Anti-CanAg antibody complex

Abstract

The present invention relates to antibody conjugates that target the CanAg antigen, and compositions (e.g., pharmaceutical compositions) that include the antibody conjugates. Methods of using the antibody conjugates and compositions, including for the treatment of cancer, are also provided.

Description

[Technical field]

[0001] The present invention relates to antibody conjugates that target the CanAg antigen, and compositions comprising the antibody conjugates. Methods of using the antibody conjugates and compositions, including for the treatment of cancer, are also provided. [Background technology]

[0002] Antibody-drug conjugate (ADC) technology is a targeting-oriented technology that exploits the ability of antibodies to sensitively discriminate between healthy and diseased tissues to selectively deliver a cytotoxic payload. ADCs are defined by three key components: an antibody, a cytotoxic drug (also called a payload), and a linker that connects the drug to the antibody. Known for their use as anticancer drugs, ADCs work by using antibodies to target specific antigens associated with cancer cells and release the drug payload under certain conditions to induce cell death. This allows for the targeted delivery of highly effective drugs to tumors, thereby reducing systemic exposure and toxicity to normal tissues. ADCs therefore hold great potential for improving the treatment and survival of patients suffering from diseases such as cancer.

[0003] Typically, despite the possibility of using payloads with toxicity that cannot be tolerated by patients, a low therapeutic index (the ratio of toxic dose to effective dose) remains a problem and is the cause of the discontinuation of many ADCs during clinical development. The selection of the appropriate target, antibody, cytotoxic payload, and the method of antibody-payload conjugation have all been identified as important factors that determine the safety and efficacy of ADCs. This illustrates the level of complexity involved in developing an ADC that achieves the right combination of an appropriate target antigen, a stable linker, a potent cytotoxic payload, and an effective release technology.

[0004] The favorable expression pattern of the CanAg antigen suggests that it is a suitable target for antibody-based selective anticancer therapy. CanAg is highly expressed in most pancreatic, biliary tract and colorectal cancers, as well as in a significant proportion of gastric, uterine, non-small cell lung and bladder cancers. In contrast, CanAg expression in normal tissues has been reported to be low. Nevertheless, to date, no anti-CanAg ADCs have been approved for use in cancer therapy. Cantuzumab mertansine and cantuzumab ravtansine are the two known ADCs that target CanAg, but neither compound has progressed beyond phase 2 clinical trials, likely due to the limited efficacy observed against colorectal and pancreatic cancers.

[0005] Thus, there remains a need to identify and develop antibody-linker-drug combinations with potent efficacy, pharmacokinetic / pharmacodynamic, and broad therapeutic indexes. Summary of the Invention [Problem to be solved by the invention]

[0006] In a first embodiment, there is provided an antibody conjugate according to formula I, or a pharma- ceutically acceptable salt or solvate thereof: [ka] During the ceremony, -Ab is a humanized C242 antibody or an antigen-binding fragment thereof; -D is a pyrrolobenzodiazepine dimer prodrug; - L is a linker attaching Ab to D; - n is an integer from 1 to 20; where -D is represented by formula (II): [ka] the linker comprises a central moiety of formula III, IV, V, VI or VII: [ka] During the ceremony, - L1 comprises a first binding moiety that binds the central moiety to the Ab; -L2 comprises a second linking moiety that links the central moiety to D.

[0007] Alternatively, a first binding moiety L1 may bind the central moiety to D and a second binding moiety L2 may bind the central moiety to Ab.

[0008] The present inventors have found specific combinations of antibodies, linkers and drugs that together achieve the desired characteristics. Advantageously, the specific ADCs of the present invention surprisingly exhibit improved safety and efficacy compared to known anti-CanAg ADCs.

[0009] In particular, a pyrrolobenzodiazepine (PBD) dimer cytotoxic payload is employed in the form of a prodrug according to formula (II). PBDs are a known class of highly cytotoxic DNA crosslinkers that utilize different cellular targets than the auristatin and maytansinoid classes of tubulin inhibitors and a different mode of DNA damage than other DNA-interactive payloads such as calicheamicins. The prodrug form of the PBD dimer according to formula (II) is more stable and exhibits lower cytotoxicity compared to conventional PBD drugs that have poor blood stability after administration. The prodrug is converted to its active form by cleavage of the glucuronic acid moiety by the β-glucuronidase enzyme, which is known to be upregulated in cancer cells compared to surrounding normal tissues (Fishman, WH, J. Biol. Chem., 1947, 169(2) p:449). This is believed to result in increased tumor selectivity of the active drug and reduced occurrence of side effects due to premature degradation of the linker by normal cells.

[0010] As mentioned above, the stability of the linker is a key factor that determines the efficacy and toxicity of antibody-drug conjugates. Existing linkers, such as the widely used maleimide conjugation method, can suffer from non-specific release of payload in non-tumor tissues, leading to off-target toxicity and limited therapeutic window. Advantageously, the linker according to the present invention as described above provides a stable bond between antibody and drug while allowing efficient cleavage of the drug in tumor cells.

[0011] The central portion of the linker linking the antibody and the drug may comprise an O-substituted oxime according to formula III. When the carbon atom of the oxime is replaced with a first linking group that covalently links the oxime to the antibody, the oxygen atom of the oxime is replaced with a second linking group that covalently links the oxime to the drug (D). Alternatively, when the carbon atom of the oxime is replaced with a linking group that covalently links the oxime to the drug, the oxygen atom of the oxime is replaced with a linking group that covalently links the oxime to the antibody.

[0012] In other embodiments, the central portion of the linker may contain a substituted triazole according to formula IV, V, VI or VII instead of an oxime. Advantageously, the triazole may be formed by a click chemistry reaction carried out under mild conditions, which may be carried out in the presence of the antibody without denaturation. Furthermore, for example, an azide-alkyne click chemistry reaction may produce a triazole with high yield and high reaction specificity. Thus, even if the antibody has various functional groups (e.g., amine, carboxyl, carboxamide, guanidinium), the click chemistry reaction may be carried out without affecting, for example, the amino acid side chains of the antibody. As will be appreciated by those skilled in the art, formulae VI and VII are positional isomers of formulae IV and V, respectively.

[0013] Any humanized antibody or antigen-binding fragment thereof that can target CanAg can be used according to the present invention. Preferably, the antibody is a humanized C242 antibody or antigen-binding fragment thereof. Humanized C242 (HuC242 or cantuzumab) binds to the CA242 epitope on the extracellular domain of the CanAg antigen. Examples of humanized C242 for use in the present invention may include one or more amino acid sequences from SEQ ID NO:5-10 or 13-18.

[0014] Preferably, the linker is covalently attached to the antibody by a thioether bond. For example, the thioether bond may include a sulfur atom of a cysteine ​​of the antibody. Cysteine-based conjugation methods provide better control of drug loading, i.e., drug-to-antibody ratio (DAR), and uniformity, compared to lysine conjugation methods. It is known that improved uniformity of ADCs leads to improved pharmacokinetics and efficacy, as well as reduced off-target toxicity. Covalent thioether bonds can be formed using existing thiol groups or by introducing thiol groups at a precursor stage, for example, by reacting one or more functional groups of the antibody to generate thiol groups, or by introducing thiol groups or their precursors into the antibody. As an example, it may include a step of introducing a cysteine ​​residue at the site where it is desired to attach the linker to the antibody. This may be useful when the starting antibody or wild-type antibody does not have a cysteine ​​residue that is favorable for reaction according to the invention. Conveniently, this can be achieved using site-directed mutagenesis of antibodies, the use of which is well established in the art.

[0015] The first binding moiety has formula VIII: [ka] It can contain at least one isoprenyl unit represented by

[0016] When the first linking moiety comprises at least one isoprenyl unit of formula VIII, carbon atom (a) of the isoprenyl unit forms a thioether bond with a sulfur atom, preferably of a cysteine, of the antibody, thereby covalently linking the isoprenyl group to the antibody, and carbon atom (b) of the isoprenyl group covalently links the isoprenyl group to the central portion of the linker.

[0017] Advantageously, antibody prenylation of the C-terminal amino acid sequence to introduce modified isoprenyl units has been described, allowing the attachment of drugs or other active agents to antibodies in a gentle and site-specific manner, allowing the preparation of homogenous ADCs with a defined number of drugs, which is known to improve pharmacokinetics and efficacy, and is more desirable from a regulatory standpoint.

[0018] The antibody may comprise an amino acid motif recognized by isoprenoid transferase, preferably at the C-terminus of the antibody. The amino acid motif may be a sequence selected from CXX, CXC, XCXC, XXCC and CYYX, where C represents cysteine; Y represents, independently for each occurrence, an aliphatic amino acid; and X represents, independently for each occurrence, glutamine, glutamic acid, serine, cysteine, methionine, alanine or leucine. Suitably, the thioether bond may comprise the sulfur atom of the cysteine ​​of the amino acid motif.

[0019] Optionally, the amino acid motif may be the sequence CYYX, where Y represents, independently for each occurrence, alanine, isoleucine, leucine, methionine, or valine. For example, the amino acid motif may be CVIM or CVLL. At least one of the seven amino acids preceding the amino acid motif may be glycine. For example, at least three of the seven amino acids preceding the amino acid motif are each independently selected from glycine and proline. Suitably, each of the one, two, three, four, five, six, seven, eight, nine, or ten amino acids preceding the amino acid motif is a glycine, preferably seven. Optionally, the antibody comprises the amino acid sequence GGGGGGCVIM, preferably at the C-terminus.

[0020] Alternatively, the first binding moiety has formula IX: [ka] Represented by During the ceremony, - a indicates the attachment point to Ab; - b indicates the attachment point to the central moiety; -m is an integer from 1 to 10.

[0021] Advantageously, vinylpyridine-based linkers according to formula IX have been shown to selectively and irreversibly react with thiol groups on antibodies to form highly stable thioether bonds. As discussed above, linker stability is crucial to the efficacy and toxicity of ADCs.

[0022] Suitably, the second binding moiety has the formula X: [ka] and may contain at least one polyethylene glycol unit represented by In the formula, o is an integer of 1 to 10.

[0023] Alternatively, the second binding moiety has formula XI: [ka] It can also be expressed as During the ceremony, p is an integer from 1 to 10; q is an integer from 1 to 20; r is an integer from 1 to 10; s is an integer from 1 to 10.

[0024] Preferably, the antibody conjugate is selected from the following structures: [ka] or [ka] or [ka] or [ka] During the ceremony, m is an integer from 0 to 20; o is an integer from 0 to 10.

[0025] In a second aspect, there is provided a pharmaceutical composition comprising an antibody conjugate according to the first aspect; and one or more pharma- ceutically acceptable excipients, diluents or carriers.

[0026] Suitably, the pharmaceutical composition can be used as a medicine.For example, the medicine can be used for treating cancer.Optionally, the cancer can be selected from the group consisting of lung cancer, small cell lung cancer, digestive cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary tract cancer, cervical cancer and uterine cancer.For example, the cancer can be pancreatic cancer.

[0027] In another aspect, there is provided use of a pharmaceutical composition according to the second aspect in the preparation of a medicament for the treatment of cancer.

[0028] In another aspect, a method for cancer treatment is provided for a subject in need thereof, comprising administering a therapeutic amount of the pharmaceutical composition according to the second aspect to the subject.Optionally, the cancer can be selected from the group consisting of lung cancer, small cell lung cancer, digestive cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary tract cancer, cervical cancer and uterine cancer.For example, the cancer can be pancreatic cancer.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention will now be described by way of example only and not by way of limitation with reference to the accompanying figures.

[0030] The accompanying drawings illustrate presently exemplary embodiments of the disclosure and, together with the general description given above and the detailed description of the embodiments given below, serve to explain, by way of example, the principles of the disclosure. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 shows binding of humanized antibodies to CanAg-positive Colo205 cells. [Diagram 2] PLRP chromatogram (A214 nm) of ADC-1 with a DAR of 2.3. Numbers indicate the amount of drug bound to the light chain (L) or heavy chain (H). [Diagram 3] SEC chromatogram (A214 nm) of ADC-1 with a DAR of 2.3. [Figure 4] HIC chromatogram (A214 nm) of ADC-2 with a DAR of 2 after coupling of compound 2 to the prenylated HC1+LC1 intermediate and purification by semi-preparative HIC. [Diagram 5] SEC chromatogram (A214 nm) of purified ADC-2 having a DAR of 2. [Figure 6] HIC chromatogram (A214 nm) of ADC-3 with a DAR of 2 after coupling of compound 5 to the prenylated HC1LC1 intermediate and purification by semi-preparative HIC. [Figure 7]SEC chromatogram (A214 nm) of purified ADC-3 having a DAR of 2. [Figure 8] FIG. 1 shows SEC analysis of cantuzumab ravtansine at 280 nm. [Figure 9] FIG. 1 shows CanAg expression levels in SNU-16, Colo-205, HT29, BxPC3 and NCI-N87 cells. [Figure 10] FIG. 1 shows β-glucuronidase activity in Colo-205, HT29, BxPC3 and NCI-N87 cells. [Figure 11] FIG. 1 shows the in vitro activity of ADC-2 and a non-ADC binding control against Colo-205 cells. [Figure 12] FIG. 1 shows the in vitro activity of ADC-2 and a non-ADC binding control against BxPC-3 cells. [Figure 13] FIG. 1 shows the in vitro activity of ADC-2 and a non-ADC binding control on HT-29 cells. [Figure 14] FIG. 1 shows the in vitro activity of ADC-2 and a non-ADC binding control against N87 cells. [Figure 15] FIG. 1 shows the in vitro activity of ADC-1, ADC-2 and ADC-3 against Colo-205 cells. [Figure 16] FIG. 1 shows the in vitro activity of ADC-3, cantuzumab ravtansine and a non-ADC binding control against Colo-205 cells. [Figure 17] (A) is a graph showing the in vivo activity of ADC-2 and ADC-3 in Colo-205 xenografts, and (B) is a graph showing changes in body weight. [Figure 18] (A) is a graph showing the in vivo activity of ADC-1, ADC-2 and ADC-3 in Colo-205 xenografts, and (B) is a graph showing changes in body weight. [Figure 19]FIG. 1A shows the in vivo activity of cantuzumab ravtansine in Colo-205 xenografts, and FIG. 1B shows the changes in body weight. [Figure 20] (A) is a graph showing the in vivo activity of ADC-2, ADC-3, and cantuzumab ravtansine in BxPC-3 xenografts, and (B) is a graph showing changes in body weight. [Figure 21] (A) is a graph showing the in vivo activity of cantuzumab ravtansine in BxPC-3 xenografts, and (B) is a graph showing changes in body weight. [Figure 22] (A) In vivo activity of ADC-3 and non-conjugated ADC control in NCI-N87 xenografts; (B) Change in body weight. [Figure 23] (A) is a graph showing the in vivo activity of cantuzumab ravtansine ADC in NCI-N87 xenografts, (B) is a graph showing the in vivo activity of Enhertu ADC in NCI-N87 xenografts, and (C) and (D) are graphs showing body weight change, respectively. [Figure 24] Alignment of heavy chain (HC) and light chain (LC) sequences for antibody humanization. CDR regions are underlined. Amino acids belonging to the signal peptide are shown in bold. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present invention relates to antibody conjugates that target the CanAg antigen, and compositions (e.g., pharmaceutical compositions) that include the antibody conjugates. Methods of using the antibody conjugates and compositions, including for the treatment of cancer, are also provided.

[0033] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, through at least one antigen recognition site in the variable region of the immunoglobulin molecule. Immunoglobulins include antibodies whether they are natural or partially or wholly synthetic. The term also includes polypeptides or proteins that contain an antigen-binding domain. As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, Fv fragments), single-chain Fv (scFv) variants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, fusion proteins containing an antigenic determinant of an antibody, and other modified immunoglobulin molecules that contain an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM (based on the identity of the heavy chain constant domains, called α, δ, ε, γ, and μ, respectively), or their subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.

[0034] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, nanobodies, single-chain antibodies, bispecific and multispecific antibodies formed from antibody fragments, etc.

[0035] "Prodrug" refers to a compound that is metabolized, e.g., hydrolyzed, in a host after administration to form a biologically active molecule. Typical examples of prodrugs include compounds that have biologically labile or cleavable protecting groups on a functional moiety of an active compound.

[0036] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals in which a cell population is characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, germinoma, sarcoma, and leukemia. More specific examples of such cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, fallopian tube cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various head and neck cancers.

[0037] "Tumor" refers to a mass of tissue resulting from excessive cell growth or proliferation, whether benign (non-cancerous), including precancerous, or malignant (cancerous).

[0038] The term "subject" refers to any animal (e.g., mammal) that is the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, etc. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to human subjects.

[0039] The term "pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0040] An "effective amount" as disclosed herein is an amount sufficient to accomplish a specifically described purpose. An "effective amount" can be determined empirically and routinely in relation to the described purpose.

[0041] The term "therapeutically effective amount" refers to an amount of an ADC or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, and in some embodiments, stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, and in some embodiments stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with cancer. See the definition of "treat" below. To the extent a drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. A "prophylactically effective amount" refers to an amount effective at dosages and for periods of time necessary to obtain the desired prophylactic result. Generally, but not necessarily, a prophylactic dose is used in subjects at a pre-stage or earlier stage of disease, so the prophylactically effective amount will be less than the therapeutically effective amount.

[0042] The terms "treating" or "treatment" or "treating" or "palliative" or "alleviating" and the like refer to both 1) therapeutic measures that cure, slow, lessen the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and 2) prophylactic or preventative measures that prevent and / or delay the onset of the pathological condition or disorder of interest. Thus, those in need of treatment include those already with the disorder; those susceptible to having the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject has been successfully "treated" for cancer according to the methods of the invention if the patient exhibits one or more of the following: a reduction in the number or complete elimination of cancer cells; a reduction in tumor size; inhibition or elimination of cancer cell invasion into peripheral organs, including, for example, spread of cancer to soft tissue and bone; inhibition or elimination of tumor metastasis; inhibition or elimination of tumor growth; mitigation of one or more symptoms associated with a particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in the tumorigenicity, tumorigenic frequency or tumorigenic potential of the tumor; a reduction in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenic cells to a non-tumorigenic state; or some combination of effects.

[0043] The present disclosure provides an antibody-drug conjugate of an antibody that binds to the CanAg antigen. CanAg is strongly expressed in most pancreatic, biliary and colorectal cancers. It is also expressed in a significant proportion of gastric, uterine, non-small cell lung and bladder cancers. In contrast, CanAg expression in normal tissues has been reported to be low. Therefore, CanAg appears to be a suitable candidate for mAb-based anticancer drugs. However, there are currently no commercially available ADCs targeting CanAg. Cantuzumab mertansine and cantuzumab ravtansine are two known ADCs targeting CanAg, but neither compound has progressed beyond phase 2 clinical trials. The present invention has surprisingly found that certain claimed ADCs provide improved ADCs targeting CanAg.

[0044] Any humanized antibody or antigen-binding fragment thereof capable of targeting CanAg can be used according to the present invention. To maintain binding affinity, the humanization process may include identifying CDR regions and residues that interact with the CDRs or residues at the VH-VL interface, and preserving these regions in the humanized antibody. Suitably, the antibody according to the present invention is a humanized antibody in which the CDRs underlined in Figure 24 are preserved, or an antigen-binding fragment comprising these regions. An example of a humanized C242 for use in the present invention may comprise one or more amino acid sequences from SEQ ID NO:5-10 or 13-18.

[0045] The antibody conjugate according to the present invention may be useful in a variety of applications, including, but not limited to, therapeutic treatment methods, such as the treatment of cancer. In certain embodiments, the agent is useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume and / or reducing tumorigenicity of tumors. The method of use may be an in vitro method, an ex vivo method, or an in vivo method. In certain embodiments, the disease treated with the antibody conjugate or the composition comprising the antibody conjugate is cancer. In certain embodiments, the cancer is characterized by tumors expressing CanAg.

[0046] The present invention provides a method of treating cancer, comprising administering a therapeutically effective amount of an antibody conjugate or composition thereof to a subject (e.g., a subject in need of treatment). In certain embodiments, the cancer is selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary tract cancer, cervical cancer, and uterine cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is colon cancer. In certain embodiments, the subject is a human.

[0047] The pharmaceutical composition of the present invention can be administered in any number of ways, either for local or systemic treatment.Administration can be pulmonary (e.g., by inhalation or insufflation of powder or aerosol, including by nebulizer; intratracheal, intranasal, epidermal and transdermal administration);oral;or parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion;or intracranial (e.g., intrathecal or intraventricular).

[0048] For the treatment of disease, the appropriate dosage of the antibody or agent of the present invention will depend on the type of disease being treated, the severity and course of the disease, the responsiveness of the disease, whether the antibody conjugate is administered for therapeutic or prophylactic purposes, previous treatments, the patient's clinical history, etc., all at the discretion of the treating physician. The antibody conjugate or composition thereof can be administered once or over a series of treatments ranging from several days to several months, or until a cure is achieved or a reduction in disease symptoms (e.g., a reduction in tumor size) is achieved. Optimal administration schedules can be calculated from measurements of drug accumulation in the patient's body and will vary with the relative potency of individual antibodies or agents. The administering physician can easily determine optimal dosages, administration methods, and repetition rates.

[0049] Experimental data and considerations The present invention is further illustrated by the following examples, which should not be construed as imposing limitations on the scope of the invention, which is defined by the appended claims. EXAMPLES

[0050] Example 1. Development of humanized anti-CanAg antibody Humanization design of the parent antibody was performed using in silico analysis. Homology modeling was used to generate the 3D structure of the parent antibody. Acceptor frameworks were identified based on overall framework sequence identity, matching interface positions, and canonical positions of similarly classified CDRs. Three heavy chain (HC) frameworks and three light chain (LC) frameworks were selected for humanization design.

[0051] Humanized antibodies were designed by creating multiple hybrid sequences in which selected portions of the parent antibody sequence were fused with human framework sequences. Using the 3D structure, these humanized sequences were systematically analyzed by eye and by computer modeling to isolate sequences most likely to retain antigen binding (focusing on key residues supporting the CDR loops and the VH-VL interface). The goal was to maximize the amount of human sequence in the final humanized antibody while retaining the specificity of the original antibody.

[0052] Three humanized VH and three humanized VL sequences were designed: "HV1-18BM (HC1)", "HV1-46BM (HC2)" and "HV1-8BM (HC3)", "KV2-40BM (LC1)" and "KV2-28BM (LC2)" and "KV2D-29BM (LC3)". The humanness score (T20 score) of the humanized antibodies was calculated by analyzing the primary sequences of the variable regions using the method described by Gao et al. (Monoclonal antibody humanness score and its applications; BMC Biotechnology, 13:55, 2013).

[0053] The humanized heavy and light chains were then combined to generate a fully humanized antibody. Nine combinations of humanized heavy and light chains were tested for their expression levels and antigen binding affinity to identify antibodies that function similarly to the chimeric parent antibody. The antibodies tested are shown in Table 1 below. Transient production (TunaCHO™ 7day) of 0.01L was performed for the nine humanized variants and the chimeric parent antibody. All clones were purified with Protein A (see Table 1 for production yields).

[0054] [Table 1]

[0055] The affinity of the nine humanized antibody combinations for CanAg-positive Colo-205 cells was assessed by flow cytometry as follows: 20 × 10 5 Colo205 cells were aliquoted into each well and washed once with FACS buffer (2% FBS in PBS). Humanized and isotype control antibodies (anti-HEL human IgG1 (N297A), Catalog: B109801, Brand: BIOINTRON) were diluted in FACS buffer (starting at 100μg / mL and diluted 2-fold for 8 concentrations) and added 100uL per well containing cells. Cells were incubated at 4℃ for 60 minutes and then washed twice with FACS buffer. Secondary antibody AF488 goat anti-human IgG (H+L) (Cat: A11013, Brand: Invitrogen) was diluted 1:1000 in FACS buffer and added 100uL per well and incubated at 4℃ for 30 minutes. After incubation, cells were resuspended in 200uL FACS buffer and analyzed by flow cytometry. MFI (mean fluorescence intensity) was used to calculate EC50. The EC50 values ​​of the binding between the humanized antibodies and Colo205 cells determined by FACS are shown in Table 2.

[0056] [Table 2]

[0057] As can be seen from Table 2, all humanized antibodies tested (HC1+LC1, HC1+LC2, HC1+LC3, HC2+LC1, HC2+LC2, HC2+LC3, HC3+LC1, HC3+LC2 and HC3+LC3) showed comparable affinity and expression yields in the TunaCHO™ cell transient expression system.

[0058] Example 2. ADC-1 ADC-1 was produced by vinylpyridine-mediated cysteine ​​modification using Compound 1 and Compound 2 below.

[0059] Synthesis of compound 1 [ka]

[0060] Lithium 3-(6-methyl-4-vinylpyridin-2-yl)propanoate (17.5 g, 87.6 mmol) and 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine (23.0 g, 87.6 mmol, commercially available from BroadPharm, catalog number: BP-21615) were dissolved in dimethylformamide (525.0 mL). N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (33.6 g, 175 mmol) was added in small portions at 0° C. Diisopropylethylamine (61.0 mL, 350.0 mmol) was added dropwise and the mixture was stirred for 12 h. The reaction was quenched by the addition of aqueous LiCl (1.0 L, 5.0% wt / vol) and washed with ethyl acetate (3×500 mL). The organic extracts were combined and washed with MgSO 4 The crude product was purified by column chromatography (Si 2 0, dichloromethane:methanol gradient 98:2-96:4) to give compound 1 as an orange oil (21.5 g, 56% yield). 1 H NMR (CDCl 3 , 400 MHz) δ ppm: 6.97 (2H, d) 6.53 (1H, t), 5.90 (1H, d), 5.42 (1H, d), 3.63 (14H, m), 3.44 (2H, d), 3,40 (4H, m), 3.04 (2H, t), 2.62 (2H, t), 2.45 (3H, s). LCMS (ESI+): Compound 1 (C 21 H 33 N 5 O 5 ) Theoretical: 436.25 [M+1] 1+ . Found: 436.28 [M+1] 1+ .

[0061] Compound 2, shown below, was synthesized according to the method described in WO 2018 / 182341, the entirety of which is incorporated herein by reference.

[0062] [ka]

[0063] ADC-1 generation The HC1+LC1 antibody was partially reduced with 2.8 molar equivalents of TCEP and conjugated to compound 1 in a 4-fold molar excess in the presence of 1.2% (v / v) dimethylacetamide (DMA) at pH 7.4 for ≥18 h at 25 °C. After isolation by desalting, the resulting intermediate was conjugated to compound 2 in a 2.5-fold molar excess in the presence of 0.7% (v / v) dimethylacetamide (DMA) at pH 7.4 for ≥4 h at 25 °C. The conjugate was purified on a desalting column to remove excess free drug and solvent, and rebuffered in phosphate-buffered saline (PBS), pH 7.4.

[0064] Measurement of Drug-to-Antibody Ratio (DAR) of ADC-1 Measurement of the mean drug loading and drug loading distribution is crucial in the generation of ADCs, as these factors affect the potency and pharmacokinetics of the ADC. DAR measurement of ADC-1 was achieved by polymer-linked reversed-phase (PLRP) chromatography using an Agilent PLRP-S (1000A, 2.1 × 50 mm, 5 μm) column. Separation of the dithiothreitol (DTT)-reduced complex using the PLRP column resulted in good resolution of the peaks corresponding to the light and heavy chains of the unbound or drug-bound antibody, as shown in Figure 2. The DAR value of ADC-1 was measured to be 2.3. Peak separation was performed as follows; Buffer A (H 2 HO+0.1% TFA) and buffer B (MeCN+0.1% TFA); gradient; 0-3 min 25% buffer B; 3-28 min = 25-50% buffer B; 28-31 min = 95% buffer B; 31-40 min = 25% buffer B.

[0065] Measurement of monomer content of ADC-1 Size exclusion chromatography (SEC) was used to measure the extent of aggregation upon binding of the complex using a MAbPac™ SEC-1 column (5 μM, 300 Å, 7.8×300 mm). Elution was performed with 20 mM MES sodium salt, 150 mM NaCl, 5% MeCN, pH 6.0. As shown in FIG. 3, ADC-1 exhibited a monomer content of >96%.

[0066] Example 3. ADC-2 ADC-2 is prepared using protein prenylation of the C-terminal amino acid sequence, and a modified isoprenoid unit is introduced to allow gentle and site-specific attachment of drugs to the antibody. Such prenylation of antibodies is described, for example, in US Patent Publication No. 2012 / 0308584, US Patent No. 9919057, PCT Publication No. WO 2017 / 089890 and PCT Publication No. WO 2017 / 089895, the contents of which are incorporated herein by reference in their entirety.

[0067] In this example, the antibody was modified with an isoprenoid derivative functionalized with an azide group, shown below as compound 3, for coupling to compound 2 via click chemistry using the procedure shown below, which was prepared as described in U.S. Patent Application Publication No. 2012 / 0308584.

[0068] [ka]

[0069] ADC-2 generation 0.2μM FTase, 100μM DTT, 500mM Tris-HCl, 0.1mM ZnCl 2 and 50 mM MgCl 2The CAAX-tagged HC1LC1 antibody was prenylated with 8.3 molar equivalents of compound 3 in the presence of 0.01% DMA at pH 7.4 for 4 h at 30 °C. The resulting intermediate was desalted into PBS, pH 7.4. The prenylated intermediate was combined with 2.5 molar equivalents of compound 2 in the presence of 0.45% DMA at 30 °C for 2 h. ADC-2 was purified by semi-preparative HIC using a phenyl-phase HIC column (Tosoh Biosciences, L x I.D 7.5 cm x 7.5 mm) with the following procedure; Buffer A (50 mM potassium phosphate + 0.5 M ammonium sulfate, pH 7.0) and Buffer B (50 mM potassium phosphate + 30% (v / v) MeCN, pH 7.0); Gradient-0-30 min=0-100% Buffer B; 30-32 min=100% Buffer B; 32-32.1 min=100-0% Buffer B; 32.1-49 min=0% Buffer B; Flow rate-0.8 mL / min. Samples were desalted into phosphate-buffered saline (PBS), pH 7.4.

[0070] Measurement of Drug-to-Antibody Ratio (DAR) of ADC-2 DAR measurements of ADC-2 were performed by hydrophobic interaction chromatography (HIC) using a Tosoh Bioscience HIC column (butyl phase, L×I.D 3.5 cm 4 4.6 mm, 2.5 μM particle size). Separation of the conjugate sample using the HIC column yielded one peak corresponding to the antibody bound to the two drugs, as shown in Figure 4. Elution was performed as follows: Buffer A (25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0) and Buffer B (25 mM sodium phosphate, 25% isopropanol, pH 7.0); gradient-0-30 min = 0-100% buffer B; 30-35 min = 0% buffer B.

[0071] Measurement of ADC-2 monomer content Size exclusion chromatography (SEC) was performed using a MAbPac™ SEC-1 column (5 μM, 300 Å, 7.8×300 mm) to measure the extent of aggregation upon binding of the complex. Elution was performed with 20 mM MES sodium salt, 150 mM NaCl, 5% MeCN, pH 6.0. As shown in FIG. 5, ADC-2 showed a monomer content of >99%.

[0072] Example 4. ADC-3 In this example, an antibody was modified with a ketone functionalized isoprenoid derivative shown above as compound 4 for coupling to compound 5 via oxime formation chemistry using the procedure shown below. Compound 4 was prepared as described in U.S. Patent Publication No. 2012 / 0308584. Compound 5 was synthesized according to the method described in WO2018182341.

[0073] ADC-3 generation 0.2μM FTase, 250μM DTT, 500mM Tris-HCl, 0.1mM ZnCl 2 , 50 mM MgCl 2 The CAAX-tagged HC1LC1 antibody was prenylated with 6.25 molar equivalents of compound 4 in the presence of 10% DMSO at pH 7 for >18 hours at 30 °C. The resulting intermediate was desalted into PBS, pH 7.4. The prenylated intermediate was conjugated with 10 molar equivalents of compound 5 in the presence of 10% DMSO at 30 °C for 6 hours. ADC-3 was purified by semi-preparative HIC using a phenyl-phase HIC column (Tosoh Biosciences, L x I.D 7.5 cm x 7.5 mm) with the following procedure; Buffer A (50 mM potassium phosphate + 0.5 M ammonium sulfate, pH 7.0) and Buffer B (50 mM potassium phosphate + 30% (v / v) MeCN, pH 7.0); Gradient-0-30 min=0-100% Buffer B; 30-32 min=100% Buffer B; 32-32.1 min=100-0% Buffer B; 32.1-49 min=0% Buffer B; Flow rate-0.8 mL / min. Samples were desalted into phosphate-buffered saline (PBS), pH 7.4.

[0074] Measurement of Drug-to-Antibody Ratio (DAR) of ADC-3 DAR measurements of ADC-3 were performed by hydrophobic interaction chromatography (HIC) using a Tosoh Bioscience HIC column (butyl phase, L × ID 3.5 cm × 4.6 mm, 2.5 μM particle size). Separation of the conjugate sample using the HIC column yielded one peak corresponding to the antibody conjugated with two drugs, as shown in Figure 6. Elution was performed as follows: Buffer A (25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0) and Buffer B (25 mM sodium phosphate, 25% isopropanol, pH 7.0); gradient-0-30 min = 0-100% buffer B; 30-35 min = 0% buffer B. Samples were desalted into phosphate-buffered saline (PBS), pH 7.4.

[0075] Measurement of monomer content of ADC-3 Size exclusion chromatography (SEC) was performed using a MAbPac™ SEC-1 column (5 μM, 300 Å, 7.8×300 mm) to measure the extent of aggregation upon binding of the complex. Isocratic elution was performed with 20 mM MES sodium salt, 150 mM NaCl, 5% MeCN, pH 6.0. As shown in FIG. 7, ADC-3 exhibited >99% monomer content.

[0076] Example 5. Control drug Cantuzumab ravtansine As mentioned above, cantuzumab ravtansine is a humanized antibody-drug conjugate that targets CanAg. Cantuzumab ravtansine contains cantuzumab linked to the cytotoxic maytansinoid drug ravtansine, shown below as compound 6.

[0077] Preparation of Cantuzumab Ravtansine HuC242 (cantuzumab antibody) at 5.35 mg / mL in PBS buffer was pretreated for binding with 5% v / v 0.5 M borate, 25 mM EDTA, pH 8.2.

[0078] Six equivalents of compound 6 relative to antibody were added as a 50 mM stock in DMA, with additional DMA added beforehand to give a final DMA of 5% v / v. The reaction was incubated at 20°C for 3 hours and then quenched with 6 equivalents of glycine relative to antibody from a 50 mM stock solution in water. The DAR4.0 conjugate was purified by G25 desalting and then buffer exchanged for 6 volumes into 25 mM His / Cl, pH 6.0, 200 mM sucrose using a 30KDa ViVa membrane concentrator. 2% w / v polysorbate 20 (PS20) was added to give a final PS20 of 0.02% w / v in 25 mM His / Cl, pH 6.0, 200 mM sucrose.

[0079] [ka]

[0080] Determination of Drug-Antibody Ratio (DAR) of Cantuzumab Rabtansine by UV-VIS DAR values ​​of cantuzumab ravtansine were measured using UV-VIS analysis at 252 nm, 280 nm, and 320 nm. Dilutions were performed in formulation buffer using a 1 cm path length quartz cuvette with blank correction with formulation buffer only. The molar concentration of DM4 in the ADC sample was calculated as follows: [DM4] = DM4 molar concentration, A280 = absorbance at 280 nm - absorbance at 320 nm x dilution, A252 = absorbance at 252 nm - absorbance at 320 nm x dilution, εDM4 252 =26159M -1 c -1 , εDM4 280 =5180M -1 c -1 was calculated according to Equation 1.

[0081]

number

[0082] The molar concentration of protein in an ADC sample is given by Equation 1: [Ab] = protein molar concentration, A280 = absorbance at 280 nm - absorbance at 320 nm x dilution, εDM4 280 =5180M -1 c -1 , εAb 280 =223400M -1 c -1 , [DM4] = molar concentration of DM4 in the ADC sample, calculated according to Equation 2.

[0083]

number

[0084] To calculate the DAR by UV analysis, divide the molar concentration of DM4 in the ADC sample by the molar protein concentration of the ADC: Equation 3.

[0085] From Equation 1, [DM4] = molar concentration of DM4 in the ADC sample, and from Equation 2, [Ab] = molar protein concentration. DAR4.04 was calculated using Equations 1-3, and the results are shown in Table 3.

[0086]

number

[0087] [Table 3]

[0088] Table 3 summarizes the triplicate UV-vis measurements of cantuzumab ravtansine and the calculated DAR values ​​based on Eqs. 1, 2, and 3.

[0089] Determination of monomer content of the reference drug cantuzumab ravtansine The ADCs were assessed for monomer content and the presence of high molecular weight (HMW) aggregates, dimers and fragments (LMW) using size exclusion chromatography (TOSOH TSKgel G3000SWXL 7.8 mm x 30 cm, 5 μm column). Run conditions: 10% IPA, 0.2 M potassium phosphate, 0.25 M potassium chloride, pH 6.95, flow rate 0.5 mL / min. Analysis of cantuzumab ravtansine showed 98.6% monomeric ADC, as shown in Figure 8.

[0090] Example 6. Measurement of CanAg expression levels in SNU-16, Colo205, BxPC3, HT29 and N87 cell lines using flow cytometry Expression of CanAg in five different cell lines was assessed using a flow cytometry-based binding assay by comparison of phycoerythrin (PE)-conjugated HC1LC1 antibody with a phycoerythrin-conjugated isotype-matched control antibody (Biolegend, No: 403504). Phycoerythrin-conjugated HC1LC1 antibody was prepared using the PE / R-Phycoerythrin Binding Kit-Lightning-Link® Kit (Abcam, ab102918) according to the manufacturer's instructions. The five cell lines tested were SNU-16 (gastric cancer cell line), Colo-205 (human colon cancer cell line), BxPC-3 (human pancreatic epithelial adenocarcinoma cells), N87 (human gastric cancer cells) and HT29 (human colon adenocarcinoma cells). Cells were harvested, resuspended in FACS buffer and counted. 2 × 10 5 Cells were aliquoted and washed once with 3 mL FACS buffer. Cells were then resuspended in 100 μL FACS buffer containing 2 μL (20 μg / mL) HC1LC1 antibody-PE or isotype control-PE. Cells were incubated with PE-conjugates for 30 min at 4°C and then washed twice with 3 mL FACS buffer. Cells were resuspended in 500 μL FACS buffer and subjected to flow cytometry analysis. BD Quantibrite™ PE beads (BD Biosciences, 340495) were reconstituted in 0.5 mL 0.5% BSA and analyzed according to the manufacturer's instructions.

[0091] The PE geometric mean values ​​for different HC1LC1 antibody-PE concentrations were exported and Log10 values ​​were calculated. Log10 values ​​for the number of PE molecules per bead were also calculated based on lot-specific values ​​provided by the manufacturer. A linear regression of the Log10 values ​​of the PE geometric mean against the number of PE molecules per bead was created. To determine the PE molecules per cell, the Log10PE geometric mean values ​​were substituted into the equation and the anti-Log was determined. As shown in Figure 9 and Table 4, SNU-16 and Colo-205 cells were found to be the highest expressers of CanAg with an average of 11.5 million and 2.6 million PE molecules per cell. HT-29 showed moderate CanAg expression with an average of 626,000 PE molecules per cell. BxPC-3 showed low expression of CanAg with an average of 75,000 PE molecules per cell. N87 cells showed very low expression of CanAg with an average of 2,500 PE molecules per cell.

[0092] [Table 4]

[0093] Table 4 shows the calculation of PE molecules in SNU-16, Colo205, BxPC3 and HT29 cell lines.

[0094] Example 7. Measurement of β-glucuronidase activity in Colo205, BxPC3, HT29 and N87 cell lines using a fluorometric method β-glucuronidase enzyme activity was measured in the cell lines using a β-glucuronidase activity measurement kit (Abcam, Ab234625). Cells were counted and diluted to 1 × 10 7Cells were harvested. Cells were washed once with 1 mL DPBS and centrifuged at 400 × g for 5 min. The supernatant was discarded and cells were lysed in 500 μL assay buffer (Colo205, HT-29 and BxPC3 cells) or 300 μL assay buffer (N87 cells) and homogenized with an ultrasonic cell disrupter. The lysates were centrifuged at 10,000 × g for 5 min at 4 °C and the supernatants were collected. 50 μL of the supernatants were placed in wells of a black 96-well plate. The volume was adjusted to 90 μL with β-glucuronidase assay buffer. The positive control was mixed with β-glucuronidase assay buffer to a 90 μL solution. A 200 μM 4-methylumbelliferone (4-MU) standard solution was prepared in β-glucuronidase assay buffer and 0, 0.5, 1, 2, 4, 6, 8, 10 μL of 200 μM 4-MU standard was placed into a series of wells and the volume of each reaction was adjusted to 100 μL with β-glucuronidase assay buffer to yield 0, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6 and 2.0 nmol of 4-MU per well, respectively. The substrate solution was diluted 10-fold with β-glucuronidase assay buffer. 10 μL of substrate was added to the positive control and test samples. Fluorescence (Ex / Em=330 / 450 nm) was measured immediately after substrate addition for 60 min at 37 °C and recorded every 2 min.

[0095] The results are shown in Figure 10, which shows the volume-normalized β-glucuronidase levels in the four cell lines. HT29 cells were found to have the highest activity of β-glucuronidase enzyme. Colo-205 and BxPC3 cells showed similar β-glucuronidase activity, while N87 cells showed the lowest enzyme activity.

[0096] Example 8. In vitro activity assay In vitro activity was assessed using the luminescence-based Cell Titre-Glo (CTG) assay (Promega, No:G7572), which quantifies ATP abundance as a measure of viable cells. Specificity of cell killing was demonstrated by incubating cells with a non-binding ADC control consisting of the same payload.

[0097] The in vitro activity of the anti-CanAg ADC was evaluated in colon cancer cell lines Colo-205 (ATCC: CCL-222) and HT-29 (ATCC: HTB-38), pancreatic adenocarcinoma cell lines BxPC-3 (ATCC: CRL-1687) and N87 gastric carcinoma (ATCC: CRL-5822). The non-ADC conjugated control was an anti-CD19 ADC (DAR2.0) consisting of the same linker-drug combination as ADC-3.

[0098] Cells were trypsinized and cultured in appropriate complete medium (Colo-205, BxPC-3, N87 - RPMI-1640 with 20% heat-inactivated fetal bovine serum (FBS); HT-29 - McCoy's-5A medium with 20% heat-inactivated FBS) in 96-well microplates at 37 °C and 5% CO. 2 Cells were seeded at a density of 4000 (Colo-205) or 5000 (BxPC-3, HT-29, N87) cells per well in a volume of 100 μL. After incubation, the medium was removed and replaced with 100 μL of fresh appropriate growth medium. ADC-2 was serially diluted 3- or 5-fold in the appropriate growth medium to give the following range of ADC concentrations in 100 μL: BxPC-3 and HT-29-1000nM, 333.3nM, 111.1nM, 37nM, 12.34nM, 4.1nM, 1.37nM, 0.45nm, 0.15nm, 0.051nm; Colo-205-100nm, 20nm, 4nm, 0.8nm, 0.16nm, 0.032nm, 0.0064nm, 0.0013nm, 0.00026nm, 0.000051nm; N87-1000nm, 200nm, 40nm, 8nm, 1.6nm, 0.32nm, 0.06nm, 0.013nm, 0.0026nm, 0.0013nm.

[0099] For comparison of ADC-1, ADC-2, and ADC-3, cells were trypsinized and cultured in appropriate complete medium (RPMI-1640 with 10% FBS) in 96-well microplates at 37 °C and 5% CO. 2Cells were seeded at 100 μL for 24 hours. Cells were seeded at a density of 4000 (Colo-205) cells per well in a volume of 100 μL. After incubation, the medium was removed and replaced with 100 μL of fresh appropriate growth medium. ADCs were serially diluted 5-fold in the appropriate growth medium to give the following range of ADC concentrations in 100 μL: 50 nm, 10 nm, 2 nm, 0.40 nm, 0.08 nm, 0.016 nm, 0.0032 nm, 0.00064 nm, 0.000128 nm, 0.0000256 nm, 0.0000051 nm, 0.000001 nm.

[0100] For comparison of ADC-3 with cantuzumab ravtansine and non-binding ADC control cells, cells were trypsinized and cultured in appropriate complete medium (RPMI-1640 with 10% FBS) in 96-well microplates at 37°C and 5% CO. 2 Cells were seeded at 4000 (Colo-205) cells per well in a volume of 100 μL. After incubation, the medium was removed and replaced with 100 μL of fresh appropriate growth medium. All ADCs were serially diluted 3-fold in the appropriate growth medium to give the following range of ADC concentrations in 100 μL: 50 nM, 16.67 nM, 5.56 nM, 1.85 nM, 0.62 nM, 0.206 nM, 0.069 nM, 0.023 nM, 0.0076 nM, 0.0025 nM, 0.00085 nM, 0.00028 nM. Cells were incubated with ADCs for 3 days (72 h). After ADC treatment, the CTG reagent and cell plates were kept at RT for 30 min before adding the CTG reagent. 100 μL of CTG reagent was added to each well and the plate was shaken for 30 seconds, after which the plate was incubated at RT for 20 minutes before luminescence measurement.

[0101] The results are shown in Figures 11-16. The IC50 of ADC-2 correlates with the CanAg expression levels in Colo205, BxPC3 and N87 cells (see Figures 11, 12 and 14). Colo205 and BxPC3 cell lines have the same activity of β-glucuronidase (see Figure 10). N87 cells have the lowest amount of β-glucuronidase activity and CanAg expression levels (see Figures 10 and 9), and ADC-2 showed very limited activity against N87 cells (see Figure 14). The activity of ADC-2 against HT-29 was similar to that against Colo205 cells (see Figure 13), despite the low expression levels of CanAg in HT-29, but β-glucuronidase activity was highest in HT-29 (see Figure 10). This indicates that β-glucuronidase activity is also a contributing factor to the observed ADC activity. ADC-2 induced 100% cell death in Colo-205, HT-29 and BcPC3. The specific activity of ADC-2 was confirmed by incubating cells with a non-binding ADC (see Figures 11-14).

[0102] As shown in Figure 15, ADC1, ADC-2 and ADC-3 all showed similar potent activity against Colo205 cells, indicating that the in vitro activity of the ADCs is independent of the conjugation chemistry used in this cell line. Importantly, cantuzumab ravtansine was less efficient at killing Colo205 cancer cells, killing only 75% of the cells (see Figure 16).

[0103] Example 9. In vivo efficacy study in the Colo-205 xenograft model ADC1, ADC-2, ADC-3 and cantuzumab ravtansine were evaluated in female CB17 SCID mice bearing Colo-205 xenografts. Mice received 5 × 10 6 Colo-205 cells were inoculated subcutaneously into the right flank. Tumor-bearing mice were randomly assigned to groups of 5 mice each, and the mean tumor volume was approximately 170 mm. 3(Cantuzumabu Rabbutanshin) or 190mm 3 Once tumor size reached 100%, tumors were treated with a single intravenous dose of ADC or alternatively with vehicle solution (30 mM histidine, 200 mM sorbitol, 0.02% PS20 (w / v)). In the Colo-205 xenograft study, a conjugate dose of 1 mg / kg (13 nmol drug / kg) was used for ADC-1, ADC-2, and ADC-3, and a conjugate dose of 2 mg / kg (53 nmol drug / kg) was used for cantuzumab ravtansine. Tumor size was measured bidimensionally with calipers three times weekly, and volume was calculated according to the formula: [Number 4] V = 0.5a × b 2 (where a and b are the long and short diameters of the tumor, respectively) 3 The tumor size was then used to calculate the TGI (%) value (see Table 5). The TGI, which represents the antitumor effect, was calculated using the formula: [Number 5] TGI(%)=[1-(V 処置-t -V 処置-1 ) / (V 対照-t -V 対照-1 )] x 100 Calculation was performed using the formula: 処置-1 and V 対照-1 is the average volume of the treatment and control groups on the day of grouping; V 処置-t and V 対照-t is the mean volume of the treatment and control groups on a given day. 3 Animals were euthanized when body weight was measured three times a week as an indicator of compound toxicity (see Figures 17B, 18B and 19B).

[0104] The in vivo efficacy of ADCs on Colo205 tumor xenografts is shown in Figures 17, 18 and 19. ADC-1, ADC-2 and ADC-3 induced substantial tumor growth inhibition (79%, 103% and 102% TGI, respectively) without observable toxicity at 1 mg / kg (payload 13 nmol / kg (ADC-2 and ADC-3) or 15 nmol / kg (ADC-1) (see, e.g., Figures 17 and 18 and Table 5). Cantuzumab ravtansine showed 40% tumor growth inhibition at 2 mg / kg (payload 53 nmol / kg), which corresponds to a drug loading of 4 mg / kg for ADC-2 and ADC-3 (see Figure 19A and Table 5).

[0105] [Table 5]

[0106] Table 5 shows the tumor growth inhibition (TGI) at study day 15 for ADCs tested in the Colo-205 xenograft model. *TGI calculation for cantuzumab ravtansine is based on day 16.

[0107] Example 10. In vivo efficacy studies in the BxPC3 xenograft model ADC-2, ADC-3 and cantuzumab ravtansine were evaluated in female CB17 SCID mice bearing BxPC3 xenografts. Mice were inoculated with 5 × 10 6 BxPC3 cells were subcutaneously inoculated into the right flank. Tumor-bearing mice were randomly assigned to groups of 5 mice each, and the mean tumor volume was approximately 150–180 mm. 3Once tumor size reached 100 mg / kg, tumors were treated with a single intravenous dose of ADC or, alternatively, with a vehicle solution containing PBS pH 7.4 (see FIG. 20) or 30 mM histidine, 200 mM sorbitol, 0.02% PS20 (w / v) (see FIG. 21). Conjugate doses of 0.4 mg / kg (5.3 nmol / kg of binding agent) and 1 mg / kg (13 nmol / kg of binding agent) were used for ADC-2 and ADC-3, and conjugate doses of 0.2 mg / kg (5.3 nmol / kg of binding agent) and 0.5 mg / kg (13 nmol / kg of binding agent) for cantuzumab ravtansine. Tumor size was measured three times a week in two dimensions using calipers and volume was calculated using the formula: [Number 6] V = 0.5a × b 2 (where a and b are the long and short diameters of the tumor, respectively) Using mm 3 The tumor size was then used to calculate the TGI (%) value (see Table 6). The TGI, which represents the antitumor effect, was calculated using the formula: [Number 7] TGI(%)=[1-(V 処置-t -V 処置-1 ) / (V 対照-t -V 対照-1 )] x 100 Calculation was performed using the formula: 処置-1 and V 対照-1 is the average volume of the treatment and control groups on the day of grouping; V 処置-t and V 対照-t is the mean volume of the treatment and control groups on a given day. 3 Animals were euthanized when body weight was measured three times a week as an indicator of compound toxicity (see Figures 20B and 21B).

[0108] The in vivo effects of ADCs on BxPC3 tumor xenografts are shown in Figures 20-21. ADC-2 induced tumor growth inhibition (66.7 and 107% TGI) at doses such as 0.4 mg / kg (5.3 nmol / kg of conjugate) and 1 mg / kg (13 nmol / kg of conjugate) (see Figure 20A and Table 6). Treatment of BxPC3 xenografts with ADC-3 showed lower activity (57% and 101% TGI) than ADC-2 at doses of 0.4 mg / kg (5.3 nmol / kg of conjugate) and 1 mg / kg (13 nmol / kg of conjugate). The control drug cantuzumab ravtansine, 0.2 mg / kg (5.3 nmol / kg of conjugate) and 0.5 mg / kg (13 nmol / kg of conjugate), showed weaker antitumor responses compared to the same doses of ADC-2 and ADC-3 treatment (see Figure 20).

[0109] The control drug cantuzumab ravtansine induced complete tumor suppression at a dose of 2 mg / kg (53 nmol / kg drug), but the anticancer activity was not sustained as tumors began to regrow after 32 days (see FIG. 21A).

[0110] Table 6 shows the tumor growth inhibition (TGI) at study days 29 or 32 for ADCs tested in the BxPC3 xenograft model.

[0111] [Table 6]

[0112] Example 11. In vivo efficacy study using the NCI-N87 xenograft model ADC-3, a non-ADC binding control, cantuzumab ravtansine and Enhertu were evaluated in female CB17 SCID mice bearing NCI-N87 gastric cancer xenografts.

[0113] Enhertu is the trade name for trastuzumab deruxtecan, an ADC consisting of the humanized anti-Her2 antibody trastuzumab (Herceptin) covalently linked to the topoisomerase I inhibitor deruxtecan (DAR 8.0). Enhertu is approved by the U.S. Food and Drug Administration (FDA) for the treatment of breast cancer, gastric cancer, or gastric or gastroesophageal adenocarcinoma.

[0114] The non-ADC binding control was an anti-CD19 ADC (DAR 2.0) consisting of the same linker and drug combination as ADC-3. Mice were injected with 5 × 10 6 NCI-N87 cells were inoculated subcutaneously into the right flank. Tumor-bearing mice were randomly assigned to groups of 5 mice each, and the mean tumor volume was approximately 170–175 mm. 3 Once this was achieved, mice were treated with a single intravenous dose of ADC or alternatively with vehicle solution (30 mM histidine, 200 mM sorbitol, 0.02% PS20 (w / v)). Conjugate doses of 0.2 mg / kg (2.6 nmol / kg of conjugate), 0.5 mg / kg (6.7 nmol / kg of conjugate) and 1 mg / kg (13 nmol / kg of conjugate) were used for ADC-3; 0.5 mg / kg (6.7 nmol / kg of conjugate) and 1 mg / kg (13 nmol / kg of conjugate) for non-ADC conjugate controls; 0.2 mg / kg (10 nmol / kg of conjugate), 0.5 mg / kg (26 nmol / kg of conjugate) and 1 mg / kg (53 nmol / kg of conjugate) for Enhertu; and 2 mg / kg (53 nmol / kg of conjugate) for cantuzumab ravtansine. Tumor size was measured bidimensionally using calipers three times a week, and volume was calculated using the formula: [Number 8] V = 0.5a × b 2 (where a and b are the long and short diameters of the tumor, respectively) 3 The tumor size was then used to calculate the TGI (%) value (see Table 7). The TGI, which represents the antitumor effect, was calculated using the formula: [Number 9] TGI(%)=[1-(V 処置-t -V処置-1 ) / (V 対照-t -V 対照-1 )] x 100 Calculation was performed using the formula: 処置-1 and V 対照-1 is the average volume of the treatment and control groups on the day of grouping; V 処置-t and V 対照-t is the mean volume of the treatment and control groups on a given day. 3 Animals were euthanized when body weight was measured three times a week as an indicator of compound toxicity (see Figures 22B, 23C and D).

[0115] The in vivo efficacy of ADCs against NCI-N87 tumor xenografts is shown in Figures 22 and 23. Interestingly, the in vitro analysis of ADC-2 against NCI-N87 cells described above showed no specific activity due to the lack of CanAg expression in in vitro cell culture (see Figures 14 and 9). However, in this in vivo xenograft, ADC-3 showed target-specific activity in NCI-N87 compared to the response of the non-ADC binding control. This indicates a difference in CanAg expression between in vitro cultured N87 cells and in vivo xenografts.

[0116] ADC-3 induced 53% specific tumor growth inhibition at a dose as low as 0.2 mg / kg (2.6 nmol / kg of conjugate drug) (see FIG. 22A and Table 7). In contrast, cantuzumab ravtansine and Enhertz required higher doses (i.e., 2 mg / kg (53 nmol / kg drug) for cantuzumab ravtansine; 0.5 mg / kg (26 nmol / kg drug) for Enhertz) to achieve 43% and 46% tumor growth inhibition, respectively (see FIG. 23A-B, Table 7). Furthermore, a 1 mg / kg dose of Enhertz induced 99% TGI, but tumor regrowth was observed after 28 days. In contrast, ADC-3 doses of 0.5 mg / kg (6.7 nmol / kg binding drug) and 1 mg / kg (13 nmol / kg drug) induced 100% TGI by day 40, with no subsequent tumor recurrence observed at the 1 mg / kg dose (see Figure 22A and Table 7).

[0117] [Table 7]

[0118] Table 7 shows the tumor growth inhibition (TGI) of the ADCs tested in the NCI-N87 xenograft model on day 33 of the study.

[0119] Thus, in all cell lines tested, the ADC according to the present invention showed superior TGI compared to the control anti-CanAg ADC cantuzumab ravtansine. Furthermore, ADC-3 showed comparable TGI in gastric cancer xenografts at approximately 8-fold lower conjugate concentrations compared to Enhertz, an ADC approved by the FDA for use in the treatment of gastric cancer. Furthermore, although Enhertz induced 99% TGI at higher conjugate concentrations (i.e., 1 mg / kg, 53 nmol / kg), no observable tumor regrowth was detected even 55 days after administration of ADC-3 at a dose of 1 mg / kg (13 nmol / kg drug), whereas tumor regrowth was observed 28 days after administration. This result is even more surprising when considering the relative concentrations of drugs used, i.e., 13 nmol / kg conjugate for ADC-3 and 53 nmol / kg conjugate for Enhertz.

[0120] Those skilled in the art will appreciate that the above embodiments have been described by way of example and not in a limiting sense, and that various changes and modifications may be made without departing from the scope of the invention as defined by the appended claims.

[0121] Sequence Listing In the sequence listing below, the amino acids belonging to the signal peptide are shown in bold.

[0122] 1. Mouse parent sequence used for humanization SEQ ID NO 1 - Mouse c242 HC (CDR regions are underlined) JPEG2025509152000026.jpg16121

[0123] SEQ ID NO 2-Mouse c242LC (CDR regions are underlined) JPEG2025509152000027.jpg17122

[0124] 2. Mouse c242 sequence in the hIgG1(G1m17) backbone SEQ ID NO 3-pLEV123-Parent Mouse HC-hIgG1-Mouse Ab(Mu) JPEG2025509152000028.jpg49122

[0125] SEQ ID NO 4-pLEV123-Parent Mouse LC-hKappa JPEG2025509152000029.jpg27122

[0126] 3. Humanized antibodies against CanAg targets SEQ ID NO 5-pLEV123-HC1-hIgG1(G1m17) JPEG2025509152000030.jpg37123 JPEG2025509152000031.jpg12123

[0127] SEQ ID NO 6-pLEV123-HC2-hIgG1(G1m17) JPEG2025509152000032.jpg49122

[0128] SEQ ID NO 7-pLEV123-HC3-hIgG1(G1m17) JPEG2025509152000033.jpg49123

[0129] SEQ ID NO 8-pLEV123-LC1-hKappa JPEG2025509152000034.jpg27122

[0130] SEQ ID NO 9-pLEV123-LC2-hKappa JPEG2025509152000035.jpg27122

[0131] SEQ ID NO 10-pLEV123-LC3-hKappa JPEG2025509152000036.jpg27122

[0132] 4. HuC242 (cantuzumab) antibody SEQ ID NO 11-H-GAMMA-1 JPEG2025509152000037.jpg49123

[0133] SEQ ID NO 12-L-KAPPA JPEG2025509152000038.jpg11121 JPEG2025509152000039.jpg10120

[0134] 5. Humanized antibody with CAAX tag (GGGGGGCVIM) at the C-terminus of LC SEQ ID NO 13-pLEV123-HC1-hIgG1(G1m17) JPEG2025509152000040.jpg49123

[0135] SEQ ID NO 14-pLEV123-HC2-hIgG1(G1m17) JPEG2025509152000041.jpg50123

[0136] SEQ ID NO 15-pLEV123-HC3-hIgG1(G1m17) JPEG2025509152000042.jpg21123 JPEG2025509152000043.jpg27122

[0137] SEQ ID NO 16-pLEV123-LC1-hKappa JPEG2025509152000044.jpg27122

[0138] SEQ ID NO 17-pLEV123-LC2-hKappa JPEG2025509152000045.jpg27122

[0139] SEQ ID NO 18-pLEV123-LC3-hKappa JPEG2025509152000046.jpg27122

Claims

1. 1. An antibody conjugate of formula I, or a pharma- ceutically acceptable salt or solvate thereof: 【Chemistry 1】 During the ceremony, - Ab is a humanized C242 antibody or an antigen-binding fragment thereof; -D is a pyrrolobenzodiazepine dimer prodrug; -L is a linker connecting Ab to D; - n is an integer from 1 to 20; where -D is a member of the formula (II): 【Chemistry 2】 Represented by said linker comprising a central moiety represented by formula III, IV, V or an isomer thereof: 【Chemistry 3】 During the ceremony, - L1 comprises a first binding moiety that binds said central moiety to an Ab; - L2 comprises a second linking moiety linking said central moiety to D; An antibody conjugate or a pharma- ceutically acceptable salt or solvate thereof.

2. 2. The antibody conjugate of claim 1, wherein L is covalently attached to Ab by a thioether bond, optionally wherein the thioether bond includes a sulfur atom of a cysteine ​​of the Ab.

3. The first binding moiety has formula VIII: 【Chemistry 4】 The antibody conjugate of claim 1 or claim 2, which comprises at least one isoprenyl unit represented by:

4. The first binding moiety has formula IX: 【Chemistry 5】 (In the formula, - ａ indicates the point of attachment to the Ab; - ｂ indicates a point of attachment to the central moiety; -m is an integer from 1 to 20. The antibody complex according to claim 1 or 2.

5. The second binding moiety has the formula X: 【Chemistry 6】 (wherein o is an integer from 1 to 10). The antibody conjugate according to any one of claims 1 to 4, comprising at least one polyethylene glycol unit represented by the formula:

6. The second binding moiety has formula XI: 【Chemistry 7】 Formula XI (In the formula, p is an integer from 1 to 10; q is an integer from 0 to 20; r is an integer from 1 to 10; s is an integer from 1 to 10. The antibody complex according to any one of claims 1 to 4, wherein

7. The antibody conjugate of any one of claims 1 to 6, wherein the Ab comprises one or more amino acid motifs that can be recognized by an isoprenoid transferase.

8. The antibody conjugate of claim 7 , wherein the isoprenoid transferase is FTase (farnesyl protein transferase) or GGTase (geranylgeranyl transferase).

9. The antibody conjugate of claim 7 or claim 8, wherein the amino acid motif is CYYX, XXCC, XCXC, CXC or CXX, where C represents cysteine, Y represents an aliphatic amino acid, and X represents an amino acid that determines the substrate specificity of an isoprenoid transferase.

10. below: 【Chemistry 8】 or 【Chemistry 9】 or 【Chemistry 10】 or 【Chemistry 11】 (In the formula, m is an integer from 0 to 20; o is an integer from 0 to 10. The antibody conjugate of claim 1, comprising a structure selected from:

11. An antibody conjugate according to any one of claims 1 to 10; and one or more pharma- ceutical compositions comprising a pharma- ceutical agent, diluent, or carrier.

12. A pharmaceutical composition according to claim 11 for use as a medicament.

13. 13. The pharmaceutical composition of claim 12, wherein the medicament is for use in the treatment of cancer.

14. The pharmaceutical composition of claim 13, wherein the cancer is selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary tract cancer, cervical cancer and uterine cancer.

15. The pharmaceutical composition of claim 14, wherein the cancer is pancreatic cancer.

16. 12. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 11.

17. 17. The method of claim 16, wherein the cancer is selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary tract cancer, cervical cancer and uterine cancer.

18. 18. The method of claim 17, wherein the cancer is pancreatic cancer.

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