 - represents; z represents an integer selected from 2 to 20; X 1 represents -S- or -N(H)-; Y 1 represents a bond, -C(O)NH-, or -O-; Y 2 represents a bond, -O-, or -NHC(O)-; F is galactosyl-α-1,3-galactosyl-β-1,4-N-acetylglucosamine, α1-3 galactobiose, α1-3-β1-4-galactotriose, galili pentasaccharide, or a structure represented by one of the following formulas [Chemical Formula 2] (In the formula, S 2 represents the attachment point to the S 2 group) represents; m represents an integer selected from 1 to 4; and Cy represents phenyl, biphenyl, or triphenyl. When Cy represents biphenyl or triphenyl, the -Y 1 -S 1 -X 1 -L group may be present on any of the phenyl rings and one or more of the [F-S 2-Y 2 m -group may be present on any of the phenyl rings). **Claim 2** Y 1 represents -C(O)NH-, the immunoconjugate according to claim 1. **Claim 3** Y 2 represents a bond or -O-; or -O-, the immunoconjugate according to claim 1 or 2. **Claim 4** m represents an integer selected from 1 to 3; 1 or 3; or 2 or 3; or 1 or 2; or 1; or 2; or 3; or 4, the immunoconjugate according to any one of claims 1 to 3. **Claim 5** z represents 2; or 4.9; or 5; or 7; or 8; or 10; or 11; or 14; or 15; or 17; or 20, the immunoconjugate according to any one of claims 1 to 4. **Claim 6** Cy represents phenyl or biphenyl; or biphenyl or triphenyl; or phenyl or triphenyl; or biphenyl, the immunoconjugate according to any one of claims 1 to 5. **Claim 7** The compound of formula (I) a or a pharmaceutically acceptable salt thereof, an immunoconjugate: **Chemical Formula 3** (wherein, L represents a binding site selected from an antibody or an antigen-binding fragment thereof; S 1 is -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 -3-pyrrolidine-2,5-dione-, -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 -3-pyrrolidine-2,5-dione-S-(CH 2 ) 3 -C(=NH)-, -(CH​ 2 ) 2 -NHCO-(CH 2 ) 3 -CO-、 or -(CH 2 ) 2 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -3-pyrrolidine-2,5-dione-; S 2 is -(CH 2 ) 3 -NHCO-CH 2 -、-(CH 2 ) 3 -、-(CH 2 ) 3 -NHCO-(CH 2 ) 4 -CONH-CH 2 -、-(CH 2 ) 3 -NH-CH 2 -、-(CH 2 ) 3 -NHCO-(CH 2 ) 3 -NHCO-CH 2 -、-(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2 -、-(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 12 -(CH 2 ) 2 -NHCO-CH 2 -、 or -(CH 2 ) 3 -NHCO-(CH 2 ) 3 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -NHCO-CH 2represents -; z represents an integer selected from 2 to 20; X 1 represents -S- or -N(H)-; Y 1 represents -C(O)NH-; Y 2 represents -O-; F is galactosyl-α-1,3-galactosyl-β-1,4-N-acetylglucosamine, α1-3 galactobiose, α1-3-β1-4-galactotriose, galili pentasaccharide, or a structure represented by one of the following formulas 【Chemical Formula 4】 (wherein S 2 is the attachment point to the S 2 group) represents; m represents an integer selected from 1 or 3; and Cy represents biphenyl, and the -Y 1 -S 1 -X 1 -L group may be present on either of the phenyl rings, and one or more of the [F-S 2 -Y 2 m -group may be present on either of the phenyl rings).

8. The immunoconjugate according to any one of claims 1 to 7, wherein the antibody is a polyclonal antibody.

9. The immunoconjugate according to any one of claims 1 to 8, wherein the antibody is a humanized antibody, a human antibody, a mouse antibody, or a chimeric antibody.

10. The immunoconjugate according to any one of claims 1 to 9, wherein the antigen-binding fragment is an antigen-binding fragment (Fab) or a single-chain variable fragment (scFv).

11. The immunoconjugate according to claim 10, wherein the fragment is selected from the group consisting of Fab, Fab′, F(ab)2, F(ab′)2, and scFv.

12. ​ The immunoconjugate according to any one of claims 1 to 11, wherein the antibody or its antigen-binding fragment is selected from an EGFR antibody or its fragment.

13. The immunoconjugate according to claim 12, wherein the EGFR antibody or its fragment is cetuximab, cetuximab Fab, or nimotuzumab.

14. The immunoconjugate according to claim 12, wherein the EGFR antibody or its fragment has at least 80% sequence identity to SEQ ID NOs: 1 to 4.

15. The immunoconjugate according to any one of claims 1 to 11, wherein the antibody or its antigen-binding fragment is selected from a CD20 antibody or its fragment.

16. The immunoconjugate according to claim 15, wherein the CD20 antibody or its fragment is rituximab or rituximab Fab.

17. The immunoconjugate according to any one of claims 1 to 11, wherein the antibody or its antigen-binding fragment is selected from a pathogen-specific antibody or its fragment.

18. The immunoconjugate according to any one of claims 1 to 17, wherein F is selected from galactosyl-α-1,3-galactosyl-β-1,4-N-acetylglucosamine, α1-3 galactobiose, α1-3-β1-4-galactotriose, or galili pentasaccharide.

19. The immunoconjugate according to claim 1 or 7, which is a compound selected from any one of the following, or a pharmaceutically acceptable salt thereof: [Chemical Formula 5] [Chemical Formula] [Chemical Formula] [Chemical Formula] [Chemical Formula] [Chemical Formula] [Chemical] 。

20. A pharmaceutical composition comprising the immunoconjugate according to any one of claims 1 to 19.

21. The pharmaceutical composition according to claim 20, further comprising one or more additional therapeutic agents.

22. A pharmaceutical composition comprising the immunoconjugate according to any one of claims 1 to 19 for use in therapy.

23. A pharmaceutical composition comprising the immunoconjugate according to any one of claims 12 to 14 for use in the treatment of cancer.

24. A pharmaceutical composition comprising the immunoconjugate according to claim 15 or 16 for use in the treatment of cancer.

25. The pharmaceutical composition according to claim 24, wherein the cancer is a blood cancer.

26. The pharmaceutical composition according to claim 25, wherein the blood cancer is leukemia or lymphoma.

27. A pharmaceutical composition comprising the immunoconjugate according to claim 17 for use in the treatment of a bacterial infection.

28. A process for preparing the immunoconjugate according to claim 1, comprising: (a) reacting the compound of formula (III) in which the antibody or antigen-binding fragment has at least one reactive thiol group with the compound of formula (II) to form S 1 being -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 -3-pyrrolidine-2,5-dione- or -(CH 2 ) 2 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 -3-pyrrolidine-2,5-dione- and X 1 represents -S- to prepare a compound of formula (IA): 【Chemical Formula 6】 (wherein F, S 2 , Y 2 , m, Cy, Y 1 , and L are as defined in claim 1, and S 1a is -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 - or -(CH 2 ) 2 -NHCO-(CH 2 CH 2 O) 4 -(CH 2 ) 2 - represents); or (b) reacting a compound of formula (IIIA) in which the antibody or antigen-binding fragment has at least one reactive thiol group with a compound of formula (II) so that S 1 is -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 -3-pyrrolidine-2,5-dione-S-(CH 2 ) 3 -C(=NH)- represents, and X 1 represents -NH- to prepare a compound of formula (IC): 【Chemical Formula 7】 (wherein F, S 2 , Y 2 , m, Cy, Y 1 , and L are as defined in claim 1, and S 1b is -(CH 2 ) 2 -NHCO-cyclohexyl-CH 2 - represents); or (c) reacting a compound of formula (IIB) with a compound of formula (IIIB) in which the antibody or antigen-binding fragment has at least one reactive amino group so that S 1 is -(CH 2 ) 2 -NHCO-(CH 2 ) 3 represents -CO-, and preparing a compound of formula (IB) wherein X 1 represents -NH-: 【Chemical Formula 8】 (wherein F, S 2 , Y 2 , m, Cy, Y 1 , and L are as defined in claim 1, and S 1c represents -(CH 2 ) 2 -NHCO-(CH 2 ) 3 -); and / or (d) the interconversion of a compound of formula (I) or a protected derivative thereof to a further compound of formula (I) or a protected derivative thereof The process as described above, which includes this step.

29. A compound of formula (II) or formula (IIB) according to claim 28.

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