Application of targeted folate receptor antibody coupling medicine in colorectal cancer and humanization of targeted folate receptor antibody coupling medicine

Abstract

The invention provides application of a targeted folate receptor antibody coupling medicine in colorectal cancer and humanization of the targeted folate receptor antibody coupling medicine. Specifically, the invention provides an application of a drug conjugate formed by coupling an anti-FOLR1 antibody and VcMMAE in treatment of colorectal cancer. In addition, the invention also provides application of the combined combination of the drug conjugate and gemcitabine in colorectal cancer treatment, and compared with independent administration, the combined combination of the drug conjugate and gemcitabine has a better inhibition effect on colorectal cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and more particularly, the present application relates to the use of a folate receptor targeting antibody conjugate drug in colorectal cancer and its humanization. BACKGROUND

[0002] Folic acid (vitamin B9) is an important single-carbon donor for purine and thymidine, which are important components of nucleic acid synthesis, and indirectly provides a donor for protein and DNA methylation modification through S-adenosyl methionine. In adult normal tissues, folic acid is mainly absorbed by Reduced Folate Carrier (RFC). RFC is a kind of anion channel that is universally expressed in the human body, responsible for the transport of folic acid by most normal cells, but has low affinity for folic acid binding (Km=1-10 μM).

[0003] Folate receptors (FR, gene name FOLR) are a class of cysteine-rich glycoproteins that bind folic acid with high affinity to mediate the absorption of folic acid, and are divided into α, β, γ three subtypes. Folate receptor alpha (FR1 for short, gene name FOLR1) is a glycosyl phosphatidylinositol (GPI) anchored membrane protein composed of a single chain polypeptide, which contains three domains: N-terminal domain, middle domain and C-terminal domain. Among them, the N-terminal domain is the main site of folic acid binding. FR1 is a key protein involved in the uptake of folic acid by cells. Folic acid is essential for DNA synthesis, repair and methylation processes, so FR1 is essential for rapidly dividing cells, such as developing embryonic cells and some cancer cells. FR1 is essential for folic acid homeostasis and has important significance in oncology, neurology and developmental biology. Although FR1 has very low expression levels in most normal tissues, it is highly expressed in many cancer cells of epithelial origin, which is used to seize the limited folic acid molecules in the environment to meet the needs of rapid division and growth of cancer cells.

[0004] FOLR1 is highly expressed on the surface of various tumor cells, such as ovarian cancer, lung cancer, breast cancer, etc., while the expression level in normal tissues is low or not expressed. This differential expression in normal cells and cancer cells provides an important basis for targeting FR1 for molecular diagnosis and treatment. Among the various forms of FR1-targeted drugs, only one antibody conjugate drug targeting folate receptor (Mirvetuximab soravtansine, MIRV) has been successfully used for clinical treatment of ovarian cancer. At present, there are still many deficiencies in the research on FOLR1 monoclonal antibodies, and there is an urgent need in the current field to develop new monoclonal antibodies against FR1 and explore their potential applications in various tumor targeted therapies, diagnostic markers and other medical fields. SUMMARY

[0005] The present application aims to provide an application of a humanized antibody drug conjugate against folate receptor alpha (FOLR1) in the treatment of colorectal cancer.

[0006] In the first aspect of the present application, a humanized antibody against folate receptor alpha is provided, which comprises: 1) a VH region as shown in SEQ ID NO. 9; and 2) a VL region as shown in SEQ ID NO. 10.

[0007] In another preferred embodiment, the VH region and VL region of the humanized antibody comprise the following CDRs: HCDR1 as shown in SEQ ID NO. 3; HCDR2 as shown in SEQ ID NO. 4; HCDR3 as shown in SEQ ID NO. 5; and LCDR1 as shown in SEQ ID NO. 6; LCDR2 as shown in SEQ ID NO. 7; LCDR3 as shown in SEQ ID NO. 8.

[0008] In another preferred embodiment, the humanized antibody comprises murine CDR regions and humanized framework regions (FRs).

[0009] In another preferred embodiment, the humanized antibody further comprises a heavy chain constant region.

[0010] In another preferred embodiment, the humanized antibody further comprises a light chain constant region.

[0011] In another preferred embodiment, the heavy chain constant region or light chain constant region is human, murine or rabbit, preferably human.

[0012] In another preferred embodiment, the humanized antibody specifically binds to FOLR1.

[0013] In the second aspect of the present application, a drug conjugate comprising the humanized antibody of the first aspect of the present application is provided, which comprises the humanized antibody of the first aspect of the present application and a drug molecule conjugated thereto.

[0014] In another preferred embodiment, the drug conjugate is an antibody conjugate against folate receptor alpha, MMAE, MMAF (microtubule inhibitors), DM1 (microtubule inhibitors), PBD dimers (DNA cross-linking agents), DXd (Topo I inhibitors).

[0015] In another preferred embodiment, the drug conjugate is an anti- folate receptor alpha antibody conjugated to MMAE.

[0016] In another preferred embodiment, the drug conjugate has killing activity against colorectal cancer cells SW620, HT29 and / or Caco-2.

[0017] In another preferred embodiment, the drug conjugate inhibits tumor growth of colorectal cancer.

[0018] In a third aspect of the present application, there is provided a pharmaceutical composition comprising: a humanized antibody according to the first aspect of the present application or a drug conjugate according to the second aspect of the present application; and a pharmaceutically acceptable carrier.

[0019] In a fourth aspect of the present application, there is provided use of a humanized antibody according to the first aspect of the present application, a drug conjugate according to the second aspect of the present application or a pharmaceutical composition according to the third aspect of the present application, in the manufacture of a medicament for treating colorectal cancer.

[0020] In a fifth aspect of the present application, there is provided a combination comprising: 1) a drug conjugate according to the second aspect of the present application; and 2) an anti-tumor drug which is a nucleoside analogue.

[0021] In another preferred embodiment, the anti-tumor drug is gemcitabine, capecitabine, decitabine, cytarabine, azacitidine, trifluridine or 5-FU.

[0022] In a sixth aspect of the present application, there is provided use of a combination for treating colorectal cancer; wherein the combination comprises: 1) a drug conjugate according to the second aspect of the present application; and 2) an anti-tumor drug which is a nucleoside analogue.

[0023] In another preferred embodiment, the anti-tumor drug is gemcitabine, capecitabine, decitabine, cytarabine, azacitidine, trifluridine or 5-FU.

[0024] In a seventh aspect of the present application, there is provided a method of treating colorectal cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a humanized antibody according to the first aspect of the present application or a drug conjugate according to the second aspect of the present application.

[0025] It should be understood that, in the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (such as the examples) can be combined with each other to constitute a new or preferred technical scheme. Limited by the length, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 HIC hydrophobic column chart of antibody 106 (106-Ab) and drug conjugate of antibody 106-VcMMAE (106-ADC) is shown. (Conditions: Proteomix HIC Butyl (Sepax Technologies). Buffer A: 1.5M (NH4)2SO4, 25mM Na2HPO4, Buffer B: 25mM Na2HPO4, 25% IPA. The flow rate is 0.8 mL / min, and linear gradient elution is performed between buffer A and buffer B between 3-16 min.) Figure 2 The killing effect of 106-ADC on different colorectal cancer cells is shown.

[0027] Figure 3 The effect of 106-ADC on the tumor volume of SW620 tumor-bearing mice is shown.

[0028] Figure 4 The effect of 106-ADC on the body weight of SW620 tumor-bearing mice is shown.

[0029] Figure 5 The effect of 106-ADC on the tumor weight of SW620 tumor-bearing mice is shown.

[0030] Figure 6 The effect of 106-ADC combined with gemcitabine on the tumor volume of HT29 tumor-bearing mice is shown. Gem in the figure represents gemcitabine.

[0031] Figure 7 The effect of 106-ADC combined with gemcitabine on the body weight of HT29 tumor-bearing mice is shown.

[0032] Figure 8 The effect of 106-ADC combined with gemcitabine on the tumor weight of HT29 tumor-bearing mice is shown.

[0033] Figure 9 The flow cytometry binding experiment of humanized antibody-106 with hela cells is shown. DETAILED DESCRIPTION

[0034] After extensive and in-depth research, the application develops an application of an anti-FOLR1 antibody-VcMMAE drug conjugate (106-ADC) in the treatment of colorectal cancer. The present application verifies the killing ability of 106-ADC on colorectal cancer cells (SW620, HT29 and Caco-2), and verifies the tumor inhibition activity of 106-ADC in a colorectal cancer tumor model mouse. In addition, compared with single administration, the combination of 106-ADC and gemcitabine antitumor drugs of the present application is proved to have better tumor inhibition effect on colorectal cancer.

[0035] Terms In order to facilitate the understanding of the present application, certain technical and scientific terms are defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary.

[0036] As used herein, the terms "comprising", "including", "containing", are used interchangeably and mean that the composition includes the recited elements, but not exclusively. In other words, the terms include "consisting of", "consisting essentially of".

[0037] Anti-folate receptor alpha humanized antibody The humanized antibody of the present application is obtained by humanizing the antibody 106, which is described with reference to CN2025113584273, the contents of which are incorporated herein by reference.

[0038] Since the antibody 106 is a murine antibody, it is easy to cause adverse immune reactions in the human body in clinical application. Therefore, it needs to be humanized.

[0039] Humanized antibodies (humanized antibodies), also known as CDR-grafted antibodies (CDR-grafted antibodies), are antibodies produced by grafting murine CDR sequences into human antibody variable region frameworks, i.e. different types of human germline antibody framework sequences. Humanized antibodies can overcome the heterogeneity reaction induced by chimeric antibodies due to the presence of a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. In order to avoid the decrease of immunogenicity while causing the decrease of activity, the human antibody variable region framework sequence can be subjected to minimum reverse mutation or back mutation to maintain the activity.

[0040] There are two kinds of selection of human framework region in antibody humanization, one is known mature antibody and the other is human germline sequence. The known mature antibody framework region usually contains somatic mutation sites, which may bring potential immunogenicity. Compared with the mature antibody, the human germline sequence framework region is theoretically less immunogenic, and the structure is more flexible, plastic and easy to accept different CDR regions. The frequency of use of human antibody germline genes in the human body has a certain bias, and the antibody humanized by selecting the high-frequency germline framework region has the advantages of low immunogenicity, high expression, stable structure and the like.

[0041] Through a large number of screening, the application unexpectedly obtains an anti- folate receptor alpha humanized antibody with an affinity comparable to that of wild-type murine antibody.

[0042] In addition, the humanized antibody of the application has almost the same binding curve as the wild-type murine antibody. Figure 9 This indicates that the two antibodies have almost the same binding performance. Based on the excellent performance of the murine antibody ADC in mouse animal experiments, therefore, the humanized antibody ADC with the same structure also has excellent performance in primates (such as monkeys or humans), wherein the "same structure" refers to the same or substantially same drug molecules (such as MMAE), linkers, linkage sites (C-lock or K-lock) and DAR in the two ADCs.

[0043] Preferably, the humanized antibody of the application comprises: 1) a VH region as shown in SEQ ID NO. 9; and 2) a VL region as shown in SEQ ID NO. 10.

[0044] The VH region and the VL region of the humanized antibody comprise murine CDRs and human FR framework regions, and the sequence of the murine CDRs is as follows: HCDR1 as shown in SEQ ID NO. 3; HCDR2 as shown in SEQ ID NO. 4; HCDR3 as shown in SEQ ID NO. 5; and LCDR1 as shown in SEQ ID NO. 6; LCDR2 as shown in SEQ ID NO. 7; LCDR3 as shown in SEQ ID NO. 8.

[0045] In addition, the anti-folate receptor alpha humanized antibody further comprises a heavy chain constant region and a light chain constant region, and the light chain constant region or the heavy chain constant region is human-derived.

[0046] The humanized antibody of the application is an IgG type antibody.

[0047] Drug conjugate (ADC) A drug conjugate comprises the antibody and a drug molecule, which is conjugated, and preferably chemically conjugated, to the antibody. The drug molecule is preferably a therapeutically active drug. Further, the drug molecule can be one or more of a toxin, a chemotherapeutic drug, a small molecule drug, or a radionuclide. The ADC comprises a linker between the drug and the antibody. The linker can be a degradable linker or a non-degradable linker. A degradable linker is typically susceptible to degradation in the intracellular environment, e.g., the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, e.g., enzymatically degradable linkers, including peptide-based linkers that are degradable by intracellular proteases, e.g., lysosomal or endosomal proteases, or saccharide linkers, e.g., glucuronide-containing linkers that are degradable by glucuronidases. Peptide-based linkers can include, e.g., dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. A non-degradable linker is typically susceptible to release of the drug under conditions in which the antibody is proteolytically cleaved.

[0048] The drug conjugate of the present application comprises the humanized antibody of the present application and a drug molecule conjugated thereto. Preferably, the drug conjugate of the present application is a conjugate of the humanized antibody and VcMMAE. Further, the drug conjugate of the present application also includes a conjugate of the humanized antibody and a drug selected from the group consisting of DM4, Exatecan, VcMMAF (Val-Cit-Monomethyl Auristatin F), and a-Amanitin.

[0049] In another preferred embodiment, the drug conjugate of the present application further comprises a conjugate of a drug molecule and a murine anti-folate receptor alpha antibody (i.e., antibody 106 of the present application, comprising a VH region as set forth in SEQ ID NO. 1; and a VL region as set forth in SEQ ID NO. 2). In the present application, "antibody 106" and "106-Ab" are used interchangeably.

[0050] The present application also provides a method of making an ADC, which can further comprise: combining the antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0051] In certain embodiments, the method of the present application comprises: combining the antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present application further comprises: combining the antibody-linker conjugate with a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0052] In some embodiments, the drug conjugate of the application is represented by the following formula:

[0053] wherein: Ab is a humanized antibody or a murine antibody, LU is a linker; D is a drug; and subscript p is a value from 1 to 8.

[0054] Drug As used herein, "drug" refers broadly to any compound having a desired biological activity and having a reactive functional group so as to be capable of being conjugated to the conjugate of the application. Desired biological activity includes diagnosing, curing, mitigating, treating, or preventing a disease in humans or other animals. Thus, the term "drug" as used herein encompasses compounds that are recognized as drugs by the official compendia, such as the United States Pharmacopeia, and, for example, the official compendium of homoeopathic medicines, the official National Formulary, or any supplement to either of these compendia. Typical drugs are listed in the Physicians' Desk Reference (PDR) and the Orange Book of the U.S. Food and Drug Administration (FDA). It is understood that as new drugs are discovered and developed, these drugs are also encompassed by "drug" in the conjugate of the application.

[0055] Drugs useful in the ADCs of the application include, but are not limited to, cytotoxic agents (e.g., cytotoxic small molecule drugs).

[0056] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression of an activity, a cell function, and / or causes destruction of a cell. The term includes radioisotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to, a ricin, (e.g., ricin E, ricin F, MMAE and MMAF), a duocarmycin, a calicheamicin, a dolastatin, a maytansinoid, a combrestatin, a discodermolide, a duocarmycin, a maytansinoid, a dolastatin, a doxorubicin, a daunorubicin, a taxol, a cisplatin, cc1065, ethidium bromide, a mitomycin, an etoposide, a tenoposide, a vincristine, a vinblastine, a colchicine, a dihydroxy anthracin dione, a dactinomycin, a diptheria toxin, a Pseudomonas exotoxin (PE) A, PE40, a gelonin, a gelonin A chain, a calein A chain, an alpha-sarcin, a saporin, a mitogellin, a retstrictocin, a phenomycin, an enomycin, a curicin, a phoratoxin, a calicheamicin, a sapaonaria officinalis inhibitor, and a glucocorticoid and other chemotherapeutic agents, and radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and radioisotopes of Lu including Lu177. Antibodies can also be conjugated to anti-cancer prodrug activating enzymes that convert a prodrug to its active form.

[0057] VcMMAE refers to a "warhead" combination in an antibody conjugate, which can be understood as a standardized, modular unit of toxicity for building specific ADC drugs. Vc: stands for valine-citrulline, a cleavable linker. MMAE: stands for monomethyl auristatin E, a cytotoxin that can kill cancer cells.

[0058] When VcMMAE is attached to an antibody that targets specific cancer cells, forming a complete ADC drug, its mechanism of action is as follows: Targeted delivery: The ADC finds and binds to specific antigens on the surface of cancer cells through its antibody part.

[0059] Internalization: The ADC-antigen complex is engulfed by the cancer cell into the interior of the cell.

[0060] Lysosomal degradation: In the lysosome, the antibody and linker are degraded or cleaved.

[0061] Linker cleavage: The Vc linker is specifically cleaved by lysosomal cathepsins.

[0062] Toxin release: MMAE is released into the cytoplasm.

[0063] Killing of cells: MMAE is a microtubulin inhibitor that prevents microtubulin polymerization in cells, thereby disrupting the cytoskeleton and mitotic processes, and ultimately inducing apoptosis in cancer cells.

[0064] Bystander effect: Because MMAE is a small molecule and is hydrophobic, it can penetrate the cell membrane, diffuse to surrounding tumor cells, and kill them, even if those cells do not express the target antigen. This is advantageous for clearing heterogeneous tumor tissue.

[0065] In another preferred embodiment, the MMAE can also be replaced by MMAF, DM1, a PBD dimer, or DXd.

[0066] Pharmaceutical composition The present application also provides a composition. Preferably, the composition is a pharmaceutical composition, which comprises the humanized antibody or active fragment thereof described above, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumorally, intraperitoneally, intravenously, or topically.

[0067] The pharmaceutical composition of the present application comprises a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above in the present application, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as a needle, solution should be manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day.

[0068] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0069] Combination The combination therapy of this invention refers to the combination of the drug conjugate (ADC) of this invention with an antitumor drug. Preferably, the combination therapy is a combination of ADC and gemcitabine. Compared with single administration, the combination of the ADC of this invention with an antitumor drug shows superior tumor suppression effects in cancer treatment, such as colorectal cancer.

[0070] Similarly, the ADC of the present invention can also be used in combination with other antitumor drugs, such as capecitabine, decitabine, cytarabine, azacitidine, trifluuridine, or 5-FU.

[0071] Compared with the prior art, the main advantages of the present invention include: 1. The humanized antibody (Hu106-Ab) of the present invention not only has low clinical immunogenicity, but also has an affinity almost identical to that of the wild-type mouse antibody (106-Ab) and a nearly identical binding curve. Figure 9 This suggests that the humanized antibody of the present invention has almost the same binding properties as the mouse antibody (including binding site, binding strength, etc.).

[0072] 2. The drug conjugate (106-ADC) of the present invention has killing ability against different colorectal cancer cell lines, such as SW620, HT29 and Caco-2, and has tumor inhibitory activity against colorectal cancer tumor model mice.

[0073] 3. The combination of the present invention (106-ADC and gemcitabine) has a better inhibitory effect on tumor growth in colorectal cancer compared with 106-ADC or gemcitabine alone.

[0074] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0075] Example 1. Humanization of antibody 106 The variable region genes of antibody 106 were used to generate humanized monoclonal antibodies. The amino acid sequences of the VH and VK of antibody 106 were compared with existing human Ig gene sequence databases to find the overall best matching human germline Ig sequences, and the humanized sequences of antibody 106 are shown in Table 1.

[0076] To achieve humanization of antibody 106, IgHV1-206, IgHV1-301, IgHV1-4601, IgHV1 / OR15-104, IgHV1 / OR15-1*01 were first selected as the reference sequences for the framework backbones of the heavy chain of antibody 106 for humanization; on this basis, the framework and constant region amino acid residues in the heavy chain of antibody 106 that are not of human origin were replaced with the corresponding human-derived residues, thereby obtaining a humanized heavy chain. Similarly, for the light chain of 106, the sequences of IgKV7-301, IgKV4-101, IgKV3D-2002, IgKV3-1101, IgKV3-20*01 were used as the reference sequences for humanization of the framework, and the non-human framework / constant region residues of the light chain of antibody 106 were replaced to construct a humanized light chain. To verify that the humanization replacement does not destroy the conformation of the antibody, three-dimensional structure modeling and comparative analysis of the humanized antibodies were performed, and the conformational maintenance and structural stability of the key regions (such as the complementarity determining regions CDR) and the overall framework were evaluated.

[0077] Table 1. Sequences of humanized antibody 106

[0078] The humanized antibodies are designated Hu106-Ab.

[0079] Example 2. Flow cytometry detection of the binding activity of humanized antibody 106 1) Take 1x10 5Hela cells were placed in 1.5 mL EP tubes, and 20 nM of the highest concentration of humanized antibody 106 was added for 2-fold gradient dilution, and incubated at 4°C in the dark for 30 min in a 100 μL system; after incubation, centrifugation was performed at 300g at 4°C for 5 min; after resuspension in 500 μL of PBS (containing 0.1% BSA), centrifugation was performed at 300g at 4°C for 5 min; the supernatant was discarded, and secondary antibody anti-human IgG H+L (brand: invitrogen; item number: A21445; 1:2000) was added in a 100 μL system, and incubated at 4°C in the dark for 30 min; after incubation, centrifugation was performed at 300g at 4°C for 5 min; after resuspension in 500 μL of PBS (containing 0.1% BSA), centrifugation was performed at 300g at 4°C for 5 min; the supernatant was discarded, and 300 μL of PBS was added; after filtration of the cells through a 300-mesh filter screen, the cells were subjected to flow detection.

[0080] The results are shown in Table 2 and FIG. 2. Figure 9 As shown in Table 2 and FIG. 2, the humanized antibody 106 still has specific binding activity to Hela cells expressing folate receptor alpha.

[0081] Table 2 shows the specific values of the binding rates of Figure 9

[0082] Unexpectedly, the affinity of one strain of humanized antibody is almost completely consistent with that of the wild-type murine antibody, in contrast to the decrease in affinity of most humanized antibodies. Moreover, the humanized antibody of the present application has almost the same binding curve as the wild-type murine antibody (FIG. 2), which indicates that the two antibodies have almost the same binding performance. Figure 9

[0083] Example 3. Preparation of ADC In this example, ADCs based on murine antibodies and humanized antibodies were prepared. In mouse experiments, ADCs using murine antibodies were used for experiments.

[0084] 1. In the preparation of 106-ADC, the sequence of the antibody (106-Ab) used is shown in Table 3: Table 3 Sequence of 106-Ab antibody

[0085] 2. Preparation process of 106-ADC: 1) TCEP (tris (2-carboxyethyl) phosphine hydrochloride) was used to reduce 106-Ab antibody at about 8 times the molar concentration of 106-Ab antibody, and incubated at room temperature for 3 h; ​​2) Add VcMMAE (brand: MCE; catalog: HY-15575) small molecule toxin reagent solution at about 4-8 times the molar concentration of 106-Ab antibody, react for 1-2 h at room temperature; 3) Add NAC reagent (Acetylcysteine; MCE, HY-B0215) at 8 times the molar concentration of 106-Ab antibody, incubate on ice for 30 min; 4) Perform buffer exchange on the prepared 106-ADC with 1xPBS solution using a desalting column (Zeba™ Desalt Spin Column, 7K MWCO, 5 mL; ThermoScientific, 89891); 5) Concentrate the 106-ADC using a 30KDa concentrator tube and determine the concentration; 6) Identify the prepared 106-ADC using an analytical HIC hydrophobic column (Proteomix HIC Butyl); The identification map is as follows Figure 1 The results show that 106-Ab and 106-ADC can be well separated.

[0086] The preparation method of the ADC of the humanized antibody is the same as that of 106-ADC, except that the humanized antibody replaces the murine antibody Example 4. 106-ADC killing activity assay on colorectal cancer cells Take different colorectal cancer cells (SW480, HT29 and Caco-2), inoculate 5-8x10 3 cells / well in a 96-well plate, 100 μl per well, pre-culture for 16-24 h at 37°C in a 5% CO2 incubator, then add different concentrations of 106-ADC according to Table 4, incubate at 37°C in a 5% CO2 incubator, incubate for 72 h, then place the culture plate at room temperature for 30 min to balance to room temperature, and use Promega CellTiter-Glo™ Luminescent Cell Viability Assay Kit to detect the killing activity of 106-ADC antibody-drug conjugate on cells.

[0087] Table 4 Drug concentration table for testing 106-ADC killing activity on colorectal cancer cells

[0088] The results are as follows Figure 2As shown, compared with the 106-Ab and VcMMAE treatment groups, the cell viability (SW480, HT29 and Caco-2) in the 106-ADC treatment group decreased with increasing 106-ADC concentration, and SW480 was the most sensitive to 106-ADC.

[0089] Example 5. Pharmacodynamic evaluation of 106-ADC in SW620 subcutaneous tumor graft model Six-week-old female C-NKG mice (Cyagen Biosciences) were subcutaneously inoculated with 2 x 10 6 Human colon cancer cells (SW620) were randomly divided into two groups of 6 mice each after the tumors grew to approximately 100 mm³. The control group received a blank control group and a treatment group. The treatment group received a 5 mg / kg intravenous injection (10⁶-ADC) once a week via tail vein. Tumor volume and mouse weight were measured simultaneously with the administration of the drug. Tumors exceeding 1500 mm³ were treated. 3 The experiment was stopped before the mice were euthanized.

[0090] The results are as follows Figure 3 and Figure 5 As shown, compared with the control group, administration of 106-ADC significantly inhibited tumor growth and reduced tumor size.

[0091] And such as Figure 4 As shown, the drug administration had no significant effect on the weight of mice, indicating that the antibody-drug conjugate had no significant toxicity to mice.

[0092] Example 6. Pharmacodynamic evaluation of the combination of 106-ADC and gemcitabine in the HT29 subcutaneous tumor model. Six-week-old female C-NKG mice (Cyagen Biosciences) were subcutaneously inoculated with 2 x 10 6 Human colon cancer cells (HT29), when the tumor grows to 100 mm 3 Six mice were randomly assigned to four groups: a blank control group, a 106-ADC administration group, a chemotherapy drug administration group, and a combination therapy group. The control group received PBS. The 106-ADC administration group received 5 mg / kg of 106-ADC via tail vein injection once weekly. The chemotherapy drug administration group received gemcitabine at 0.5 mg / kg intraperitoneally every three days. The combination therapy group received 106-ADC at 5 mg / kg via tail vein injection once weekly and chemotherapy drug at 0.5 mg / kg intraperitoneally every three days. Tumor volume and mouse weight were measured throughout the experiment. Tumors exceeding 1500 mm² were treated accordingly. 3 The experiment was stopped before the mice were euthanized.

[0093] The results are as follows Figure 6 , 7As shown in Figures 8 and 5, 106-ADC significantly inhibited tumor growth, and its monotherapy effect was comparable to or even more significant than that of the chemotherapy drug gemcitabine. Furthermore, data showed that the combination therapy exhibited a significant synergistic therapeutic effect in in vivo anti-tumor models, with a tumor inhibition rate significantly superior to either monotherapy group. Simultaneously, the combination therapy regimen had no significant effect on the weight gain curve of tumor-bearing mice, indicating that while effectively enhancing anti-tumor activity, the combination therapy did not exhibit significant additional toxicity and possessed excellent safety characteristics.

[0094] Table 5 Correspondence Figure 6 mean tumor volume

[0095] Based on the excellent performance of murine antibody ADCs in mouse animal experiments, humanized antibody ADCs with the same structure also have excellent performance in primates (such as monkeys or humans). "Same structure" means that the drug molecules (such as MMAE), linkers, linker sites (C-lock or K-lock), and DARs conjugated in the two ADCs are the same or substantially the same.

[0096] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-Folate Receptor a humanized antibody, characterized in that, The antibody comprises: 1) a VH region as shown in SEQ ID NO. 9; and 2) a VL region as shown in SEQ ID NO.

10.

2. A drug conjugate comprising the antibody of claim 1, wherein, The conjugate comprises the antibody of claim 1 and a drug molecule coupled thereto, the drug molecule being MMAE.

3. The drug conjugate of claim 2, wherein, The drug conjugate has killing activity on colon cancer cell lines SW620, HT29 and / or Caco-2.

4. The use of the humanized antibody according to claim 1 or the drug conjugate according to claim 2, wherein A medicament for treating colorectal cancer.

5. A combination of ingredients, characterized in that, The combination comprises: 1) the drug conjugate of claim 2; and 2) an anti-tumor drug, the anti-tumor drug being a nucleoside analogue.

6. The combination of claim 5, wherein The nucleoside analogue is gemcitabine, capecitabine, decitabine, cytarabine, azacitidine, trifluridine or 5-FU.

7. The combination of claim 5, wherein The nucleoside analogue is gemcitabine.

8. Use of a combination of a compound of formula I ###0002### and a compound of formula II ###0003### wherein the variables are as defined in claim 1. A medicament for treating colorectal cancer. The combination comprises: 1) the drug conjugate of claim 2; and 2) an anti-tumor drug, the anti-tumor drug being a nucleoside analogue.

9. Use according to claim 8, characterized in that, The nucleoside analogue is gemcitabine, capecitabine, decitabine, cytarabine, azacitidine, trifluridine or 5-FU.

10. The use according to claim 8, characterized in that, The nucleoside analogue is gemcitabine.

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