Use of folate receptor-targeted antibody conjugate drugs in colorectal cancer and humanization thereof

Abstract

The present application provides a kind of targeting folate receptor antibody conjugated drug in colorectal cancer and its humanization.The specific application provides the application of the drug conjugate of anti-FOLR1 antibody and VcMMAE conjugation in the treatment of colorectal cancer.In addition, the present application also provides the application of the combination of the drug conjugate and gemcitabine in the treatment of colorectal cancer, and the combination of the drug conjugate and gemcitabine has better inhibitory effect on colorectal cancer than single administration.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, this invention relates to the use of a folic acid receptor antibody-drug conjugate in colorectal cancer and its humanization. Background Technology

[0002] Folic acid (vitamin B9) is an important single-carbon donor of purines and thymidines, essential components in the synthesis of nucleic acids, and indirectly provides a donor for protein and DNA methylation modification via S-adenosylmethionine. In normal adult tissues, folic acid is mainly absorbed by Reduced Folate Carriers (RFCs). RFCs are anion channels widely expressed in the human body, responsible for the transport of folic acid in most normal cells, but they have a low affinity for folic acid binding (Km = 1-10 μM).

[0003] Folic acid receptors (FRs, genetically named FOLR) are a class of cysteine-rich glycoproteins that mediate folate uptake by binding to folate with high affinity. They are classified into three subtypes: α, β, and γ. Folate receptor α (FR1, genetically named FOLR1) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein composed of a single-chain polypeptide containing three domains: an N-terminal domain, a central domain, and a C-terminal domain. The N-terminal domain is the primary site of folate binding. FR1 is a key protein involved in cellular folate uptake. Folate is essential for DNA synthesis, repair, and methylation processes; therefore, FR1 is crucial for rapidly dividing cells, such as developing embryonic cells and certain cancer cells. FR1 is vital for folate homeostasis and has significant implications in oncology, neurology, and developmental biology. Although FR1 is expressed at very low levels in most normal tissues, it is highly expressed in many epithelial-derived cancer cells, competing for limited folate molecules in the environment to meet the needs of rapid cancer cell division and growth.

[0004] FOLR1 is highly expressed on the surface of various tumor cells, such as those in ovarian cancer, lung cancer, and breast cancer, while its expression level is low or absent in normal tissues. This differential expression between normal and cancer cells provides important evidence for molecular diagnosis and treatment targeting FR1. Among various drug formulations targeting FR1, only one antibody-drug conjugate targeting the folate receptor (Mirvetuximab soravtansine, MIRV) has been successfully used clinically to treat ovarian cancer. Currently, research on FOLR1 monoclonal antibodies still has many shortcomings. There is an urgent need in this field to develop novel monoclonal antibodies targeting FR1 and explore their potential applications in various tumor targeted therapies, diagnostic biomarkers, and other medical fields. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a humanized antibody-drug conjugate against folate receptor α (FOLR1) in the treatment of colorectal cancer.

[0006] In a first aspect of the invention, a humanized antibody against folic acid receptor α is provided, the antibody comprising:

[0007] 1) The VH region as shown in SEQ ID NO.9; and

[0008] 2) VL region as shown in SEQ ID NO.10.

[0009] In another preferred embodiment, the VH and VL regions of the humanized antibody include the following CDRs:

[0010] HCDR1 as shown in SEQ ID NO.3;

[0011] HCDR2, as shown in SEQ ID NO.4;

[0012] HCDR3 as shown in SEQ ID NO.5; and

[0013] LCDR1 as shown in SEQ ID NO.6;

[0014] LCDR2 as shown in SEQ ID NO.7;

[0015] LCDR3, as shown in SEQ ID NO.8.

[0016] In another preferred embodiment, the humanized antibody comprises a mouse-derived CDR region and a humanized frame region (FR).

[0017] In another preferred embodiment, the humanized antibody further includes a heavy chain constant region.

[0018] In another preferred embodiment, the humanized antibody further includes a light chain constant region.

[0019] In another preferred embodiment, the heavy chain constant region or the light chain constant region is of human, mouse, or rabbit origin, preferably of human origin.

[0020] In another preferred embodiment, the humanized antibody specifically binds to FOLR1.

[0021] In a second aspect of the invention, a drug conjugate comprising the humanized antibody described in the first aspect of the invention is provided, the drug conjugate comprising the humanized antibody described in the first aspect of the invention and a drug molecule conjugated thereto.

[0022] In another preferred embodiment, the drug conjugate is an antibody against folic acid receptor α conjugate with MMAE, MMAF (microtubule inhibitor), DM1 (microtubule inhibitor), PBD dimer (DNA cross-linking agent), or DXd (Topo I inhibitor).

[0023] In another preferred embodiment, the drug conjugate is an antibody-conjugate MMAE against folic acid receptor α.

[0024] In another preferred embodiment, the drug conjugate has cytotoxic activity against colorectal cancer cells SW620, HT29, and / or Caco-2.

[0025] In another preferred embodiment, the drug conjugate inhibits the growth of colorectal cancer tumors.

[0026] In a third aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0027] Humanized antibodies as described in the first aspect of the present invention or drug conjugates as described in the second aspect of the present invention; and pharmaceutically acceptable carriers.

[0028] In a fourth aspect of the invention, the use of the humanized antibody as described in the first aspect of the invention, the drug conjugate as described in the second aspect of the invention, or the pharmaceutical composition as described in the third aspect of the invention is provided for the preparation of a medicament for treating colorectal cancer.

[0029] In a fifth aspect of the invention, a combination of ingredients is provided, the combination comprising:

[0030] 1) The drug conjugate as described in the second aspect of the present invention; and

[0031] 2) Antitumor drugs, wherein the antitumor drugs are nucleoside analogs.

[0032] In another preferred embodiment, the antitumor drug is gemcitabine, capecitabine, decitabine, cytarabine, azacitidine, trifluridine, or 5-FU.

[0033] In a sixth aspect of the invention, a combination of these ingredients is provided for the treatment of colorectal cancer;

[0034] The combination of these ingredients includes:

[0035] 1) The drug conjugate as described in the second aspect of the present invention; and

[0036] 2) Antitumor drugs, wherein the antitumor drugs are nucleoside analogs.

[0037] In another preferred embodiment, the antitumor drug is gemcitabine, capecitabine, decitabine, cytarabine, azacitidine, trifluridine, or 5-FU.

[0038] In a seventh aspect of the invention, a method for treating colorectal cancer is provided, the method comprising administering to a subject in need a therapeutically effective amount of a humanized antibody as described in the first aspect of the invention or a drug conjugate as described in the second aspect of the invention.

[0039] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0040] Figure 1 The HIC hydrophobic column plots of antibody 106 (106-Ab) and the drug conjugate of antibody 106-VcMMAE (106-ADC) are shown. (Conditions: Proteomix HIC Butyl (Sepax Technologies). Buffer A: 1.5M (NH4)2SO4, 25mM Na2HPO4; Buffer B: 25mM Na2HPO4, 25% IPA. Flow rate: 0.8 mL / min; linear gradient elution from buffer A to buffer B over 3–16 min.)

[0041] Figure 2 The study demonstrated the killing effect of 106-ADC on different colorectal cancer cells.

[0042] Figure 3 The effect of 106-ADC on tumor volume in SW620 tumor-bearing mice was shown.

[0043] Figure 4 The effect of 106-ADC on body weight in SW620 tumor-bearing mice was shown.

[0044] Figure 5 The effect of 106-ADC on tumor weight in SW620 tumor-bearing mice was shown.

[0045] Figure 6 The effect of 106-ADC combined with gemcitabine on tumor volume in HT29 tumor-bearing mice is shown. Gem in the figure represents gemcitabine.

[0046] Figure 7 The effect of 106-ADC combined with gemcitabine on body weight in HT29 tumor-bearing mice was shown.

[0047] Figure 8The effect of 106-ADC combined with gemcitabine on tumor weight in HT29 tumor-bearing mice was shown.

[0048] Figure 9 The flow cytometry binding assay of humanized antibody-106 to HeLa cells is shown. Detailed Implementation

[0049] Following extensive and in-depth research, this application develops a drug conjugate (106-ADC) of an anti-FOLR1 antibody-VcMMAE for the treatment of colorectal cancer. This invention validates the killing ability of 106-ADC against colorectal cancer cells (SW620, HT29, and Caco-2), and its tumor-suppressive activity in mouse models of colorectal cancer. Furthermore, compared to monotherapy, the combination of the 106-ADC of this invention with the antitumor drug gemcitabine has been shown to have a better tumor-suppressive effect in colorectal cancer.

[0050] the term

[0051] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly 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 this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0052] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0053] Anti-folate receptor α humanized antibody

[0054] The humanized antibody of the present invention is obtained by humanizing antibody 106, which is described in CN2025113584273, all of which are incorporated herein by reference.

[0055] Because antibody 106 is a murine antibody, it is prone to causing adverse immune responses in humans during clinical application. Therefore, it needs to be humanized.

[0056] Humanized antibodies, also known as CDR-grafted antibodies, are antibodies created by grafting mouse CDR sequences into the variable region framework of human antibodies, resulting in different types of human germline antibody framework sequences. Humanized antibodies can overcome the heterologous reactions induced by chimeric antibodies, which carry a large amount of mouse protein components. These framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0057] In antibody humanization, there are generally two choices for the human framework region: known mature antibodies and human Germline sequences. Known mature antibody framework regions often contain somatic mutation sites, potentially introducing immunogenicity. Compared to mature antibodies, human Germline sequence framework regions theoretically have lower immunogenicity, are more structurally flexible and malleable, and readily accept different CDR regions. The frequency of use of human antibody Germline genes in the human body exhibits a certain bias; antibodies humanized from frequently used Germline framework regions have advantages such as low immunogenicity, high expression levels, and structural stability.

[0058] Through extensive screening, this invention unexpectedly yielded a humanized antibody against folic acid receptor α with an affinity comparable to that of wild-type mouse antibodies.

[0059] Furthermore, the humanized antibody of the present invention has almost the same binding curve as the wild-type mouse antibody. Figure 9 This suggests that the two antibodies have almost identical binding properties. 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). Here, "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.

[0060] Preferably, the humanized antibody of the present invention comprises: 1) a VH region as shown in SEQ ID NO. 9; and 2) a VL region as shown in SEQ ID NO. 10.

[0061] The humanized antibody contains a mouse CDR and a human FR frame region in its VH and VL regions, and the sequence of the mouse CDR is shown below:

[0062] HCDR1 as shown in SEQ ID NO.3;

[0063] HCDR2, as shown in SEQ ID NO.4;

[0064] HCDR3 as shown in SEQ ID NO.5; and

[0065] LCDR1 as shown in SEQ ID NO.6;

[0066] LCDR2 as shown in SEQ ID NO.7;

[0067] LCDR3, as shown in SEQ ID NO.8.

[0068] In addition, the anti-folate receptor α humanized antibody also includes a heavy chain constant region and a light chain constant region, wherein the light chain constant region or the heavy chain constant region is human-derived.

[0069] The humanized antibody of this invention is an IgG type antibody.

[0070] Drug conjugates (ADCs)

[0071] Drug conjugates (ADCs) include the antibody and a drug molecule, wherein the antibody is conjugated to the drug molecule, preferably chemically conjugated. The drug molecule is preferably a drug with therapeutic activity. Furthermore, the drug molecule can be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide. The ADC includes a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers typically degrade readily in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0072] The drug conjugates of the present invention comprise the humanized antibody described herein and a drug molecule conjugated thereto. Preferably, the drug conjugates of the present invention are conjugates of the humanized antibody and VcMMAE. Furthermore, the drug conjugates of the present invention also include conjugates of the humanized antibody and the following group of drugs: DM4, Exatecan, VcMMAF (Val-Cit-MonomethylAuristatin F), and α-Amanitin.

[0073] In another preferred embodiment, the drug conjugate of the present invention further includes conjugation of the drug molecule with a murine antifolate receptor α antibody (i.e., antibody 106 of the present invention, including the VH region as shown in SEQ ID NO.1 and the VL region as shown in SEQ ID NO.2). In the present invention, "antibody 106" and "106-Ab" are used interchangeably.

[0074] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0075] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0076] In some embodiments, the drug conjugates of the present invention are shown in the following molecular formula:

[0077]

[0078] in:

[0079] Ab is a humanized antibody or a murine antibody.

[0080] LU stands for connector / connector;

[0081] D is a drug;

[0082] Furthermore, the subscript p is a value from 1 to 8.

[0083] Drug

[0084] As used herein, "drug" refers to any compound having the desired biological activity and possessing reactive functional groups for the preparation of the conjugates described herein. The desired biological activity includes diagnosing, curing, alleviating, treating, and preventing diseases in humans or other animals. Therefore, the term "drug" refers to compounds identified in official national pharmacopoeias, as well as those confirmed by, for example, the United States Pharmacopeia of Allotherapy, the National Formulary, or any of its supplements, provided they possess the necessary reactive functional groups. Typical drugs are listed in the Physician's Desk Reference (PDR) and the Orange Book of the U.S. Food and Drug Administration (FDA). It should be understood that as new drugs are discovered and developed, these drugs should also be included in the term "drug" in the conjugates described herein.

[0085] Drugs that can be used in the ADC of this invention include, but are not limited to, cytotoxic agents (e.g., small molecule cytotoxic drugs).

[0086] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression of cellular activity, cellular function, and / or causes cellular damage. This term includes radioactive isotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzyme-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: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, phenytoxin, phenytoxin A-chain, cobustatin, docalimcin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxetine dione, actinomycin, diphtheria toxin, pseudomonadoxocytoxin (PE)A, PE40, absinthecin, absinthecin A-chain, arbuscular mycotoxin A-chain, α- Antibodies can be conjugated with aspirin, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachomycin, Sapaonaria officinalis inhibitors, glucocorticoids and other chemotherapeutic agents, as well as radioactive isotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and Lu radioactive isotopes including Lu177. Antibodies can also be conjugated to anticancer prodrug activating enzymes capable of converting prodrugs to their active forms.

[0087] VcMMAE refers to a "warhead" assembly in an antibody-drug conjugate (ADC). It can be understood as a standardized, modular toxicity unit used to construct specific ADC drugs. Vc: stands for valine-citrulline, a cleavable linker. MMAE: stands for monomethyl auristatin E, a cytotoxic agent that can kill cancer cells.

[0088] When VcMMAE is linked to an antibody targeting specific cancer cells, forming a complete ADC drug, its mechanism of action is as follows:

[0089] Targeted delivery: ADCs precisely locate and bind to specific antigens on the surface of cancer cells through their antibody portion.

[0090] Internalization: The ADC-antigen complex is engulfed by cancer cells and enters the cell.

[0091] Lysosomal degradation: In lysosomes, antibodies and linkers are degraded or cleaved.

[0092] Linker breakage: The Vc linker is specifically cleaved by cathepsins in lysosomes.

[0093] Toxin release: MMAE is released into the cytoplasm.

[0094] Kills cells: MMAE is a microtubule inhibitor that prevents microtubule polymerization within cells, thereby disrupting the cytoskeleton structure and mitosis, and ultimately inducing apoptosis in cancer cells.

[0095] Bystander effect: Because MMAE is a small molecule and highly hydrophobic, it can penetrate cell membranes, diffuse into surrounding tumor cells, and kill them, even if those cells do not express the target antigen. This is highly advantageous for clearing heterogeneous tumor tissue.

[0096] In another preferred embodiment, the MMAE may also be replaced by MMAF, DM1, PBD dimer, or DXd.

[0097] Pharmaceutical Composition

[0098] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the above-described humanized antibody or its active fragment, and a pharmaceutically acceptable carrier. Typically, these substances are 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 may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0099] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day.

[0100] 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.

[0101] Combination

[0102] 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.

[0103] 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.

[0104] Compared with the prior art, the main advantages of the present invention include:

[0105] 1. The humanized antibody (Hu106-Ab) of the present invention not only has low immunogenicity in clinical applications, 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.).

[0106] 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.

[0107] 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.

[0108] 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.

[0109] Example 1. Humanization of 106 antibody

[0110] The variable region gene of antibody 106 was used to generate a humanized monoclonal antibody. The amino acid sequences of VH and VK of antibody 106 were compared with existing human Ig gene sequence databases to find the best-matching human germline Ig sequence overall. The humanized sequence of antibody 106 is shown in Table 1.

[0111] To achieve the humanization of antibody 106, the following sequences were first selected as the humanization framework reference sequences for its heavy chain: IgHV1-206, IgHV1-301, IgHV1-4601, IgHV1 / OR15-104, and IgHV1 / OR15-1*01. Based on these, non-human framework and constant region amino acid residues in the antibody 106 heavy chain were replaced with corresponding human residues to obtain a humanized heavy chain. Similarly, for the 106 light chain, the sequences IgKV7-301, IgKV4-101, IgKV3D-2002, IgKV3-1101, and IgKV3-20*01 were used as humanization framework references. Non-human framework / constant region residues in the antibody 106 light chain were replaced to construct a humanized light chain. To verify that humanization replacement does not disrupt the antibody conformation, three-dimensional structural modeling and comparative analysis were performed on the humanized antibody, and the conformational retention and structural stability of key regions (such as complementarity-determining regions, CDRs) and the overall framework were evaluated.

[0112] Table 1. Sequence of humanized antibody 106

[0113]

[0114] The humanized antibody was named Hu106-Ab.

[0115] Example 2. Flow cytometry detection of the binding activity of humanized antibody 106

[0116] 1) Take 1x10 5One HeLa cell was placed in a 1.5 mL EP tube, and the highest concentration of 20 nM humanized antibody 106 was added. The cells were serially diluted 2-fold and incubated in 100 μL at 4°C in the dark for 30 min. After incubation, the cells were centrifuged at 300g at 4°C for 5 min. The cells were then resuspended in 500 μL of PBS (containing 0.1% BSA) and centrifuged again at 300g at 4°C for 5 min. The supernatant was discarded, and the secondary antibody anti-huamn IgG H+L (brand: Invitrogen; catalog number: A21445; 1:2000) was added and incubated in 100 μL at 4°C in the dark for 30 min. After incubation, the cells were centrifuged at 300g at 4°C for 5 min. The cells were then resuspended in 500 μL of PBS (containing 0.1% BSA) and centrifuged again at 300g at 4°C for 5 min. The supernatant was discarded, and 300 μL of PBS was added. The cells were filtered through a 300-mesh filter and then analyzed by flow cytometry.

[0117] The results are as follows Figure 9 As shown in Table 2, the humanized antibody 106 still exhibits specific binding activity against HeLa cells expressing folate receptor α.

[0118] Table 2 Corresponding Figure 9 Specific numerical value of the binding rate

[0119]

[0120] Surprisingly, contrary to the decrease in affinity observed in most humanized antibodies, the affinity of one humanized antibody was almost identical to that of the wild-type mouse antibody. Furthermore, the humanized antibody of this invention exhibits almost identical binding curves to the wild-type mouse antibody. Figure 9 This suggests that the two antibodies have almost identical binding properties.

[0121] Example 3. Preparation of ADC

[0122] In this embodiment, an ADC based on murine antibodies and humanized antibodies was prepared. In mouse experiments, the ADC based on murine antibodies was used.

[0123] 1. The sequence of the antibody (106-Ab) used in the preparation of 106-ADC is shown in Table 3:

[0124] Table 3. Sequences of 106-Ab antibodies

[0125]

[0126] 2. Fabrication process of 106-ADC:

[0127] 1) Reduce the 106-Ab antibody with approximately 8 times the molar concentration of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) and incubate at room temperature for 3 hours;

[0128] 2) Add approximately 4-8 times the molar concentration of VcMMAE (brand: MCE; catalog number: HY-15575) small molecule toxin reagent solution, and react at room temperature for 1-2 hours;

[0129] 3) Add 8 times the molar concentration of 106-Ab antibody NAC reagent (Acetylcysteine; MCE, HY-B0215) and incubate on ice for 30 min;

[0130] 4) The prepared 106-ADC was buffer-replaced with 1xPBS solution using a desalting column (Zeba™ Desalt Spin Column, 7K MWCO, 5 mL; ThermoScientific, 89891);

[0131] 5) Concentrate 10⁶-ADC using a 30 kDa concentrator and determine its concentration;

[0132] 6) The prepared 106-ADC was identified using an analytical HIC hydrophobic column (Proteomix HIC Butyl);

[0133] Identification chart as follows Figure 1 The results showed that 106-Ab and 106-ADC could be separated well.

[0134] The preparation method of humanized antibody ADC is the same as that of 106-ADC, the difference being that the humanized antibody replaces the mouse antibody.

[0135] Example 4. Assay of the killing activity of 106-ADC against colorectal cancer cells

[0136] Different colorectal cancer cells (SW480, HT29, and Caco-2) were collected at a ratio of 5-8 x 10⁻⁶. 3 Cells were seeded per well in 96-well plates at 100 μl per well and pre-cultured at 37°C in a 5% CO2 incubator for 16-24 hours. Then, different concentrations of 106-ADC were added according to Table 4, and the plates were incubated at 37°C in a 5% CO2 incubator for 72 hours. After incubation, the plates were placed at room temperature for 30 minutes to allow them to equilibrate to room temperature. The cell-killing activity of the 106-ADC antibody-drug conjugate was detected using the Promega CellTiter-Glo™ Luminescent Cell Viability Assay Kit.

[0137] Table 4. Drug concentrations used to test the killing activity of 106-ADC against colorectal cancer cells.

[0138]

[0139] The results are as follows Figure 2 As 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.

[0140] Example 5. Pharmacodynamic evaluation of 106-ADC in SW620 subcutaneous tumor graft model

[0141] 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.

[0142] 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.

[0143] 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.

[0144] Example 6. Pharmacodynamic evaluation of the combination of 106-ADC and gemcitabine in the HT29 subcutaneous tumor model.

[0145] 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. 3The experiment was stopped before the mice were euthanized.

[0146] The results are as follows Figure 6 , 7 As 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.

[0147] Table 5 Correspondence Figure 6 mean tumor volume

[0148]

[0149] 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.

[0150] 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. A humanized antibody against folic acid receptor α, characterized in that, The antibodies include: 1) The VH region as shown in SEQ ID NO.9; and 2) VL region as shown in SEQ ID NO.

10.

2. A drug conjugate comprising the antibody of claim 1, characterized in that, The conjugate comprises the antibody of claim 1 and a drug molecule conjugated thereto, wherein the drug molecule is MMAE.

3. The drug conjugate as described in claim 2, characterized in that, The drug conjugates exhibit killing activity against colon cancer cell lines SW620, HT29, and / or Caco-2.

4. The use of the humanized antibody as described in claim 1 or the drug conjugate as described in claim 2, characterized in that, Used to prepare drugs for the treatment of colorectal cancer.

5. A combination of ingredients, characterized in that, The combination of drugs includes: 1) The drug conjugate as described in claim 2; and 2) Gemcitabine.

6. The use of a combination thereof, characterized in that, Used in the preparation of drugs for treating colorectal cancer; The combination of these ingredients includes: 1) The drug conjugate as described in claim 2; and 2) Gemcitabine.

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