Anti-5T4 antibody and application thereof

Abstract

Relates to anti-5T4 antibodies, in particular humanized antibodies. The invention also relates to pharmaceutical compositions comprising these antibodies and pharmaceutical uses thereof. The antibody has relatively high affinity, relatively good cell binding force and relatively high internalization rate, and can be used for treating 5T4 overexpression diseases, such as cancers.

Description

Anti-5T4 antibodies and their applications Technical Field

[0001] The present invention relates to anti-5T4 antibodies, in particular humanized antibodies, and also to pharmaceutical compositions comprising these antibodies and their medical uses. Background Art

[0002] 5T4 (trophoblast glycoprotein), first discovered in embryonic cells 20 years ago, is a 72 kDa glycoprotein identified by indirect immunofluorescence staining using the mouse monoclonal antibody 5T4. It is expressed in human trophoblasts and is highly expressed in various tumor tissues, but is absent or expressed at low levels in normal adult tissues. The 5T4 gene is located on chromosome 6, between the 6q14-q15 region, and encodes 420 amino acids. The human and mouse 5T4 protein sequences share up to 81% homology. The 5T4 protein is extensively glycosylated, but its biological function remains unclear.

[0003] 5T4 is overexpressed in some tumors, such as breast, lung, and renal cancers, but its expression is very low in normal tissues. Furthermore, high 5T4 expression is positively correlated with poor prognosis in head and neck cancer and colorectal cancer. Recent studies have shown that 5T4-positive B cell populations are more prevalent in lymphocytic leukemias in relapsed patients. 5T4 expression in cancer stem cells (CSCs) is associated with a poor overall prognosis in non-small cell lung cancer, potentially contributing to tumor metastasis, drug resistance, and relapse. Under normal circumstances, 5T4 is primarily expressed in embryonic cells to enhance chemotaxis and play a key role in embryonic development and cell differentiation. Pathologically, 5T4 overexpression is associated with downregulation of E-cadherin, leading to metastatic spread of epithelial tumors. 5T4 also participates in regulating β-catenin-independent Wnt signaling by modulating LRP6 subcellular localization. Furthermore, 5T4 modulates CXCL12 / CXCR4 chemokine-mediated chemotaxis, which may promote the spread of 5T4-positive tumor cells.

[0004] In vitro experiments show that the first generation of human T cells expressing chimeric antigen receptors targeting 5T4 have the ability to promote the release of specific cytokines and certain cytotoxicity against 5T4-positive cells. This chimeric antigen receptor targeting 5T4 was used to modify T cells derived from patients with renal cell carcinoma (RCC) and can effectively kill renal cancer cells in vitro. In addition, mice with B16 tumors that are positive for 5T4 expression have prolonged survival after receiving CAR-T cell therapy; the prolonged survival is the result of the synergistic effect of CAR-T cells and vaccines targeting 5T4, which also provides evidence for the synergistic effect of the two when used together. These research results indicate that 5T4 is a potential target for tumor treatment.

[0005] Summary of the Invention

[0006] In a first aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that binds to 5T4 (trophoblast glycoprotein), the monoclonal antibody comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2, LCDR3, and the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein: the LCDR1, LCDR2, and LCDR3 respectively comprise or are the CDR1, CDR2, and CDR3 of the light chain variable region with an 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, or equivalent variants thereof, and the HCDR1, HCDR2, and HCDR3 respectively comprise or are the CDR1, CDR2, and CDR3 of the heavy chain variable region with an 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, or equivalent variants thereof.

[0007] In some embodiments, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are defined based on any one of the IMGT, Kabat, Chothia, Contact, or Martin definitions.

[0008] In some embodiments, the LCDR1 comprises or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, the LCDR2 comprises or is the sequence shown in SEQ ID NO: 2 or an equivalent variant thereof, the LCDR3 comprises or is the sequence shown in SEQ ID NO: 3 or an equivalent variant thereof, and the HCDR1 comprises or is the sequence shown in SEQ ID NO: 4 or an equivalent variant thereof, the HCDR2 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, and the HCDR3 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof.

[0009] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 8 or an equivalent variant thereof.

[0010] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0011] In some embodiments, the antibody is a humanized antibody. In some embodiments, the light chain variable region comprises or is any sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and equivalent variants thereof, and the heavy chain variable region comprises or is any sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and equivalent variants thereof.

[0012] 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 also provides a vector comprising the aforementioned polynucleotide. In another aspect, the present invention also provides a non-human host cell comprising the aforementioned vector. Furthermore, the present invention also 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.

[0013] In a second aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the above antibodies, polynucleotides, vectors or host cells, and a pharmaceutically acceptable carrier.

[0014] In a third aspect, the present invention provides use of any of the above antibodies, polynucleotides, vectors or host cells in the preparation of a drug for treating cancer.

[0015] In a fourth aspect, the present invention provides any one of the above antibodies, polynucleotides, vectors or host cells for use in treating cancer.

[0016] In a fifth aspect, the present invention provides a method for treating cancer, comprising administering to a subject an effective amount of any one of the antibodies, polynucleotides, vectors or host cells described above.

[0017] In related aspects of the above, the cancer includes, but is 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, leukemia (e.g., acute myeloid leukemia).

[0018] The anti-5T4 monoclonal antibody of the present invention has high affinity, good cell binding ability and high internalization rate, and can be used to treat diseases that overexpress 5T4, such as cancer.

[0019] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1: Internalization rate test of humanized antibodies.

[0021] Figure 2: ELISA affinity determination.

[0022] Figure 3: Biacore affinity determination.

[0023] Figure 4: ELISA assay for cross-reactivity.

[0024] Figure 5: FACS determination of cell binding capacity.

[0025] Figure 6: Internalization rate determined by FACS. DETAILED DESCRIPTION

[0026] definition

[0027] In the present invention, "about" means that a value is within an acceptable error range for the specific value determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (i.e., the limitations of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. In addition, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0028] Compositions or methods described herein as "comprising" one or more recited elements or steps are open-ended, meaning that the recited elements or steps are required, but other elements or steps may be added within the scope of the composition or method. It should also be understood that any composition or method described as "comprising" one or more recited elements or steps also describes a corresponding, more limited composition or method that "consists essentially of" the recited elements or steps, meaning that the composition or method includes the recited elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method.

[0029] As used herein, the term "antibody" refers to any form of an antibody that exhibits a desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signal transduction). "Antibody fragment" and "antigen-binding fragment" refer to antigen-binding fragments of antibodies and antibody analogs, which typically 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.

[0030] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homologous antibody population, i.e., the individual antibodies constituting the population are identical except for possible natural mutants that may be present in small amounts. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include a variety of different antibodies directed against a plurality of different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homologous antibody population and is not to be construed as requiring the preparation of the antibody by any particular method. For example, the monoclonal antibodies used in the present invention can be prepared by hybridoma or recombinant DNA methods.

[0031] Monoclonal antibodies can include "chimeric," humanized, or fully human antibodies. In some embodiments, antibodies form part of a larger biomolecule, such as a fusion protein or antibody-drug conjugate. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, antibody fragments retain at least 10% of the parent binding activity. Preferably, antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody for the target.

[0032] As used herein, the term "humanized antibody" refers to an antibody comprising CDRs of an antibody derived from mammals other than humans, and framework regions (FRs) and constant regions of a human antibody.

[0033] An "equivalent variant" of an antibody or polypeptide refers to an antibody or polypeptide that has a certain degree of homology or sequence identity to the amino acid sequence of the antibody or polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some aspects, an equivalent variant has one, two, three, four or five additions, deletions, substitutions and combinations thereof compared to a reference antibody or polypeptide. In some aspects, an equivalent variant of an antibody or polypeptide retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0034] As used herein, a "variant" of a sequence is one that differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0035] As used herein, "% identity" between two sequences refers to a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of % identity between two sequences can be accomplished using a mathematical algorithm.

[0036] "Conservative substitutions" refer to amino acid substitutions known to those skilled in the art that are made without generally altering the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter or substantially change the biological activity. "No alteration or no substantial alteration" means that one or more aspects of the polypeptide differ by no more than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% when compared to the subject being compared, when measured by the same or similar method.

[0037] Generally, CDR3 and CDR3 are considered to play a more important role in antigen recognition than other CDRs. Therefore, in the case of substitution, the present invention preferably performs conservative substitutions on CDRs other than CDR3. In some embodiments, CDR3 is not substituted. Preferred amino acid substitutions include, but are not limited to, substitutions that (1) reduce sensitivity to proteolysis, (2) reduce sensitivity to oxidation, (3) change the binding affinity for forming protein complexes, and (4) provide or modify other physicochemical or functional properties of these analogs. Analogs may include mutations of various sequences other than naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be performed in naturally occurring sequences (preferably in polypeptide portions outside the region forming intermolecular contacts). Conservative amino acid substitutions should not substantially change the structural characteristics of the parent sequence (e.g., amino acid substitutions should not tend to destroy the helix present in the parent sequence, or characterize other secondary structure types that destroy the parent sequence).

[0038] It is anticipated that the binding domain of the antibody or antigen-binding fragment thereof of the present invention may carry a signal peptide, which is typically located at the N-terminus of the secretory protein and generally consists of 15 to 30 amino acids. After the signal peptide sequence is synthesized, it is recognized by the signal recognition particle (SRP), protein synthesis is paused or slowed, and the signal recognition particle carries the ribosome to the endoplasmic reticulum, where protein synthesis resumes. Under the guidance of the signal peptide, the newly synthesized protein enters the endoplasmic reticulum lumen, and the signal peptide sequence is removed by the action of signal peptidase. If the termination transport sequence is present at the C-terminus of the nascent peptide chain, it may not be removed by the signal peptidase, such as ovalbumin, which contains an internal signal peptide. Neither its precursor nor its mature form is removed by the signal peptidase.

[0039] According to the present invention, the term "binding" preferably refers to specific binding. When referring to a ligand / receptor, antibody / antigen or other binding pair, "specific" binding refers to the determination of the presence or absence of a binding reaction for the protein in a heterogeneous population of proteins and / or other biological reagents. Thus, under specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind to other proteins present in the sample in significant amounts. "Specific binding" refers to the ability of a monoclonal antibody or antigen-binding fragment thereof of the present invention to specifically interact with at least two, three, four, five, six, seven, eight or more amino acids of a respective target molecule. The "specific binding" of an antibody is primarily characterized by two parameters: a qualitative parameter (binding epitope or antibody binding site) and a quantitative parameter (binding affinity or binding strength). Antibody binding epitopes can be determined by FACS, peptide dot epitope mapping, mass spectrometry or peptide ELISA. Biacore and / or ELISA methods can determine the binding strength of an antibody to a specific epitope. Signal-to-noise ratio is typically used as a representative measurement and calculation method for binding specificity. In such a signal-to-noise ratio, the signal represents the strength of the antibody binding to the target epitope, while the noise represents the strength of the antibody binding to other non-target epitopes. Preferably, a signal-to-noise ratio of about 50 for the target epitope can be considered to indicate that the antibody being evaluated binds to the target epitope in a specific manner, i.e., "specifically binds". An antigen binding protein (including an antibody) "specifically binds" to an antigen if the antigen binding protein (including an antibody) binds to the antigen with a high binding affinity as determined by an affinity constant (KD) value. In some embodiments, the affinity constant KD is less than 10 -9 M. As used herein, the term "KD" refers to the affinity constant for a specific antibody-antigen interaction.

[0040] Humanized antibodies are antibody molecules derived from non-human species antibodies, which combine with the desired antigen having one or more complementary determining regions (CDRs) from non-human species and from the framework regions of human immunoglobulin molecules. Typically, the framework residues in the human framework region will be replaced by the corresponding residues from the CDR donor antibody to change, preferably improve antigen binding. These framework replacements are identified by methods well known in the art, for example, by modeling the interaction of CDR and framework residues to identify framework residues that are more important for antigen binding and sequence comparison to identify unusual framework residues at specific positions. Antibody humanization can be performed using a variety of techniques known in the art, including, for example, CDR-grafting, finishing or resurfacing, and chain shuffling.

[0041] Fully human antibodies are particularly desirable for therapeutic treatment of human patients.Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0042] Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells, either randomly or through homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be rendered functional separately or simultaneously upon introduction into the human immunoglobulin locus through homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal manner with a selected antigen, such as all or part of a desired target polypeptide. Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange themselves during B cell differentiation, subsequently undergoing class switching and somatic mutation. Thus, using this technology, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies.

[0043] Fully human antibodies that recognize a selected epitope can also be generated using a technique called "guided selection." In this method, a non-human monoclonal antibody, such as a mouse antibody, is selected to guide the selection of a fully human antibody that recognizes the same epitope.

[0044] Conventional procedures (for example, by using oligonucleotide probes that can be specifically combined with the genes encoding mouse antibody heavy and light chains) can be used to easily separate and sequence the DNA encoding the desired monoclonal antibody. The hybridoma cells separated and subcloned are used as the preferred source of this type of DNA. After separation, DNA can be placed in an expression vector, which is then transfected into a prokaryotic or eukaryotic host cell, such as an Escherichia coli cell, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins. More specifically, the DNA isolated can be used to clone constant region and variable region sequences for the manufacture of antibodies. Essentially, this requires extracting RNA from selected cells, converting it into cDNA, and amplifying by PCR using Ig-specific primers. As described herein, the transformed cells expressing the desired antibody can be grown in a relatively large amount to provide clinical and commercial supply of immunoglobulins.

[0045] In addition, using conventional recombinant DNA technology, one or more CDRs of the antigen-binding polypeptides of the present disclosure can be inserted into the framework region, for example, inserting a human framework region to humanize a non-human antibody. The framework region can be a naturally occurring or shared framework region, and preferably a human framework region. Preferably, the polynucleotide encoding generated by the combination of the framework region and the CDR specifically binds to an antibody of at least one epitope of a desired polypeptide (e.g., LIGHT). Preferably, one or more amino acid substitutions can be made in the framework region, and preferably, the amino acid substitution improves the binding of the antibody to its antigen. In addition, such methods can be used to carry out amino acid substitutions or deletions of one or more variable region cysteine ​​residues that participate in intrachain disulfide bonds to produce antibody molecules lacking one or more intrachain disulfide bonds. Other changes to the polynucleotide are included in the present disclosure and are within the scope of the art.

[0046] As used herein, the term "patient" or "subject" refers to any organism to which a provided antibody, derivative thereof, or pharmaceutical composition is or can be administered 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 a human. In some embodiments, the subject suffers from or is susceptible to one or more disorders or conditions. The patient may exhibit one or more symptoms of a disorder or condition, or may have been diagnosed with one or more disorders or conditions. In some embodiments, the patient is receiving or has received a therapy for the diagnosis and / or treatment of such a disease, disorder, or condition.

[0047] In the present invention, the term "treatment" refers to therapeutic and preventive measures that prevent or slow the occurrence or progression of undesirable physiological changes or symptoms in a subject. Favorable or desired clinical effects include, but are not limited to, relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, alleviation or alleviation of the disease state, and partial or complete cure of the disease, regardless of whether the above effects are detectable. "Treatment" may also refer to prolonged survival compared to no treatment. Subjects in need of treatment include subjects who already have the disease or condition, as well as subjects who are likely to have the disease or condition, or subjects who are to prevent the disease or condition.

[0048] "Administering" and "treating," when used in reference to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting an agent with a cell and contacting an agent with a fluid, wherein the fluid is contacted with the cell. "Administering" and "treating" also mean the in vitro and ex vivo treatment of cells, for example, by an agent, diagnostic agent, binding composition, or by other cells.

[0049] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an antibody or derivative thereof of the present invention that is effective in preventing or alleviating the disease or condition to be treated when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. A therapeutically effective dose further refers to an amount sufficient to cause a reduction in symptoms, such as treating, curing, preventing, or alleviating a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviating of the symptoms. When administered to an individual, the therapeutically effective amount refers to that individual ingredient. When administered in combination, the therapeutically effective amount refers to the combined amount of the active ingredients that produces the therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. A therapeutically effective amount will alleviate symptoms typically by at least 10%; typically by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%.

[0050] As used herein, "pharmaceutically acceptable carriers" include materials that, when combined with the active ingredient of a composition, allow the ingredient to maintain biological activity and do not cause a destructive reaction with the subject's immune system. These carriers may include stabilizers, preservatives, salt or sugar complexes or crystals, etc. "Pharmaceutically acceptable" refers to molecules and ingredients that do not produce an allergic reaction or similar undesirable reaction when administered to a human.

[0051] Anti-5T4 antibody

[0052] The present invention immunizes mice with 5T4 antigen to screen and obtain monoclonal antibodies, and then humanizes the antibodies to obtain humanized anti-5T4 antibodies.

[0053] In one aspect of the present invention, the present invention provides a monoclonal antibody or an 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, wherein the light chain variable region comprises LCDR1, LCDR2, LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein: the LCDR1, LCDR2, and LCDR3 respectively comprise or are the CDR1, CDR2, and CDR3 of the light chain variable region with an 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, or their respective equivalent variants, and the HCDR1, HCDR2, and HCDR3 respectively comprise or are the CDR1, CDR2, and CDR3 of the heavy chain variable region with an 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, or their respective equivalent variants.

[0054] In some embodiments, the CDR1, CDR2, CDR3 (e.g., LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are defined based on any one of the IMGT, Kabat, Chothia, Contact, or Martin definitions. In some embodiments, the CDR1, CDR2, CDR3 (e.g., LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are defined based on the IMGT definition.

[0055] In some embodiments, the LCDR1 comprises or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, the LCDR2 comprises or is the sequence shown in SEQ ID NO: 2 or an equivalent variant thereof, the LCDR3 comprises or is the sequence shown in SEQ ID NO: 3 or an equivalent variant thereof, and the HCDR1 comprises or is the sequence shown in SEQ ID NO: 4 or an equivalent variant thereof, the HCDR2 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, and the HCDR3 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof.

[0056] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 8 or an equivalent variant thereof.

[0057] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0058] In some embodiments, the antibody is a humanized antibody. In some embodiments, the light chain variable region comprises or is any sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and equivalent variants thereof, and the heavy chain variable region comprises or is any sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and equivalent variants thereof.

[0059] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof.

[0060] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof.

[0061] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof.

[0062] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof.

[0063] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof.

[0064] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof.

[0065] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof.

[0066] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof.

[0067] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof.

[0068] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof.

[0069] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof.

[0070] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof.

[0071] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 16 or an equivalent variant thereof.

[0072] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 16 or an equivalent variant thereof.

[0073] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 16 or an equivalent variant thereof.

[0074] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 16 or an equivalent variant thereof.

[0075] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof.

[0076] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof.

[0077] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof.

[0078] In some embodiments, the light chain variable region comprises or is the sequence shown in SEQ ID NO: 12 or an equivalent variant thereof, and the heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof.

[0079] The humanized antibody may comprise a light chain constant region and a heavy chain constant region. 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.

[0080] In some embodiments, an equivalent variant of each of the CDR1, CDR2, CDR3 (e.g., LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) refers to a single amino acid substitution, deletion, or insertion compared to the reference sequence.

[0081] In some embodiments, the equivalent variant of the variable region refers to a sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, and having the same or equivalent CDR1, CDR2 and CDR3.

[0082] In some embodiments, the LCDR1 comprises the sequence shown in SEQ ID NO:1, the LCDR2 comprises the sequence shown in SEQ ID NO:2, the LCDR3 comprises the sequence shown in SEQ ID NO:3, and the HCDR1 comprises the sequence shown in SEQ ID NO:4, the HCDR2 comprises the sequence shown in SEQ ID NO:5, and the HCDR3 comprises the sequence shown in SEQ ID NO:6.

[0083] In some embodiments, the light chain variable region comprises the sequence shown in SEQ ID NO:7, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:8.

[0084] In some embodiments, the antibody is a humanized antibody. In some embodiments, the light chain variable region comprises any one of a 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 comprises any one of a sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0085] In some embodiments, the LCDR1 is the sequence shown in SEQ ID NO: 1, the LCDR2 is the sequence shown in SEQ ID NO: 2, the LCDR3 is the sequence shown in SEQ ID NO: 3, and the HCDR1 is the sequence shown in SEQ ID NO: 4, the HCDR2 is the sequence shown in SEQ ID NO: 5, and the HCDR3 is the sequence shown in SEQ ID NO: 6.

[0086] In some embodiments, the light chain variable region is the sequence shown in SEQ ID NO:7, and the heavy chain variable region is the sequence shown in SEQ ID NO:8.

[0087] In some embodiments, the antibody is a humanized antibody. In some embodiments, the light chain variable region is any one of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and the heavy chain variable region is any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0088] In some embodiments, the antibody is of IgG type, such as IgG1, IgG2, IgG3 or IgG4 type. In some embodiments, the antibody is of IgG1 type.

[0089] In some embodiments, the substitutions described herein are conservative substitutions.

[0090] "Conservative (amino acid) substitutions" refer to substitutions of amino acid residues with amino acids having similar side chains. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably substituted with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be substituted with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0091] It will also be understood by those skilled in the art that the antibodies disclosed herein can be modified so that their amino acid sequences differ from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a given protein can be similar to the starting sequence, such as having a certain percentage of identity, for example, it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or a range between any two of these values ​​identical to the starting sequence.

[0092] In some embodiments, the antibodies comprise amino acid sequences or one or more moieties not typically associated with antibodies. Exemplary modifications are described in more detail herein. For example, the antibodies disclosed herein may comprise 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).

[0093] The antibodies, variants or derivatives thereof of the present invention include modified derivatives, i.e., covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not limitation, the antibody can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibody can comprise one or more non-classical amino acids.

[0094] In some embodiments, the antibody may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0095] The antibodies may be conjugated or fused to therapeutic agents, which may include detectable labels (e.g., radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancing agents, non-radioactive labels, combinations thereof, and other such agents known in the art.

[0096] Antibodies can be detectably labeled by coupling them to a chemiluminescent compound. The presence of the chemiluminescent-labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, thermostable acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0097] Fluorescent emitting metals (e.g. 152 Antibodies can be detectably labeled with metals such as Eu or other metals from the lanthanide series. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known.

[0098] In another aspect, the present invention provides a nucleotide sequence comprising a polynucleotide encoding an antibody according to any of the above aspects. In another aspect, the present invention also provides a vector comprising the aforementioned polynucleotide. In another aspect, the present invention also provides a non-human host cell comprising the aforementioned vector.

[0099] Pharmaceutical composition

[0100] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the above antibodies, polynucleotides, vectors or host cells, and a pharmaceutically acceptable carrier.

[0101] In any of the above aspects, the pharmaceutical composition is used to treat a disease that overexpresses 5T4, such as cancer. In some embodiments, the cancer includes but is 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, leukemia (e.g., acute myeloid leukemia).

[0102] To prepare a pharmaceutical composition or sterile composition, the drug is mixed with a pharmaceutically acceptable carrier or excipient. Formulations can be prepared, for example, as lyophilized powders, slurries, aqueous solutions, or suspensions by mixing with physiologically acceptable carriers, excipients, or stabilizers. Pharmaceutically acceptable carriers are well known in the art. It is also known in the art how to prepare aqueous compositions containing active ingredients. Typically, these compositions are prepared as injections or sprays, such as liquid solutions or suspensions; solid forms suitable for preparation as solutions or suspensions prior to injection or spraying can also be prepared.

[0103] Methods and Applications

[0104] In one aspect, the present invention provides a method for treating cancer, comprising administering to a subject an effective amount of any one of the antibodies, polynucleotides, vectors or host cells described above.

[0105] Accordingly, another aspect of the present invention provides use of any of the above antibodies, polynucleotides, vectors or host cells in the preparation of a drug for treating cancer.

[0106] Accordingly, another aspect of the present invention provides any one of the above antibodies, polynucleotides, vectors or host cells for treating cancer.

[0107] In some embodiments, the cancer includes but is 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, leukemia (e.g., acute myeloid leukemia).

[0108] Suitable administration routes include parenteral administration (e.g., intramuscular, intravenous or subcutaneous administration) and oral administration. Other conventional administration methods include administration via endotracheal tube, oral ingestion, inhalation, topical application or transdermal, subcutaneous, intraperitoneal, intraarterial injection.

[0109] The appropriate dosage is determined by a clinician based on parameters or factors known or suspected to affect treatment or expected to affect treatment in the art. Typically, the initial dose is slightly lower than the optimal dose and then increased in small amounts until the desired or optimal effect relative to any adverse side effects is achieved. Important monitoring indicators include measuring, for example, inflammatory symptoms or the levels of inflammatory cytokines produced.

[0110] Sequence Listing

[0111] Example

[0112] Example 1: Antibody screening and sequencing

[0113] Step 1: Infect CHO cells with a lentivirus containing the 5T4 gene, and use the constructed CHO-5T4+ cells to immunize 6-week-old BALB / c mice.

[0114] Step 2: Fusion of cells to obtain multiple original cell lines;

[0115] Step 3: Screen the hybridoma supernatant secreted by the original cell line through ELISA, flow cytometry, and Biacore to obtain multiple candidate hybridoma original clones with good affinity;

[0116] Step 4: Subclone the candidate cell lines and screen monoclonal antibodies with high affinity, rapid internalization and good specificity through ELISA, flow cytometry, SPR, and Western blot.

[0117] A candidate monoclonal antibody was screened using the above method and named B5. The amino acid sequences of the light chain and heavy chain variable regions of antibody B5 were obtained by PCR, as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0118] Example 2: Affinity test of antibody B5

[0119] The affinity of the antibody B5 screened in Example 1 was determined by Biacore.

[0120] The specific measurement method is as follows:

[0121] 1) Hybridoma affinity screening: the antigen was 5T4 EDC-Fc, with a molecular weight of 65-120 kD. The antigen was directly coupled to CM5 via amino groups.

[0122] 2) According to Rmax=(MW analyte / MW ligand )×RL×Sm, the actual coupling amount is 1.5 times that of RL. The calculated suitable coupling amount for simultaneous detection of antigens is 120Ru;

[0123] 3) Coupling: Use 1× HBS-EP buffer and 50 mM NaOH as a wash buffer. Prepare the antigen to 5 μg / mL using sodium acetate solution at pH 4.5. Select amino coupling and enter 120 Ru in the Target level. Place the antigen sample, NaOH, EDC, NSH, ethanolamine, and an empty test tube in the corresponding positions on the sample tray according to the instructions.

[0124] 4) Regeneration conditions were explored, and finally pH 1.7 glycine hydrochloride for 10 seconds was selected;

[0125] 5) Sample Preparation: For the positive control antibody, dilute the B5 antibody to 8 nM, 4 nM, 2 nM, 1 nM, 0.5 nM, and 0.25 nM using 1× HBS-EP buffer and centrifuge at 13,000 rpm for 5 min. All samples were plated in duplicate. Hybridoma supernatant sample preparation: Hybridoma cell supernatant was collected and centrifuged at 13,000 rpm for 5 min. The supernatant was diluted 1 / 2 with 1× HBS-EP buffer. Each sample was plated in duplicate.

[0126] 6) Program settings: In the Kinetics / Affinity options, select 2-1 for Flow pattern; select 2 for Regeneration; set the Contact time to 180 seconds; set the Dissociation time to 600 seconds; and set the Regeneration conditions to Gly 1.7 and 10 seconds. Set the sample intervals for repeated tests, set three 0 concentrations, and three Start-Ups. Click Start to run the program.

[0127] 7) Result analysis: Select appropriate concentration-corresponding response unit change curve, fit it using a 1:2 calculation model, and perform kinetic analysis.

[0128] The test results are shown in Table 1.

[0129] Table 1. Affinity test of antibody B5

[0130] Example 3: Construction of humanized antibodies

[0131] The antibody B5 screened in Example 1 was humanized 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), and 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).

[0132] The above-mentioned light chain variable regions were combined with the heavy chain variable regions to construct 20 humanized antibodies as shown in Table 2, which were named hB5-2-1 to hB5-2-20, respectively. The constant regions of these antibodies all adopted the human Igκ light chain constant region (SEQ ID NO: 18) and the human IgG1 heavy chain constant region (SEQ ID NO: 19).

[0133] Table 2. Construction of humanized antibodies

[0134] Example 4: Affinity testing of humanized antibodies

[0135] The affinity of the 20 hB5 humanized antibodies constructed in Example 3 was rapidly tested at a single concentration using Biacore 8K, thereby comparing different humanized antibodies.

[0136] Experimental process:

[0137] (1) CM5 chip amino-coupled Anti-Human Antibody protein.

[0138] (2) hB5 humanized antibody flows over the chip surface.

[0139] (3) High concentration of TPBG-His antigen flows through the chip surface.

[0140] (4) Affinity detection.

[0141] Experimental Materials:

[0142] Antibody transfection system: 30ml; purification column: Protein A / G; purity is above 95%.

[0143] Experimental steps:

[0144] 1) Ligand capture

[0145] - Collect the cell supernatant and centrifuge at 4°C, 13,000 rpm / min for 30 min. Take 2 / 3 of the supernatant and place it in a new EP tube.

[0146] -The CM5 chip was separated into 8 channels using a microfluidic chip system (IFC). Each channel was divided into 2 channels, with channel 1 serving as a reference channel and channel 2 capturing the supernatant.

[0147] - Set the flow rate to 10 μl / min and allow the supernatant to flow for 200 s.

[0148] -More than 95% of the antibody capture capacity is basically higher than 100RU.

[0149] 2) Antigen binding

[0150] - In channels 1 and 2, TPBG-His protein was diluted to 300 nM with HBS-EP+ buffer and flowed for 120 s at a flow rate of 30 μl / min.

[0151] - In channels 1 and 2, glycine pH 1.7 was passed through at a flow rate of 30 μl / min for 40 s.

[0152] 3) Obtain the response value of each antibody.

[0153] 4) Use the 1:1 binding model of Bia-evaluation analysis software to calculate the kinetic parameters and compare the affinity of each antibody.

[0154] Experimental results:

[0155] The measurement results are shown in Table 3.

[0156] Table 3. Statistics of kinetic parameters of cell supernatants with different antibodies and TPBG-His

[0157] As can be seen from Table 3, the 20 hB5 humanized antibodies constructed in Example 3 all showed good affinity, and the affinity constant KD reached 10 -9 M, a considerable number of which reached 10 -10 M.

[0158] Example 5: Internalization rate test of humanized antibodies

[0159] In this example, the internalization rates of hB5-2-5, hB5-2-6, and hB5-2-17 among the humanized antibodies constructed in Example 3 were tested.

[0160] Experimental process:

[0161] 1) Cell collection: Remove the old culture medium, add 0.25% trypsin digestion solution to digest the cells, then add DMEM culture medium to terminate the digestion. Collect the cell suspension and centrifuge at 800 rpm for 3 minutes.

[0162] 2) Resuspend the cells in 4 ml of culture medium and perform a cell count: Mix the cell suspension with 0.4% trypan blue in a 1:1 ratio, add the mixture to a cell counting plate, and place the plate on a Countstar cell counter to automatically count the cell number and viability.

[0163] 3) The cell suspensions were divided into equal groups. The number of cells in the isotype control and negative control was 1×10 6 The number of cells in each experimental group was 2×10 6 .

[0164] 4) Centrifuge the cells at 4°C, 3500 rpm / min for 3 min and carefully remove the supernatant with a pipette.

[0165] 5) Incubation with primary antibody: Add 100 μl of mouse IgG (10 μg / ml) to the isotype control group, 100 μl of PBS to the blank control group, and 100 μl of antibody solution (final concentration: 10 μg / mL) to the experimental group. After labeling, resuspend the cells and incubate them 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.

[0166] 6) After resuspending the cells in 500 μl of PBS, divide the experimental group into two groups. Place the non-internalized group on ice or incubate at 4°C for 3 hours, and the internalized group incubate at 37°C for 3 hours. The isotype and negative control groups are temporarily placed at 4°C. During this period, gently tap the tube wall every 10 minutes to prevent cell precipitation.

[0167] 7) After incubation, centrifuge at 3500 rpm and 4°C for 3 min, discard the supernatant, and wash the cells twice;

[0168] 8) Incubate with secondary antibody. Dilute the secondary antibody (FITC-goat anti-mouse IgG antibody / FITC-goat anti-human IgG antibody) at 1:200 with PBS. Add 100 μl of secondary antibody to each EP tube and resuspend. Incubate on ice at 4°C for 40 min.

[0169] 9) After incubation, add 500 μl of PBS buffer to each group, resuspend the cells by pipetting twice, and then centrifuge the cells at 3500 rpm and 4°C for 3 min. Aspirate the supernatant and repeat 2-3 times.

[0170] 10) Add 300 μl of PBS buffer to resuspend the cells in each group, pipette them into the flow cytometry tube, and wait for detection on the flow cytometry machine.

[0171] 11) Set all parameters on the flow cytometer, use the blank control group of cells as a control after debugging, and then test each group of cell samples sequentially.

[0172] Experimental results:

[0173] Cells: CHO-5T4+; Antibody gradient: 0.1, 0.5, 1 μg / ml; Internalization time: 3 h.

[0174] The measurement results are shown in Figure 1 and Table 4.

[0175] Table 4. Internalization rates of humanized antibodies

[0176] It can be seen that the internalization rates of the three humanized antibodies are around 60%-70%, showing a relatively high internalization rate, among which hB5-2-17 performs the best.

[0177] Example 6: Performance testing of humanized antibody hB5-2-10

[0178] In this example, various properties of the humanized antibody hB5-2-10 were tested.

[0179] (1) Affinity-ELISA / Biacore

[0180] ELISA detection of affinity between hB5-2-10 and TPBG-His

[0181] Experimental process:

[0182] 1) Coating: Dilute TPBG-His to 1 μg / ml in 50 mM carbonate buffer (pH 9.6). Add 50 μl per well to the ELISA plate. Add blocking membrane and place in a refrigerator at 4°C overnight.

[0183] 2) Washing: Remove the sealing film and place the plate in a plate washer. Add 300 μl of 1× PBST to each well, shake for 20 seconds, and soak for 10 seconds. Wash the plate twice. Remove the plate and tap it gently on absorbent paper until there is no liquid residue or bubbles in the wells.

[0184] 3) Blocking: Add 200 μl of 1% BSA to each well and incubate at 37°C for 1 hour.

[0185] 4) Washing: Remove the sealing film and place the plate in a plate washer. Add 300 μl of 1× PBST to each well, shake for 20 seconds, and soak for 10 seconds. Wash the plate twice. Remove the plate and tap it gently on absorbent paper until there is no liquid residue or bubbles in the wells.

[0186] 5) Primary antibody incubation: hB5-2-10 was diluted with 0.1% BSA to a concentration of 40,000 ng / ml, and then diluted 6-fold in a series of 10 concentrations. The diluted solution was added to the plate at 100 μl / well in duplicate and incubated at 37°C for 1 hour.

[0187] 6) Washing: Remove the sealing film and place the plate in a plate washer. Add 300 μl of 1x PBST to each well, shake for 20 seconds, and soak for 10 seconds. Wash the plate four times. Once washed, remove the plate and tap gently on absorbent paper until there is no residual liquid or bubbles in the wells.

[0188] 7) Secondary antibody incubation: Dilute Goat-Anti-human IgG Fc (HRP) with 0.1% BSA at a dilution ratio of 1:10,000. Add 100 μl to each well and incubate at 37°C for 1 hour.

[0189] 8) Washing: Remove the sealing film and place the plate in a plate washer. Add 300 μl of 1x PBST to each well, shake for 20 seconds, and soak for 10 seconds. Wash the plate three times. Once washed, remove the plate and tap gently on absorbent paper until there is no residual liquid or bubbles in the wells.

[0190] 9) Color development: Add 100 μl of TMB color developer to each well and incubate at 37°C for 10 min.

[0191] 10) Add stop solution: Add 100 μl of 0.7 M oxalic acid solution to each well to terminate the reaction.

[0192] 11) OD value measurement: Use a microplate reader to measure the absorbance at a wavelength of 450 nm.

[0193] Experimental results:

[0194] The ELISA results are shown in Figure 2. Antibody hB5-2-10 binds to the EC of TPBG-His 50 It is 2.801ng / ml.

[0195] Biacore detection of affinity between hB5-2-10 and TPBG-His

[0196] Experimental process:

[0197] 1) A CM5 chip was separated into 8 channels using a microfluidic chip system (IFC), with each channel divided into 2 channels. Channel 1 served as the reference channel, and channel 2 was coupled to the ligand.

[0198] 2) pH Pre-enrichment: Use NaAC buffer (pH 4.5 / 5.0) to connect the antigen (TPBG-His) to a CM5 chip at a concentration of 10 μg / mL. Determine the appropriate antibody dilution and concentration, and then couple the antibody directly to the CM5 chip. Set the flow rate to 10 μl / min, the contact time to 2 min, and the dissociation time to 0. Use 50 mM NaOH as the regeneration reagent. Determine the coupling pH based on the results.

[0199] 3) Ligand Coupling: Dilute the TPBG-His antigen with 10 mM Acetate coupling reagent (pH determined) to the appropriate concentration determined during pre-enrichment. Activate the CM5 chip with 20% EDC and 80% NHS for 30 seconds at a flow rate of 10 μl / min. Block the coupled ligand with ethanolamine for 420 seconds to complete the ligand coupling process.

[0200] 4) Analyte binding

[0201] The antibody was diluted as the analyte to different concentration gradients at a flow rate of 30 μl / min, flowing through channel 1 and channel 2 in sequence for 120 s, and the dissociation time was set to 600 s.

[0202] In channels 1 and 2, glycine pH 1.5 regeneration buffer was flowed at a flow rate of 30 μl / min for 30 s.

[0203] 5) Obtain the response value for each analyte concentration, calculate the kinetic parameters using the 1:1 binding model of the Bia-evaluation analysis software, and compare the affinity of each antibody.

[0204] The Biacore results are shown in Figure 3 and Table 5.

[0205] Table 5. Biacore assay results for antibody hB5-2-10

[0206] (2) Cross-reactivity-ELISA

[0207] Experimental process:

[0208] 1) Coating: Dilute TPBG-His from three different species to 1 μg / ml in 1× PBS buffer. Add 50 μl to each well of the ELISA plate. Apply blocking film and refrigerate at 4°C overnight.

[0209] 2) Washing: Remove the sealing film and place the plate in a plate washer. Add 300 μl of 1× PBST to each well, shake for 3 seconds, and soak for 40 seconds. Wash the plate twice. Remove the plate and tap it gently on absorbent paper until there is no liquid residue or bubbles in the wells.

[0210] 3) Blocking: Add 250 μl of 1% BSA to each well and incubate at 37°C for 1 hour.

[0211] 4) Washing: Remove the sealing film and place the plate in a plate washer. Add 300 μl of 1× PBST to each well, shake for 3 seconds, and soak for 40 seconds. Wash the plate twice. Remove the plate and tap it gently on absorbent paper until there is no liquid residue or bubbles in the wells.

[0212] 5) Primary Antibody Incubation: hB5-2-10 was diluted with 0.1% BSA to concentrations of 20,000 ng / ml, 5,000 ng / ml, 1,000 ng / ml, 200 ng / ml, 40 ng / ml, 8 ng / ml, 1.6 ng / ml, and 0.32 ng / ml. A 0 concentration point of 1% BSA alone was set and added to the well plate at 100 μl / well in duplicate. Incubate at 37°C for 1 hour.

[0213] 6) Washing: Remove the sealing film and place the plate in a plate washer. Add 300 μl of 1× PBST to each well, shake for 3 seconds, and soak for 50 seconds. Wash the plate three times. Once washed, remove the plate and tap gently on absorbent paper until there is no residual liquid or bubbles in the wells.

[0214] 7) Secondary antibody incubation: Dilute Goat-Anti-human IgG Fc (HRP) with 0.1% BSA at a dilution ratio of 1:7000. Add 100 μl to each well and incubate at 37°C for 45 min.

[0215] 8) Washing: Remove the sealing film and place the plate in a plate washer. Add 300 μl of 1× PBST to each well, shake for 3 seconds, and soak for 50 seconds. Wash the plate three times. Once washed, remove the plate and tap gently on absorbent paper until there is no residual liquid or bubbles in the wells.

[0216] 9) Color development: Add 100 μl of TMB color developer to each well and incubate at 37°C for 10 min.

[0217] 10) Add stop solution: Add 100 μl of 2M H2SO4 stop solution to each well to terminate the reaction.

[0218] 11) OD value measurement: Use a microplate reader to measure the absorbance at a wavelength of 450 nm.

[0219] Experimental results:

[0220] The results are shown in FIG4 , indicating that antibody hB5-2-10 binds to human / monkey 5T4 antigens with similar binding activities, and does not bind to mouse 5T4 antigen.

[0221] (3) Cell binding capacity-FACS

[0222] Experimental process:

[0223] 1) Take 3×10 5 The cells were centrifuged at 3500 rpm for 5 min, the supernatant was discarded, and then 500 μl of 1× PBS buffer was added for washing, and the cells were centrifuged at 3500 rpm for 5 min and the supernatant was discarded. Repeat this process twice.

[0224] 2) Dilute the sample to be tested to a starting concentration of 20 μg / ml with 1× PBS buffer, then serially dilute 5-fold, 100 μl / sample, and incubate at 2-8°C for 1 h.

[0225] 3) Add 100 μl of each sample and incubate on ice or at 2-8°C for 1 hour, during which time the cells are flicked every 15 minutes.

[0226] 4) After incubation, wash with 500 μl of 1× PBS buffer, centrifuge at 3500 rpm for 5 minutes, and discard the supernatant. Repeat three times.

[0227] 5) Dilute FITC-anti-human IgG secondary antibody 1:100, 100 μl / sample, and incubate on ice for 40 minutes, flicking the cells every 10 minutes.

[0228] 6) After incubation, wash with 500 μl of 1× PBS buffer, centrifuge at 3500 rpm for 5 minutes, and discard the supernatant. Repeat three times.

[0229] 7) Finally, resuspend the plate in 300 μl of 1× PBS buffer and test on a microscope.

[0230] The results are shown in FIG5 , indicating that antibody hB5-2-10 exhibits good cell binding ability.

[0231] (4) Internalization-FACS

[0232] Experimental process:

[0233] 1) Collect cells and detect viability, take 3×10 5 cells;

[0234] 2) Incubation with primary antibody: Dilute hB5-2-10 to 2 μg / ml, 100 μl / sample; set up a negative control group with PBS. Incubate at 2-8°C for 1 hour. Wash once with 1× PBS buffer after incubation.

[0235] 3) Internalization Incubation: Each cell group was divided equally into two aliquots. One aliquot was incubated on ice as the non-internalization group, and the other aliquot was incubated in a 37°C incubator as the internalization group. Both aliquots were incubated simultaneously for 4 hours. After incubation, the cells were gently aspirated and washed with 500 μl of 1× PBS buffer. The cells were centrifuged at 3500 rpm for 5 minutes, and the supernatant was discarded.

[0236] 4) Incubation with secondary antibody: All groups were removed at the same time and the secondary antibody (FITC-goat anti-human IgG) was added at a dilution of 1:100, 100 μl / sample, and incubated at 2-8°C for 30 min. After incubation, the samples were washed once.

[0237] 5) Resuspend the plate in 300 μl of 1× PBS buffer and test on an instrument.

[0238] The results are shown in Figure 6 and Table 6, where internalization rate = (non-internalization group - internalization group) / non-internalization group. It can be seen that antibody hB5-2-10 exhibits a higher internalization rate.

[0239] Table 6. Internalization rate of antibody hB5-2-10

[0240] It should be understood that, although the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may make modifications, improvements and variations to the invention disclosed herein, and these modifications, improvements and variations are considered to be within the scope of the present invention. The materials, methods and examples provided herein are representative and exemplary of preferred embodiments and are not intended to be limiting of the scope of the present invention.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that binds to 5T4, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2, HCDR3, wherein: The LCDR1, LCDR2, and LCDR3 respectively comprise or are CDR1, CDR2, and CDR3 of the light chain variable region whose amino acid sequence is SEQ ID NO: 7, or their respective equivalent variants, and The HCDR1, HCDR2, and HCDR3 respectively comprise or are CDR1, CDR2, and CDR3 of the heavy chain variable region with an amino acid sequence of SEQ ID NO: 8, or their respective equivalent variants.

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

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The LCDR1 comprises or is a sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, the LCDR2 comprises or is a sequence shown in SEQ ID NO: 2 or an equivalent variant thereof, the LCDR3 comprises or is a sequence shown in SEQ ID NO: 3 or an equivalent variant thereof, and The HCDR1 comprises or is the sequence shown in SEQ ID NO:4 or an equivalent variant thereof, the HCDR2 comprises or is the sequence shown in SEQ ID NO:5 or an equivalent variant thereof, and the HCDR3 comprises or is the sequence shown in SEQ ID NO:6 or an equivalent variant thereof. 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 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The light chain variable region comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof, and The heavy chain variable region comprises or is the sequence shown in SEQ ID NO: 8 or an equivalent variant thereof.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The light chain variable region comprises or is any sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and equivalent variants of each thereof, and The heavy chain variable region comprises or is any sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and equivalent variants of each thereof. 7 . A polynucleotide comprising a polynucleotide encoding the antibody according to claim 1 . A vector comprising the polynucleotide according to claim 7.

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

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

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