Anti-5T4 antibody and its use

Abstract

This invention relates to anti-5T4 antibodies, particularly humanized antibodies. Furthermore, it relates to pharmaceutical compositions containing these antibodies and their pharmaceutical applications. The antibodies have high affinity, good cell binding ability, and high internal migration rate, and can be used to treat diseases in which 5T4 is overexpressed, such as cancer.

Description

[Technical Field]

[0001] This invention relates to anti-5T4 antibodies, particularly humanized antibodies. Furthermore, this invention relates to pharmaceutical compositions containing these antibodies and their pharmaceutical uses. [Background technology]

[0002] 5T4 (trophotrophoblast glycoprotein) is a 72kDa glycoprotein that was first discovered in fetal cells about 20 years ago and recognized by indirect immunofluorescence staining using the mouse monoclonal antibody 5T4. It is expressed in human trophotrophoblast cells and is highly expressed in various tumor tissues, but is either not expressed or is low in normal adult tissues. The 5T4 gene is located in the 6q14-q15 region of chromosome 6 and encodes 420 amino acids. The human and mouse 5T4 protein sequences have 81% homology, and the 5T4 protein has many glycosylation modifications, but its biological function has not yet been elucidated.

[0003] 5T4 is overexpressed in some tumors, such as breast, lung, and kidney cancer, but is expressed at very low levels in normal tissues. High 5T4 expression is also positively correlated with poor prognosis in head and neck tumors and colorectal cancer. Recent studies have revealed a higher proportion of 5T4-positive B cell populations in relapsed lymphocytic leukemia patients. 5T4 expression in cancer stem cells (CSCs) is associated with an overall poor prognosis in non-small cell lung cancer, potentially contributing to tumor metastasis, drug resistance, and recurrence. Under normal circumstances, 5T4 is primarily expressed in fetal cells to enhance chemotaxis and play a crucial role in embryonic development and cell differentiation. Pathologically, 5T4 overexpression is associated with the downregulation of E-cadherin, leading to metastatic spread in epithelial tumors. 5T4 is also involved in regulating β-catenin-independent Wnt signaling by modulating the intracellular localization of LRP6. Furthermore, 5T4 modulates chemotaxis via the CXCL12 / CXCR4 chemokine, which may promote the spread of 5T4-positive tumor cells.

[0004] First-generation chimeric antigen receptor T cells targeting 5T4 expressed by human T cells exhibit a degree of cytotoxicity in in vitro experiments, while promoting the release of specific cytokines against 5T4-positive cells. Such 5T4-targeting chimeric antigen receptors have been used to modify T cells derived from renal cell carcinoma (RCC) patients and can effectively kill renal cancer cells in vitro. Furthermore, mice inoculated with 5T4-expressing B16 tumors showed extended survival after CAR-T cell therapy, demonstrating a synergistic effect between CAR-T cells and the 5T4-targeting vaccine, providing evidence of synergistic efficacy when used in combination. These findings suggest that 5T4 is a potential tumor therapeutic target. [Overview of the project]

[0005] In a first embodiment, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to 5T4 (trophoblast glycoprotein), wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein LCDR1, LCDR2, and LCDR3 each comprise or are equivalent variants of the light chain variable region having the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, and HCDR1, HCDR2, and HCDR3 each comprise or are equivalent variants of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0006] In some embodiments, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are defined by one of the following definition schemes: IMGT, Kabat, Chothia, Contact, or Martin.

[0007] In some embodiments, LCDR1 includes or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, LCDR2 includes or is the sequence shown in SEQ ID NO: 2 or an equivalent variant thereof, LCDR3 includes or is the sequence shown in SEQ ID NO: 3 or an equivalent variant thereof, and HCDR1 includes or is the sequence shown in SEQ ID NO: 4 or an equivalent variant thereof, HCDR2 includes or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, and HCDR3 includes or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof.

[0008] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 8 or an equivalent variant thereof.

[0009] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0010] In some embodiments, the antibody is a humanized antibody. In some embodiments, the light chain variable region includes or is selected from sequence numbers 9, 10, 11, 12 and their respective equivalent variants, and the heavy chain variable region includes or is selected from sequence numbers 13, 14, 15, 16, 17 and their respective equivalent variants.

[0011] In another aspect, the present invention provides a polynucleotide comprising a polynucleotide encoding any of the above-described antibodies. In another aspect, the present invention further provides a vector comprising the polynucleotide. In another aspect, the present invention further provides a non-human host cell comprising the vector. The present invention also further provides a cell line that produces the antibody of the present invention, and a method for producing the antibody by culturing the antibody-producing cell line.

[0012] In a second embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the above-mentioned antibodies, polynucleotides, vectors, or host cells and a pharmaceutically acceptable carrier.

[0013] In a third embodiment, the present invention provides the use of any of the above-mentioned antibodies, polynucleotides, vectors, or host cells in the manufacture of a drug for treating cancer.

[0014] In a fourth embodiment, the present invention provides any of the above-mentioned antibodies, polynucleotides, vectors, or host cells for treating cancer.

[0015] In a fifth embodiment, the present invention provides a method for treating cancer, comprising administering an effective amount of any of the above-mentioned antibodies, polynucleotides, vectors, or host cells to a subject.

[0016] In the relevant embodiments described above, the cancers include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colorectal cancer, stomach cancer, lung cancer (e.g., non-small cell lung cancer), mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, thyroid cancer, head and neck cancer, lymphoma, and leukemia (e.g., acute myeloid leukemia). [Effects of the Invention]

[0017] The anti-5T4 monoclonal antibody of the present invention has high affinity, good cell binding ability, and high internal migration rate, and can be used to treat diseases in which 5T4 is overexpressed, such as cancer.

[0018] Other aspects and advantages of the present invention will become apparent from the following detailed description relating to the present invention. [Brief explanation of the drawing]

[0019] [Figure 1] This is a test of the internal transfer rate of humanized antibodies. [Figure 2] This involves measuring affinity using ELISA. [Figure 3] It is the measurement of affinity by Biacore. [Figure 4] It is the measurement of cross-reactivity by ELISA. [Figure 5] It is the measurement of cell binding ability by FACS. [Figure 6] It is the measurement of internalization rate by FACS.

Mode for Carrying Out the Invention

[0020] (Definition) In the present invention, "about" means that a numerical value is within the allowable error range of a specific value measured by a person skilled in the art, and the said numerical value depends to a certain extent on how it is measured or determined (i.e., the limitations of the measurement system). For example, in each practice in the art, "about" can mean within 1 standard deviation or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. Also, for a biological system or process, the term can mean up to 1 digit or up to 5 times the numerical value. Unless otherwise specified, when a specific value appears in the present specification and the claims, "about" or "substantially comprising" should be construed as being within the allowable error range of the said specific value.

[0021] As described herein, a composition or method described as "comprising" one or more elements or steps is open-ended, meaning that the said elements or steps are essential, but other elements or steps may be added within the scope of the said composition or method. Note that any composition or method described as "comprising" one or more elements or steps also describes a corresponding, more limited composition or method "consisting essentially of" the said elements or steps, meaning that the said composition or method includes the essential elements or steps and may also include additional elements or steps that have basic and novel features that do not essentially affect the said composition or method.

[0022] As used herein, the term “antibody” refers to any form of antibody that exhibits a desired physiological activity (for example, by inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signaling). “Antibody fragment” and “antigen-binding fragment” generally refer to antigen-binding fragments and antibody analogs of an antibody that include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0023] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, where, with the exception of naturally occurring variants that may exist in small amounts, each antibody constituting the population is identical. Monoclonal antibodies possess very high specificity and can target a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations which generally contain multiple different antibodies targeting multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen. The modifier "monoclonal" describes the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be understood as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used in this invention can be produced by hybridoma or recombinant DNA methods.

[0024] Monoclonal antibodies can include "chimeric" antibodies, humanized antibodies, or fully human antibodies. In some embodiments, the antibody constitutes part of a larger biomolecule, such as a fusion protein or antibody-drug conjugate. The antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when activity is expressed in moles, the antibody fragment retains at least 10% of the parent's binding activity. Preferably, the antibody fragment retains at least 20%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, or more of the parent antibody's binding affinity to the target.

[0025] As used herein, the term "humanized antibody" refers to an antibody that includes a CDR derived from an antibody of a non-human mammal, along with the framework region (FR) and constant region of a human antibody.

[0026] An "equivalent variant" of an antibody or polypeptide refers to an antibody or polypeptide that has a certain degree of homology or sequence identity with respect to the amino acid sequence of the antibody or polypeptide. In some embodiments, the sequence identity is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%. In some embodiments, the equivalent variant has one, two, three, four, or five additions, deletions, substitutions, or combinations thereof compared to the reference antibody or polypeptide. In some embodiments, the equivalent variant of an antibody or polypeptide retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0027] As used herein, the term "variant" of a sequence refers to a sequence that differs from the target sequence by one or more amino acid residues, but retains the physiological activity of the target molecule.

[0028] As used herein, the term "% identity" between two sequences refers to a function of the number of identical positions shared by these sequences (i.e., % homology = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap, which are introduced to optimally align the two sequences. Sequence comparison and determination of % identity between two sequences can be performed using mathematical algorithms.

[0029] "Conservative substitution" refers to amino acid substitutions known to those skilled in the art, and such substitutions generally do not alter the physiological activity of the target molecule. Generally, it is common knowledge among those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide substantially does not alter its physiological activity or does not essentially alter it. "Does not alter or does not essentially alter" means that, when measured by the same or similar methods, one or more aspects differ from the comparison subject by approximately 20% or less, approximately 15% or less, approximately 10% or less, approximately 9% or less, approximately 8% or less, approximately 7% or less, approximately 6% or less, approximately 5% or less, approximately 4% or less, approximately 3% or less, approximately 2% or less, or approximately 1% or less.

[0030] CDR3 is generally considered to play a more important role than other CDRs in antigen recognition. Therefore, when substitutions are made, it is preferable in the present invention to make conservative substitutions for CDRs other than CDR3. In some embodiments, CDR3 is not substituted. Preferred amino acid substitutions include, but are not limited to, (1) substitutions that reduce the hydrolysis sensitivity of the protein, (2) substitutions that reduce the oxidation sensitivity, (3) substitutions that change the binding affinity of the formed protein complex, and (4) substitutions that provide or modify other physical and chemical or functional properties of these analogs. Analogs may include a variety of sequence mutations other than naturally occurring peptide sequences. For example, one or more amino acid substitutions (preferably conservative amino acid substitutions) can be made in naturally occurring sequences (preferably in polypeptide portions other than regions that form intermolecular contacts). Conservative amino acid substitutions should not essentially alter the structural features of the parent sequence (for example, it is preferable that the amino acid substitutions are not of a type that tends to disrupt helices present in the parent sequence or that has been characterized to disrupt other secondary structures of the parent sequence).

[0031] The binding domain of the antibody or its antigen-binding fragment of the present invention is generally expected to be located at the N-terminus of a secreted protein and may contain a signal peptide consisting of 15 to 30 amino acids. When the signal peptide sequence is synthesized, it is recognized by a signal recognition particle (SRP), and protein synthesis is temporarily paused or slowed. However, when the signal recognition particle carries the ribosome to the endoplasmic reticulum, protein synthesis resumes. Guided by the signal peptide, the newly synthesized protein enters the endoplasmic reticulum lumen, but the signal peptide sequence is cleaved by the signal peptidase. Furthermore, as ovalbum contains an internal signal peptide, if a transport termination sequence is present at the C-terminus of the nascent peptide chain, it may not be cleaved by the signal peptidase. Neither the precursor nor the mature form undergoes a process of cleavage by the signal peptidase.

[0032] According to the present invention, the term “binding” preferably relates to specific binding. Where ligand-receptor, antibody-antigen, or other binding pairs are mentioned, “specific” binding refers to determining whether a binding reaction of the protein occurs in a heterogeneous population of proteins and / or other biochemical reagents. Thus, under given conditions, a particular ligand or antigen binds to a particular receptor or antibody and does not bind in significant amounts to other proteins present in the sample. “Specific binding” means that the monoclonal antibody or its antigen-binding fragment of the present invention can specifically interact with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more amino acids of each human target molecule. The “specific binding” of an antibody is primarily evaluated by two parameters: a qualitative parameter (binding epitope or antibody binding site) and a quantitative parameter (binding affinity or binding strength). The binding epitope of an antibody can be measured by FACS, epitope mapping, mass spectrometry, or peptide ELISA. The Biacore method and / or ELISA method can measure the binding strength of an antibody to a specific epitope. Generally, the signal-to-noise ratio is used as a representative method for calculating binding specificity. In such a signal-to-noise ratio, the signal represents the binding strength of the antibody to the target epitope, and the noise represents the binding strength of the antibody to other non-target epitopes. Preferably, when the signal-to-noise ratio for the target epitope is about 50, the antibody being evaluated can be considered to bind to the target epitope in a specific manner, i.e., to "specifically bind". If an antigen-binding protein (including an antibody) binds to the antigen with a high binding affinity determined from the value of its affinity constant (KD), then the antigen-binding protein (including an antibody) "specifically binds" to the antigen. In some embodiments, the affinity constant KD is 10 -9 Less than M. As used herein, the term "KD" refers to the affinity constant of a particular antibody-antigen interaction.

[0033] Humanized antibodies are antibody molecules derived from non-human species antibodies that bind to a desired antigen having one or more complementarity-determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule. Generally, framework residues in the human framework region are substituted with corresponding residues from the antibody derived from the CDR donor to alter, and preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding and sequence comparison, and further to identify unnatural framework residues at specific locations. Antibodies can be humanized using various techniques known in the art, such as CDR transplantation, veneerization or resurfacing, and chain shuffling.

[0034] Fully human antibodies are particularly ideal for treating human patients. Human antibodies can be manufactured using various methods known in this field, such as phage display technology, which uses antibody libraries derived from human immunoglobulin sequences.

[0035] Furthermore, human antibodies can be produced using genetically modified mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy-chain and light-chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy-chain and light-chain genes, human variable regions, constant regions, and diversity regions can also be introduced into mouse embryonic stem cells. By introducing human immunoglobulin gene loci by homologous recombination, the mouse heavy-chain and light-chain immunoglobulin genes can be made to lose function individually or simultaneously. In particular, homozygous deletion of the JH region inhibits the production of endogenous antibodies. Chimeric mice are obtained by amplifying modified embryonic stem cells and microinjecting them into blastulae. Next, the chimeric mice are raised to obtain homozygous offspring that express human antibodies. The genetically modified mice are immunized with a selected antigen using a conventional method, the antigen being, for example, the whole or a part of a desired target polypeptide. Monoclonal antibodies against the antigen can be obtained from the immunized genetically modified mice using conventional hybridoma technology. Human immunoglobulin genes in genetically modified mice are rearranged during B cell differentiation, followed by class switching and somatic mutations. Therefore, it is possible to use this technique to produce IgG, IgA, IgM, and IgE antibodies that are useful for therapy.

[0036] Fully human antibodies that recognize selected epitopes can also be generated using a technique called "guided selection." In this method, non-human monoclonal antibodies, such as mouse antibodies, are selected to guide the selection of fully human antibodies that recognize the same epitopes.

[0037] DNA encoding a desired monoclonal antibody can be easily isolated and sequenced using standard procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). Isolated and subcloned hybridoma cells are used as a preferred source of this type of DNA. After isolation, the DNA can be placed into an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or non-immunoglobulin-producing myeloma cells. More specifically, the isolated DNA can be used to clone constant and variable region sequences in order to produce antibodies. Essentially, this requires extracting RNA from selected cells, converting it to cDNA, and then PCR amplifying it using Ig-specific primers. As described herein, transformed cells expressing the desired antibody can be grown in relatively large quantities, thus enabling the clinical and commercial supply of immunoglobulins.

[0038] Furthermore, one or more CDRs of the antigen-binding polypeptides of this disclosure can be inserted into the framework region using conventional recombinant DNA technology, for example, by inserting them into a human framework region to humanize a non-human antibody. The framework region may be a naturally occurring or common framework region, and is preferably a human framework region. Preferably, the polynucleotide produced by the combination of the framework region and the CDR encodes an antibody that specifically binds to at least one epitope of a desired polypeptide (e.g., LIGHT). Preferably, one or more amino acid substitutions can be made within the framework region, and preferably, the amino acid substitutions improve the binding of the antibody to its antigen. Such methods can be used to produce antibody molecules lacking one or more intrachain disulfide bonds by substituting or deleting cysteine ​​residues in one or more variable regions involved in intrachain disulfide bonds. Other modifications to the polynucleotide are included in this disclosure and are within the scope of the art.

[0039] As used herein, the terms “patient” or “subject” refer to any organism that is administered, or may be administered, an antibody, its derivative, or a pharmaceutical composition provided for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the subject is human. In some embodiments, the subject has or is susceptible to one or more diseases or conditions. The patient may exhibit or be diagnosed with one or more symptoms of a disease or condition. In some embodiments, the patient has or is currently receiving a specific therapy for the diagnosis and / or treatment of this type of disease, disease, or condition.

[0040] In this invention, the term “treatment” refers to therapies and preventive measures that prevent or mitigate undesirable physiological changes or the onset or progression of a disease in a subject. Favorable or expected clinical effects include, but are not limited to, symptom reduction, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or mitigation of disease progression, reduction or mitigation of the disease state, and cure of part or all of the disease, regardless of whether such effects are detectable. “Treatment” may also refer to an extension of survival time compared to not receiving treatment. Subjects to be treated include subjects suffering from the disease or disease, subjects at risk of suffering from the disease or disease, or subjects to be prevented from suffering from the disease or disease.

[0041] When “administration” or “treatment” is used in reference to animals, humans, experimental subjects, cells, tissues, organs, or biofluids, it refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biofluid. “Administration” and “treatment” can also refer to, for example, a therapeutic method, a pharmacokinetic method, a diagnostic method, a research method, or an experimental method. Cell therapy includes contacting a reagent with cells, or contacting a reagent with a fluid, where the fluid comes into contact with the cells. “Administration” or “treatment” can also mean in vitro and ex vivo treatments performed on cells, for example, with a reagent, diagnostic agent, conjugate composition, or with other cells.

[0042] As used herein, the terms “therapeutic dose” or “effective dose” refer to the amount of the antibody or derivative thereof of the present invention administered alone or in combination with another therapeutic agent to cells, tissues, or a patient that effectively prevents or alleviates the disease or condition to be treated. The therapeutic dose further refers to an amount sufficient to alleviate the symptoms, and such alleviation of symptoms means, for example, treating, curing, preventing or alleviating the relevant medical condition, or improving the treatment rate, cure rate, prevention rate or alleviation rate for the condition. When an active ingredient is administered alone to an individual, the therapeutic dose refers to that single ingredient. When administered in combination, the therapeutic dose refers to the amount of the combined active ingredients that produce a therapeutic effect, regardless of whether it is administered concomitantly, sequentially, or simultaneously. The reduction of symptoms by the therapeutic dose is generally at least 10%, generally at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0043] As used herein, the term “pharmaceutically acceptable carrier” includes materials that, when combined with the active ingredient of a composition, preserve the physiological activity of that ingredient and do not cause a destructive reaction against the target immune system. These carriers may include stabilizers, preservatives, salts or sugar complexes or crystals, etc. “pharmaceutically acceptable” means molecules and components that do not cause an allergic reaction or a similar undesirable reaction when administered to the human body.

[0044] (anti-5T4 antibody) This invention involves immunizing mice with the 5T4 antigen, screening them to obtain monoclonal antibodies, and then humanizing these antibodies to obtain humanized anti-5T4 antibodies.

[0045] In one aspect of the present invention, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to 5T4 (trophoblast glycoprotein), wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein LCDR1, LCDR2, and LCDR3 each comprise or are equivalent variants of the light chain variable region having the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, and HCDR1, HCDR2, and HCDR3 each comprise or are equivalent variants of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0046] In some embodiments, the CDR1, CDR2, and CDR3 (e.g., LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are defined by one of the following definition schemes: IMGT, Kabat, Chothia, Contact, or Martin. In some embodiments, the CDR1, CDR2, and CDR3 (e.g., LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are defined by the IMGT definition scheme.

[0047] In some embodiments, LCDR1 includes or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, LCDR2 includes or is the sequence shown in SEQ ID NO: 2 or an equivalent variant thereof, LCDR3 includes or is the sequence shown in SEQ ID NO: 3 or an equivalent variant thereof, and HCDR1 includes or is the sequence shown in SEQ ID NO: 4 or an equivalent variant thereof, HCDR2 includes or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, and HCDR3 includes or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof.

[0048] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 8 or an equivalent variant thereof.

[0049] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0050] In some embodiments, the antibody is a humanized antibody. In some embodiments, the light chain variable region includes or is selected from sequence numbers 9, 10, 11, 12 and their respective equivalent variants, and the heavy chain variable region includes or is selected from sequence numbers 13, 14, 15, 16, 17 and their respective equivalent variants.

[0051] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof.

[0052] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof.

[0053] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof.

[0054] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof.

[0055] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof.

[0056] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof.

[0057] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof.

[0058] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof.

[0059] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof.

[0060] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof.

[0061] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof.

[0062] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof.

[0063] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 16 or an equivalent variant thereof.

[0064] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 16 or an equivalent variant thereof.

[0065] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 16 or an equivalent variant thereof.

[0066] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 16 or an equivalent variant thereof.

[0067] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof.

[0068] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof.

[0069] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof.

[0070] In some embodiments, the light chain variable region includes or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof.

[0071] The above-mentioned humanized antibody may include a light chain constant region and a heavy chain constant region, wherein the light chain constant region is, for example, the human Igκ light chain constant region shown in SEQ ID NO: 18, and the heavy chain constant region is, for example, the human IgG1 heavy chain constant region shown in SEQ ID NO: 19.

[0072] In some embodiments, each equivalent variant of CDR1, CDR2, and CDR3 (e.g., LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) refers to a variant that has a single amino acid substitution, deletion, or insertion compared to the reference sequence.

[0073] In some embodiments, the equivalent variant of the variable region refers to a sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any of the sequences of SEQ ID NOs. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17, and has the same or equivalent CDR1, CDR2, and CDR3.

[0074] In some embodiments, LCDR1 includes the sequence indicated by SEQ ID NO: 1, LCDR2 includes the sequence indicated by SEQ ID NO: 2, LCDR3 includes the sequence indicated by SEQ ID NO: 3, and HCDR1 includes the sequence indicated by SEQ ID NO: 4, HCDR2 includes the sequence indicated by SEQ ID NO: 5, and HCDR3 includes the sequence indicated by SEQ ID NO: 6.

[0075] In some embodiments, the light chain variable region includes the sequence shown in SEQ ID NO: 7, and the heavy chain variable region includes the sequence shown in SEQ ID NO: 8.

[0076] In some embodiments, the antibody is a humanized antibody. In some embodiments, the light chain variable region includes any sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and the heavy chain variable region includes any sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0077] In some embodiments, LCDR1 is the sequence indicated by sequence number 1, LCDR2 is the sequence indicated by sequence number 2, LCDR3 is the sequence indicated by sequence number 3, and HCDR1 is the sequence indicated by sequence number 4, HCDR2 is the sequence indicated by sequence number 5, and HCDR3 is the sequence indicated by sequence number 6.

[0078] In some embodiments, the light chain variable region is the sequence shown in sequence number 7, and the heavy chain variable region is the sequence shown in sequence number 8.

[0079] In some embodiments, the antibody is a humanized antibody. In some embodiments, the light chain variable region is one of the sequences selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and the heavy chain variable region is one of the sequences selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0080] In some embodiments, the antibody is of the IgG type, for example, IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is of the IgG1 type.

[0081] In some embodiments, the substitutions described herein are conservative substitutions.

[0082] A "conservative (amino acid) substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid having a similar side chain. Families of amino acid residues having similar side chains have already been defined in this field and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable that non-essential amino acid residues in immunoglobulin polypeptides be substituted with other amino acid residues from the same side chain family. In another embodiment, the amino acid string can be replaced with a string that has a similar structure but a different order and / or configuration of side chain family members.

[0083] Furthermore, those skilled in the art will understand that the antibodies disclosed herein may be modified such that their amino acid sequences differ from those of the naturally occurring conjugated polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a particular protein may be similar to the start sequence, for example, having a certain percentage of identity, such as being identical to the start sequence by 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or any two of these values.

[0084] In some embodiments, the antibody comprises an amino acid sequence or one or more parts that are not generally related to the antibody. Exemplary modifications are described in more detail here. For example, the antibodies disclosed herein may include a flexible linker sequence or may be modified to add a functional moiety (e.g., polyethylene glycol (PEG), a drug, a toxin, or a label).

[0085] The antibodies, their variants, or derivatives of the present invention include modified derivatives, i.e., those in which the antibody is covalently bonded to any type of molecule, such that the covalent bond does not inhibit the binding of the antibody to the epitope. Antibodies may be modified, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage by hydrolysis, or linkage with cell ligands or other proteins. Any of the various chemical modifications can be carried out by known techniques, including, but are not limited to, specific chemical lysis, acetylation, formylation, or metabolic synthesis of tunicamycin. Furthermore, antibodies may contain one or more non-classical amino acids.

[0086] In some embodiments, antibodies can be conjugated with therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, drugs, or PEG.

[0087] Antibodies can be combined with or fused with therapeutic agents, which may include detectable labels (e.g., radioactive labels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutics or diagnostics, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radioactive labels, combinations thereof, or other reagents of this type known in the art.

[0088] Antibodies can be detected by binding them to chemiluminescent compounds. The presence of chemiluminescently labeled antigen-binding polypeptides is then determined by detecting whether luminescence occurs during the chemical reaction. Particularly useful examples of chemiluminescent labeling compounds include luminol, isoluminol, heat-stable acridinium esters, imidazole, acridine salts, and oxalic acid esters.

[0089] Also, fluorescent metals (for example, 152Antibodies can also be detectably labeled using Eu or other lanthanide elements. These metals can be linked to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various parts of an antibody are well-known.

[0090] In another embodiment of the present invention, the present invention provides a nucleotide sequence comprising a polynucleotide encoding an antibody according to any of the above embodiments. In another embodiment, the present invention further provides a vector comprising the polynucleotide. In another embodiment, the present invention further provides a non-human host cell comprising the vector.

[0091] (Pharmaceutical composition) Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the above-mentioned antibodies, polynucleotides, vectors, or host cells and a pharmaceutically acceptable carrier.

[0092] In any of the above embodiments, the pharmaceutical composition is used to treat diseases in which 5T4 is overexpressed, such as cancer. In some embodiments, the cancers include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, thyroid cancer, head and neck cancer, lymphoma, and leukemia (e.g., acute myeloid leukemia).

[0093] To manufacture a pharmaceutical composition or a sterile composition, a drug is mixed with a pharmaceutically acceptable carrier or excipient. By mixing with a physiologically acceptable carrier, excipient, or stabilizer, formulations can be manufactured, for example, in the form of a lyophilized powder, suspension, aqueous solution, or suspending agent. Pharmaceutically acceptable carriers are well known in the art. Methods for manufacturing aqueous compositions containing active ingredients are known in the art. Generally, these compositions may be manufactured as injectable or spray preparations, such as liquids that are solutions or suspensions, or in solid form suitable for preparation as a solution or suspension before injection or spraying.

[0094] (Method and Use) One aspect of the present invention provides a method for treating cancer, comprising administering an effective amount of any of the above-mentioned antibodies, polynucleotides, vectors, or host cells to a subject.

[0095] Accordingly, another aspect of the present invention provides the use of any of the above-mentioned antibodies, polynucleotides, vectors, or host cells in the manufacture of a drug for treating cancer.

[0096] Accordingly, another aspect of the present invention provides any of the above-mentioned antibodies, polynucleotides, vectors, or host cells for treating cancer.

[0097] In some embodiments, the cancers include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colorectal cancer, stomach cancer, lung cancer (e.g., non-small cell lung cancer), mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, thyroid cancer, head and neck cancer, lymphoma, and leukemia (e.g., acute myeloid leukemia).

[0098] Appropriate routes of administration include parenteral administration (e.g., intramuscular, intravenous, or subcutaneous) and oral administration. Other usual methods of administration include via endotracheal intubation, oral, inhalation, topical, or percutaneous, subcutaneous, intraperitoneal, or intra-arterial injection.

[0099] The appropriate dose is determined by the clinician based on parameters or factors known in this field or thought to influence or expected to influence treatment. Generally, the starting dose is slightly lower than the optimal dose, and is then gradually increased until the desired or optimal effect on any adverse events is achieved. Important monitoring indicators include, for example, measuring inflammatory symptoms or the levels of inflammatory cytokines produced. (Sequence Listing) JPEG2026513841000001.jpg233168 [Examples]

[0100] Example 1: Antibody screening and sequencing Step 1 involves infecting CHO cells with a lentivirus containing the 5T4 gene, thereby constructing CHO-5T4 + Six-week-old BALB / c mice were immunized with cells. In step 2, the cells were fused to obtain multiple original cell lines. In Step 3, ELISA, flow cytometry, and Biacore were used to screen the hybridoma supernatant secreted by the original cell line, yielding several hybridoma clone candidates with good affinity. In Step 4, candidate cell lines were subcloned and further screened using ELISA, flow cytometry, SPR, and Western blot to obtain monoclonal antibodies with high affinity, high internal penetration rate, and good specificity.

[0101] The monoclonal antibody candidate obtained by screening using the method described above was named B5. Using PCR technology, the amino acid sequences of the light chain and heavy chain variable region of antibody B5 were obtained, which were sequence numbers 7 and 8, respectively.

[0102] Example 2: Affinity test of antibody B5 The affinity of antibody B5 screened in Example 1 is measured using Biacore.

[0103] The specific measurement method was as follows: 1) Screening for hybridoma affinity. The antigen was 5T4 EDC-Fc with a molecular weight of 65-120 kD. A direct method was used to couple the antigen to CM5 via an amino group. 2) Rmax = (MW analyte / MW ligand The actual coupling amount is 1.5 times RL, calculated as ) × RL × Sm. At the same time, the coupling amount suitable for antigen detection was calculated to be 120 Ru. 3) Perform coupling. The coupling buffer was 1×HBS-EP buffer, and 50 mM NaOH was used as the washing solution. A sodium acetate solution with a pH of 4.5 was prepared to contain 5 μg / mL of antigen. Select the amino group and perform coupling, input 120 Ru to the Target level, and place the antigen sample, NaOH, EDC, NSH, ethanolamine, and empty test tubes in the corresponding positions on the sample tray in the order shown. 4) Explore regeneration conditions. Ultimately, we selected 10 seconds of regeneration in glycine hydrochloride at pH 1.7. 5) Prepare the samples. As a positive control antibody, B5 antibody was diluted to 8nM, 4nM, 2nM, 1nM, 0.5nM, and 0.25nM in 1×HBS-EP buffer and centrifuged at 13,000 rpm for 5 minutes. All samples were prepared in duplicate wells. Next, prepare the hybridoma supernatant samples. The hybridoma cell supernatant was collected, centrifuged at 13,000 rpm for 5 minutes, diluted 2-fold with 1×HBS-EP buffer, and all samples were prepared in duplicate wells. 6) Set the program. In the Kinetics / Affinity options, select Flow pat 2-1, Regeneration 2, Contact time 180 seconds, Dissociation time 600 seconds, and Regeneration conditions Gly 1.7, 10 seconds. Samples were repeatedly detected at intervals, three zero concentrations and three startup values ​​were set, and the program was executed by clicking Start. 7) Analyze the results. Appropriate concentrations were selected to correspond to the response unit change curve, and pharmacokinetic analysis was performed by fitting a 1:2 calculation model.

[0104] The measurement results are shown in Table 1. [Table 1]

[0105] Example 3: Construction of a humanized antibody Humanization modifications were performed on antibody B5 screened in Example 1 to obtain four humanized light chain variable region sequences hB5-VL1 (SEQ ID NO: 9), hB5-VL2 (SEQ ID NO: 10), hB5-VL3 (SEQ ID NO: 11), hB5-VL4 (SEQ ID NO: 12), and five humanized heavy chain variable region sequences hB5-VH1 (SEQ ID NO: 13), hB5-VH2 (SEQ ID NO: 14), hB5-VH3 (SEQ ID NO: 15), hB5-VH4 (SEQ ID NO: 16), and hB5-VH5 (SEQ ID NO: 17).

[0106] By combining the light chain variable region and heavy chain variable region described above, 20 humanized antibodies shown in Table 2 were constructed and named hB5-2-1 to hB5-2-20 in order. The constant regions of these antibodies all consisted of the human Igκ light chain constant region (SEQ ID NO: 18) and the human IgG1 heavy chain constant region (SEQ ID NO: 19). [Table 2]

[0107] Example 4: Affinity test of humanized antibodies Using Biacore 8K, the affinity of the 20 hB5 humanized antibodies constructed in Example 3 will be rapidly measured at single concentrations to compare different humanized antibodies.

[0108] The experimental procedure was as follows: (1) Anti-human antibody proteins were coupled to the CM5 chip via an amino group. (2) The surface of the chip was run over with the hB5 humanized antibody. (3) The surface of the chip was run over with a high concentration of TPBG-His antigen. (4) Affinity was measured.

[0109] The experimental materials were as follows: The antibody transfection system was 30 mL, and the purification column was Protein A / G; the purity of both was 95% or higher.

[0110] The experimental steps were as follows: 1) Capture the ligand. After collecting the cell supernatant, it was centrifuged at 13,000 rpm for 30 minutes at 4°C, and two-thirds of the supernatant was set aside in a new EP tube. A microfluidic chip system (IFC) is used to separate the CM5 chip into eight channels, and each channel is further divided into two channels: channel 1 is used as the reference channel, and channel 2 captures the supernatant. The flow rate was set to 10 μL / min, and the supernatant was allowed to flow for 200 seconds. Over 95% of the antibody capture levels were effectively higher than 100 RU.

[0111] 2) Bind the antigen. In channels 1 and 2, the TPBG-His protein was diluted to 300 nM with HBS-EP+ buffer and flowed at a flow rate of 30 μL / min for 120 seconds. Glycine (pH 1.7) was flowed through channels 1 and 2 at a flow rate of 30 μL / min for 40 seconds.

[0112] 3) The response values ​​for each antibody were obtained.

[0113] 4) Pharmacokinetic parameters were calculated using the 1:1 binding model of the analysis software Bia-evaluation, and the affinity of each antibody was compared.

[0114] The experimental results were as follows. The measurement results were as shown in Table 3.

Table 3

[0115] As can be seen from Table 3, all 20 hB5 humanized antibodies constructed in Example 3 showed good affinity, and the affinity constant KD all reached 10 -9 M, and among them, those reaching 10 -10 M were not few.

[0116] Example 5: Test on the internalization rate of humanized antibodies In this example, among the humanized antibodies constructed in Example 3, the internalization rates of hB5-2-5, hB5-2-6 and hB5-2-17 were tested.

[0117] The experimental procedure was as follows. 1) Collect cells. Remove the old medium, add 0.25% trypsin digestion solution to digest the cells, then add DMEM medium to stop the digestion, collect the cell suspension and centrifuge at 800 rpm for 3 minutes. 2) Resuspend the cells in 4 mL of medium and count the cells. Obtain the cell suspension, mix it uniformly with 0.4% trypan blue at a ratio of 1:1, add it to the cell counting board, and place the counting board on the Countstar cell counter to automatically measure the number and viability of the cells. 3) Divide all the cell suspensions equally. The number of cells in the isotype control and negative control is 1×10 6 , and the number of cells in each group of the experimental group is 2×10 6 . 4) At 4°C, centrifuge the cells at 3500 rpm for 3 minutes, and carefully aspirate the supernatant using the tip of the pipette. 5) Incubate the primary antibody. Add 100 μL of mouse IgG (concentration 10 μg / mL) to the isotype control group, add 100 μL of PBS to the blank control group, and add 100 μL of antibody solution with a final concentration of 10 μg / mL to the experimental group. After labeling and resuspending the cells, incubate the cells on ice for 30-40 minutes. After incubation, resuspend the cells in 500 μL of PBS buffer, centrifuge at 3500 rpm for 3 minutes at 4°C, and wash twice. 6) After resuspending the cells in 500 μL of PBS, the experimental cells were divided into two groups. The non-internalization group was placed on ice or incubated at 4°C for 3 hours, while the internalization group was incubated in a 37°C incubator for 3 hours. The isotype control group and negative control group were initially placed at 4°C. During this time, the tube walls were gently tapped every 10 minutes to prevent cell precipitation. 7) After incubation, the cells were centrifuged at 3500 rpm for 3 minutes at 4°C, the supernatant was discarded, and the cells were washed twice. 8) Incubate the secondary antibody. The secondary antibody (FITC-goat anti-mouse IgG antibody / FITC-goat anti-human IgG antibody) was diluted 1:200 with PBS, 100 μL of the secondary antibody was added to each EP tube, and the mixture was resuspended. The mixture was incubated at 4°C on ice for 40 minutes. 9) After incubation, 500 μL of PBS buffer was added to each group, the cells were resuspended and pipetted twice, and then the cells from each group were centrifuged at 3500 rpm for 3 minutes at 4°C, and the supernatant was aspirated. This process was repeated 2-3 times. 10) In preparation for loading and measurement, 300 μL of PBS buffer was added to resuspend the cells from each group, and these were pipettered and added to the flow cytometry tubes. 11) Using a flow cytometer, parameters for each item were set, and a control was prepared using cells from the blank control group. Subsequently, cell samples from each group were tested sequentially.

[0118] The experimental results were as follows: The cells are CHO-5T4 +The antibody gradients were 0.1 μg / mL, 0.5 μg / mL, and 1 μg / mL, and the internal transfer time was 3 hours.

[0119] The measurement results are shown in Figure 1 and Table 4. [Table 4]

[0120] The internal transfer rates of the three humanized antibodies were around 60% to 70%, indicating high internal transfer rates, and among them, hB5-2-17 was found to have the best performance.

[0121] Example 6: Performance test of humanized antibody hB5-2-10 In this example, the performance of each humanized antibody hB5-2-10 was tested.

[0122] (1) Affinity test using ELISA / Biacore The affinity of hB5-2-10 for TPBG-His will be measured using ELISA. The experimental procedure was as follows: 1) Coating. TPBG-His was diluted to 1 μg / mL in 50 mM carbonate buffer (pH 9.6). After dilution, 50 μL was added per well to an ELISA plate. The plate was sealed with plate sealing film and left overnight in a refrigerator at 4°C. 2) Wash the plate. Remove the plate sealing film and place it in a plate washer. Add 300 μL of 1×PBST per well, shake for 20 seconds, and immerse for 10 seconds. Wash the plate twice. Remove the ELISA plate and gently tap it on absorbent paper until no residual liquid or air bubbles remain in the wells. 3) Blocking. Add 200 μL of 1% BSA to each well and incubate at 37°C for 1 hour. 4) Wash the plate. Remove the plate sealing film and place it in a plate washer. Add 300 μL of 1×PBST per well, shake for 20 seconds, and immerse for 10 seconds. Wash the plate twice. Remove the ELISA plate and gently tap it on absorbent paper until no residual liquid or air bubbles remain in the wells. 5) Incubate the primary antibody. Using 0.1% BSA, hB5-2-10 was diluted to a concentration of 40,000 ng / mL, and then a 6-fold serial dilution was used to create a concentration gradient of 10. This was added to a microplate at 100 μL / well in two overlapping wells and incubated at 37°C for 1 hour. 6) Wash the plate. Remove the plate sealing film and place it in a plate washer. Add 300 μL of 1×PBST per well, shake for 20 seconds, and immerse for 10 seconds. Wash the plate four times. Once complete, remove the ELISA plate and gently tap it on absorbent paper until no residual liquid or air bubbles remain in the wells. 7) Incubate the secondary antibody. Goat anti-human IgG Fc (HRP) was diluted with 0.1% BSA at a dilution ratio of 1:10000. 100 μL was used per well, and incubated at 37°C for 1 hour. 8) Wash the plate. Remove the plate sealing film and place it in a plate washer. Add 300 μL of 1×PBST per well, shake for 20 seconds, and immerse for 10 seconds. Wash the plate three times. Once complete, remove the ELISA plate and gently tap it on absorbent paper until no residual liquid or air bubbles remain in the wells. 9) Incubation. 100 μL of TMB chromogenic agent was added to each well. Incubated at 37°C for 10 minutes. 10) Add stop solution. The reaction was stopped by adding 100 μL of 0.7 M oxalic acid solution per well. 11) Measure the OD value. The absorbance at a wavelength of 450 nm was measured using a microplate reader.

[0123] The experimental results were as follows: The ELISA results are shown in Figure 2. Antibody hB5-2-10 binds to TPBG-His. 50 The value was 2.801 ng / mL.

[0124] We will use Biacore to measure the affinity of hB5-2-10 for TPBG-His. The experimental procedure was as follows: 1) Using a microfluidic chip system (IFC), the CM5 chip was separated into eight channels, and each channel was further divided into two channels. Channel 1 was used as the reference channel, and channel 2 was coupled with the ligand. 2) Pre-concentration by pH. Using NaAC buffer (pH 4.5 / 5.0), the antigen (TPBG-His) was linked to the CM5 tip at a concentration of 10 μg / mL. The appropriate pH of the antibody diluent and the antibody concentration were determined, and the antibody was directly coupled to the CM5 tip. The flow rate was set to 10 μL / min, the contact time to 2 minutes, and the dissociation time to 0. 50 mM NaOH was used as the regeneration reagent, and the coupling pH was determined according to the results. 3) Ligand coupling. The TPBG-His antigen was diluted to the appropriate concentration found by pre-concentration using a 10 mM acetate coupling reagent whose pH had already been determined. Furthermore, the CM5 tip was activated with (20%) EDC + (80%) NHS for a reaction time of 30 seconds at a flow rate of 10 μL / min. Finally, the coupled ligand was blocked with ethanolamine for a reaction time of 420 seconds, completing the ligand coupling step. 4) Binding of the analyte The antibody was diluted to different concentration gradients as the analyte, with a flow rate of 30 μL / min. The solution was flowed through channel 1 and then channel 2 in that order, with a flow time of 120 seconds and a dissociation time of 600 seconds. In channels 1 and 2, glycine regeneration buffer (pH 1.5) was flowed at a flow rate of 30 μL / min for 30 seconds. 5) The response values ​​for the concentration of each analyte were obtained, and pharmacokinetic parameters were calculated using the 1:1 binding model of the analysis software Bia-evaluation, and the affinity of each antibody was compared.

[0125] The Biacore results are shown in Figure 3 and Table 5. [Table 5]

[0126] (2) Cross-reactivity test by ELISA The experimental procedure was as follows: 1) Coating. Three different species of TPBG-His were diluted to 1 μg / mL each using 1×PBS buffer. After dilution, 50 μL was added per well to an ELISA plate. The plate was sealed with plate sealing film and left overnight in a refrigerator at 4°C. 2) Wash the plate. Remove the plate sealing film and place it in a plate washer. Add 300 μL of 1×PBST per well, shake for 3 seconds, and immerse for 40 seconds. Wash the plate twice. Remove the ELISA plate and gently tap it on absorbent paper until no residual liquid or air bubbles remain in the wells. 3) Blocking. Add 250 μL of 1% BSA per well and incubate at 37°C for 1 hour. 4) Wash the plate. Remove the plate sealing film and place it in a plate washer. Add 300 μL of 1×PBST per well, shake for 3 seconds, and immerse for 40 seconds. Wash the plate twice. Remove the ELISA plate and gently tap it on absorbent paper until no residual liquid or air bubbles remain in the wells. 5) Incubate the primary antibody. Using 0.1% BSA, hB5-2-10 was diluted to concentrations of 20000 ng / mL, 5000 ng / mL, 1000 ng / mL, 200 ng / mL, 40 ng / mL, 8 ng / mL, 1.6 ng / mL, and 0.32 ng / mL. Only 1% BSA was used to set the zero concentration point, and 100 μL / well was added to a microplate with two overlapping wells. The plates were incubated at 37°C for 1 hour. 6) Wash the plate. Remove the plate sealing film and place it in a plate washer. Add 300 μL of 1×PBST per well, shake for 3 seconds, and immerse for 50 seconds. Wash the plate three times. Once complete, remove the ELISA plate and gently tap it on absorbent paper until no residual liquid or air bubbles remain in the wells. 7) Incubate the secondary antibody. Goat anti-human IgG Fc (HRP) was diluted with 0.1% BSA at a dilution ratio of 1:7000. 100 μL was used per well, and incubation was performed at 37°C for 45 minutes. 8) Wash the plate. Remove the plate sealing film and place it in a plate washer. Add 300 μL of 1×PBST per well, shake for 3 seconds, and immerse for 50 seconds. Wash the plate three times. Once complete, remove the ELISA plate and gently tap it on absorbent paper until no residual liquid or air bubbles remain in the wells. 9) Incubation. 100 μL of TMB chromogenic agent was added to each well. Incubated at 37°C for 10 minutes. 10) Add stop solution. 100 μL of 2M H2SO4 was added as a stop solution to each well to stop the reaction. 11) Measure the OD value. The absorbance at a wavelength of 450 nm was measured using a microplate reader.

[0127] The experimental results were as follows: The results are shown in Figure 4, revealing that the antibody hB5-2-10 binds to the human / monkey 5T4 antigen, exhibiting similar binding activity, but does not bind to the mouse 5T4 antigen.

[0128] (3) Cell binding capacity test by FACS The experimental procedure was as follows: 1) 3 × 10 5 Individual cells were collected, centrifuged at 3500 rpm for 5 minutes, the supernatant was discarded, and the cells were washed with 500 μL of 1×PBS buffer, centrifuged at 3500 rpm for 5 minutes, and the supernatant was discarded. This process was repeated twice. 2) The concentration of the sample under test was diluted to 20 μg / mL with 1×PBS buffer, then serially diluted 5-fold, and incubated at 2-8°C for 1 hour in 100 μL per sample. 3) Add 100 μL of each sample and incubate on ice or at 2-8°C for 1 hour, during which time the cells were dispersed by tapping the container once every 15 minutes. 4) After incubation, wash with 500 μL of 1×PBS buffer, centrifuge at 3500 rpm for 5 minutes, and discard the supernatant. Repeat this process three times. 5) Dilute FITC-anti-human IgG secondary antibody at a 1:100 ratio, and incubate 100 μL per sample on ice for 40 minutes, during which time the cells were dispersed by tapping the container once every 10 minutes. 6) After incubation, wash with 500 μL of 1×PBS buffer, centrifuge at 3500 rpm for 5 minutes, and discard the supernatant. Repeat this process three times. 7) Finally, the sample was resuspended in 300 μL of 1×PBS buffer, loaded, and measured.

[0129] The results are shown in Figure 5, and it was found that the antibody hB5-2-10 exhibited good cell binding ability.

[0130] (4) Internal migration testing using FACS The experimental procedure was as follows: 1) Collect cells and measure their viability, and each cell is 3 × 10 5 We obtained individual cells. 2) Incubate the primary antibody. hB5-2-10 was diluted to 2 μg / mL, with 100 μL per sample. A negative control PBS group was prepared. Incubate at 2-8°C for 1 hour, and after incubation, wash once with 1×PBS buffer. 3) Internal migration by incubation. The cells of each group were divided equally into two parts. One part was incubated on ice to be designated as the non-internal migration group, and the other part was incubated in a 37°C incubator to be designated as the internal migration group. Both groups were incubated simultaneously for 4 hours. After incubation, the cells were gently pipetted out, washed, washed again with 500 μL of 1×PBS buffer, and centrifuged at 3500 rpm for 5 minutes, discarding the supernatant. 4) Incubate the secondary antibody. Each group was taken out simultaneously, the secondary antibody was added, and FITC-goat anti-human IgG was diluted 1:100. 100 μL was added per sample, and the samples were incubated at 2-8°C for 30 minutes. After incubation, the samples were washed once. 5) The sample was resuspended in 300 μL of 1× PBS buffer, loaded, and measured.

[0131] The results are shown in Figure 6 and Table 6, where the internal transfer rate was calculated as (non-internal transfer group - internal transfer group) / non-internal transfer group. It was found that the antibody hB5-2-10 showed a high internal transfer rate. [Table 6]

[0132] While the present invention is specifically disclosed in preferred embodiments and optional features, those skilled in the art should understand that modifications, improvements, and variations can be made to the invention disclosed herein, and such modifications, improvements, and variations will be considered to fall within the scope of the invention. The materials, methods, and examples provided herein are representative and illustrative of preferred embodiments and do not limit the scope of the invention.

Claims

1. A monoclonal antibody or its antigen-binding fragment that binds to 5T4, The monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3. Each of the above-mentioned LCDR1, LCDR2, and LCDR3 includes, or is equivalent to, the CDR1, CDR2, and CDR3 of the light chain variable region whose amino acid sequence is SEQ ID NO: 7, and, The HCDR1, HCDR2, and HCDR3 each include, or are equivalent variants of, the heavy chain variable region CDR1, CDR2, CDR3, or their respective amino acid sequences, which are sequence numbers 8. A monoclonal antibody or its antigen-binding fragment.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are defined by any one of the following definition methods: IMGT, Kabat, Chothia, Contact, or Martin.

3. The LCDR1 contains or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, the LCDR2 contains or is the sequence shown in SEQ ID NO: 2 or an equivalent variant thereof, the LCDR3 contains or is the sequence shown in SEQ ID NO: 3 or an equivalent variant thereof, and, The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein HCDR1 includes or is the sequence shown in SEQ ID NO: 4 or an equivalent variant thereof, HCDR2 includes or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, and HCDR3 includes or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a humanized antibody or a chimeric antibody.

5. The light chain variable region includes or is the sequence shown in Sequence ID No. 7 or an equivalent variant thereof, and The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region includes or is the sequence shown in SEQ ID NO: 8 or an equivalent variant thereof.

6. The light chain variable region includes, or is selected from, any of the sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and their respective equivalent variants, and The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region includes or is selected from any sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and their respective equivalent variants.

7. A polynucleotide comprising a polynucleotide encoding an antibody according to any one of claims 1 to 6.

8. A vector comprising the polynucleotide described in claim 7.

9. A non-human host cell comprising the vector according to claim 8.

10. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of claims 1 to 6, a polynucleotide according to claim 7, a vector according to claim 8, or a host cell according to claim 9, and a pharmaceutically acceptable carrier.

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