Anti-FOLR1 antibody and application thereof

Abstract

The present invention describes a chimeric antibody having specificity for FOLR1, a vector encoding the chimeric antibody, a recombinant host cell comprising the vector, and the use of the chimeric antibody in the preparation of bispecific antibodies and antibody drug conjugates.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an anti-folate receptor 1 (FOLR1) antibody and its uses. Background Technology

[0002] Folic acid is one of the essential vitamins for the human body, and the body must obtain the folic acid it needs from external sources. Ingested folic acid cannot function directly; it requires folate receptors to mediate its transport into the cytoplasm via endocytosis. The folate receptor family includes FRα, FRβ, FRγ, and FRδ, encoded by the genes FOLR1, FOLR2, FOLR3, and FOLR4, respectively. Among them, FRα, encoded by the FOLR1 gene, acts as a signaling molecule that promotes malignant tumor growth, similar to other glycosylated phosphatidylinositol family proteins. FOLR1, also known as FRα, initiates an intracellular regulatory signaling network upon binding to folic acid. Through phosphorylation, it initiates a series of intracellular signaling cascades, thereby activating the ERK and STAT3 signaling pathways, ultimately activating important regulatory mechanisms for cell growth. Furthermore, FRα assists in tumor invasion and spread by downregulating the intercellular adhesion molecule E-cadherin. Boshnjaku et al. reported that after folic acid uptake and internalization, FRα can localize to the cell nucleus, acting as a transcription factor binding cis-regulatory element. FRα directly regulates the expression of key developmental genes in tumor cells through this mechanism. Therefore, theoretically, inhibiting FRα could control tumor proliferation, metastasis, and invasion.

[0003] FOLR1 is known to be expressed on the plasma membranes of epithelial cells in several tissues, including the apical brush border membrane of proximal renal tubules, retinal pigment epithelium, choroid plexus, lung, ovary, fallopian tube, uterus, cervix, epididymis, submandibular salivary glands, bronchial glands, and type 1 and type 2 lung cells of the placental trophoblast. FRα is expressed on the surface of tumor cells throughout various cancers, including ovarian cancer, TNBC, endometrial cancer, mesothelioma, and lung cancer. FRα is expressed in 90% of ovarian cancer patients and has limited expression in normal tissues, thus FRα holds promise as a receptor for drug targets. Various strategies targeting FRα have been explored, including monoclonal antibodies, ADCs, FRα-specific CAR-Ts, vaccines, small molecules, and folic acid conjugates. Currently, there are 13 documented FRα-targeting drugs or therapies in preclinical stages globally, 1 on the market, and 12 in clinical trials, involving various drug types, including ADCs, monoclonal antibodies, CAR-Ts, bispecific antibodies, and fusion proteins. Currently, only ELAHERE has been approved as a viable new targeted therapy for platinum-resistant ovarian cancer, with FRα being approved. Mirvetuximabsoravtansine is also being explored for the treatment of platinum-resistant epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer.

[0004] Current treatment options do not meet the needs of FOLR1-related cancers, and therefore, many patients still cannot fully benefit from current strategies. There remains a strong need in the clinical field of oncology for new strategies to diagnose and treat FOLR1-related cancers. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, one object of the present invention is to provide antibodies that specifically target and bind to FOLR1, and their use in the preparation of humanized antibodies, bispecific antibodies, ADCs and compositions containing them or their bispecific antibodies or ADCs, and in the treatment of oncology drugs, to benefit patients with unmet needs for treatment pathways for FOLR1-positive tumors, to provide new and effective medical options for cancer, and ultimately to improve patients' quality of life.

[0006] The technical solution of this invention is as follows:

[0007] In a first aspect of the invention, an anti-folate receptor 1 (FOLR1) antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises complementarity-determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL comprises complementarity-determining regions VLCDR1, VLCDR2, and VLCDR3; wherein,

[0008] A) VHCDR1, VHCDR2, and VHCDR3 respectively comprise amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or amino acid sequences having at least 95% sequence identity with them; wherein VLCDR1, VLCDR2, and VLCDR3 respectively comprise amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or amino acid sequences having at least 95% sequence identity with them; or

[0009] B) VHCDR1, VHCDR2, and VHCDR3 respectively comprise amino acid sequences as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or amino acid sequences having at least 95% sequence identity with them; wherein VLCDR1, VLCDR2, and VLCDR3 respectively comprise amino acid sequences as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or amino acid sequences having at least 95% sequence identity with them.

[0010] The sequence of the complementarity determination region is defined according to the Kabat (10A5) or IMGT (27B10) numbering system.

[0011] Furthermore, the anti-folate receptor 1 (FOLR1) antibody may be any one or more of mouse-derived antibodies, chimeric antibodies, or humanized antibodies.

[0012] In some embodiments, the anti-FOLR1 antibody includes chimeric antibodies and humanized antibodies.

[0013] In some exemplary embodiments, the anti-FOLR1 antibody is a chimeric antibody.

[0014] Furthermore, the heavy chain variable region and light chain variable region of the chimeric antibody may be selected from any of the following:

[0015] In some embodiments, the VH comprises an amino acid sequence as shown in SEQ ID NO:13 or 15, or an amino acid sequence having at least 80% sequence identity with it.

[0016] In some embodiments, the VL comprises an amino acid sequence as shown in SEQ ID NO:14 or 16, or an amino acid sequence having at least 80% sequence identity with it.

[0017] Furthermore, in the above-mentioned antibody or its antigen-binding fragment, the chimeric antibody further includes a heavy chain constant region (CH) and a light chain constant region (CL).

[0018] Furthermore, the heavy chain constant region may be one of IgG1, IgG2, IgG3 or IgG4, and the light chain constant region may be a κ chain or a λ chain.

[0019] Furthermore, in the aforementioned antibody or its antigen-binding fragment, the heavy chain constant region may be one of human IgG1, IgG2, IgG3 or IgG4, and the light chain constant region may be of the human or mouse κ chain or λ chain.

[0020] Furthermore, in the aforementioned antibody or its antigen-binding fragment, the heavy chain constant region may be human IgG1, and the type of the light chain constant region may be human κ-type light chain.

[0021] In some embodiments, in the chimeric antibody, the C-terminus of the VH and the heavy chain constant region (CH) are linked to form the heavy chain (HC).

[0022] In some embodiments, the antibody or its antigen-binding fragment has a heavy chain (HC) as shown in SEQ ID NO:17 or 19 or has an amino acid sequence that is at least 80% sequence identical to that shown in SEQ ID NO:17 or 19.

[0023] In some embodiments, in the chimeric antibody, the C-terminus of the VL and the light chain constant region (CL) are connected to form a light chain (LC).

[0024] In some embodiments, the antibody or its antigen-binding fragment has a light chain (LC) having an amino acid sequence as shown in SEQ ID NO:18 or 20 or having at least 80% sequence identity with it.

[0025] In some embodiments, the antigen-binding fragment of the antibody or its antigen-binding fragment described above includes, but is not limited to, one or any combination of Fab fragment, Fv fragment, Fab′ fragment, F(ab′)2 fragment, single-chain antibody (ScFv), nanobody (single-domain antibody), bispecific antibody or minimum recognition unit (MRU).

[0026] In another aspect of the invention, a nucleic acid molecule or an expression cassette comprising the present invention is provided, comprising a nucleic acid molecule encoding a heavy chain variable region and / or a light chain variable region encoding an anti-FOLR1 antibody or an antigen-binding fragment thereof as described in the foregoing aspects.

[0027] Or it may contain heavy and / or light chains encoding the anti-FOLR1 antibody or its antigen-binding fragment as described above.

[0028] In another aspect of the invention, a construct (such as an expression vector) is provided that comprises the said polynucleotide or an expression cassette containing the polynucleotide.

[0029] In another aspect of the invention, an antibody expression system is provided, the expression system comprising the aforementioned construct or genome in which an exogenous nucleic acid molecule or an expression cassette containing the aforementioned is integrated.

[0030] In another aspect of the present invention, a method for preparing the anti-FOLR1 antibody is provided, comprising the steps of: expressing the antibody using the antibody expression system under conditions suitable for expressing the antibody, thereby expressing the antibody.

[0031] In some preferred embodiments, the preparation method further includes the step of purifying and separating the antibody.

[0032] In another aspect of the invention, the use of the antibody in the preparation of humanized antibodies, bispecific antibodies, or antibody-drug conjugates is provided.

[0033] In another aspect of the invention, a pharmaceutical composition (including a therapeutic composition) is provided, comprising the antiFOLR1 antibody or its antigen-binding fragment as described above.

[0034] In a preferred embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0035] In another aspect of the invention, the use of the said anti-FOLR1 antibody or its antigen-binding fragment, or the said pharmaceutical composition, in the preparation of a medicament for treating or preventing FOLR1-positive malignant tumors is provided.

[0036] In some embodiments, the malignant tumor is choriocarcinoma, ovarian cancer, fallopian tube cancer, endometrial cancer, primary peritoneal cancer, or lung cancer.

[0037] The beneficial effects of this invention are as follows:

[0038] The anti-FOLR1 antibody of this invention can bind to FOLR1 with high specificity and affinity, without cross-binding with other proteins in the folate receptor family. Its high specificity makes it promising for further development of humanized anti-FOLR1 antibodies, bispecific antibodies, and ADCs for inhibiting the proliferation of FRα-expressing tumor cells, as well as for the preparation of drugs for treating or preventing tumors. The tumor is a tumor expressing folate receptor 1; the tumor includes, but is not limited to, choriocarcinoma, ovarian cancer, lung cancer, endometrial cancer, breast cancer, cervical cancer, or combinations thereof. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 Identification of target protein expression in the CHO-hFOLR1 mixed pool (1A) and monoclonal 1B6 (1B).

[0041] Figure 2 The binding curve of the chimeric antibody of the present invention to the recombinant FORL1 protein was detected by ELISA.

[0042] Figure 3 .FACS method for detecting the binding curves of chimeric antibodies ch5G12 and ch14H4 to FOLR1 on the cell membrane (3A. binding to CAL51 cells; 3B. binding to CHO-hFOL1 cells).

[0043] Figure 4 ELISA method to detect the binding activity of antibody to FOLR1 protein of other species (4A. binding to recombinant cynomolgus monkey FOLR1 protein; 4B. binding to recombinant mouse FOLR1 protein).

[0044] Figure 5ELISA was used to detect the binding activity of the antibody to other members of the FOLR1 family (5A. Binding results with recombinant human FOLR2 protein; 5B. Binding results with recombinant human FOLR2 protein; 5C. Binding results with recombinant human FOLR4 protein).

[0045] Figure 6 Biomembrane thin-layer interferometry (BLI) was used to determine the equilibrium dissociation constant (KD) of the chimeric antibodies ch5G12 and ch14H4 bound to recombinant human FOLR1 protein (6A is the reference antibody, 6B is the chimeric antibody ch5G12, and 6C is ch14H4).

[0046] Figure 7 Results of antibody endocytosis activity assay. Detailed Implementation

[0047] Unless otherwise indicated, the practice disclosed herein will employ conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of the art.

[0048] The terms “antibody” or “immunoglobulin” are used herein as general terms, including full-length antibodies, single-chain antibodies, and all portions, domains, or fragments thereof (including, but not limited to, antigen-binding domains or fragments). Furthermore, the term “sequence” as used herein (e.g., in the terms “immunoglobulin sequence,” “antibody sequence,” “single variable domain sequence,” “protein sequence,” etc.) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said sequence, unless a more specific interpretation is required herein.

[0049] "Monoclonal antibody" refers to an antibody molecule composed of a single molecule. Monoclonal antibodies exhibit single-molecule binding specificity and affinity for a specific epitope.

[0050] "Sequence identity" between polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically analyzed using sequence analysis software. For instance, the BLAST program in the NCBI database can be used to determine identity.

[0051] For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be calculated by using blastp as a procedure, and then the identity value (%) can be obtained.

[0052] In this document, the at least 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% identity.

[0053] In this document, the at least 90% identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0054] "Bispecific antibodies" refer to bispecific antibodies obtained by selecting appropriate antibody constant regions, Ig types, and two arms from two sets of light and heavy chain genes using various in vivo or in vitro assembly methods, resulting in high yields, uniformity, and purity. Alternatively, bispecific antibodies can also be obtained using chemical cross-linking techniques.

[0055] The "Minimum Recognition Unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0056] As is well known to those skilled in the art, the antigen-binding fragment can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody to produce an antigen-binding fragment of the antibody.

[0057] "Pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredient contained therein is effective and which does not contain any other ingredients that would have unacceptable toxicity to a subject who would receive the composition.

[0058] "Pharmaceutically acceptable carriers" refer to components in a drug composition that are non-toxic to the subjects, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0059] The “effective amount” of a drug refers to the amount necessary to induce physiological changes in the cells or tissues to which it is administered.

[0060] "Treatment / prevention" (and its grammatical variations) refers to an attempt to alter the natural course of a disease in an individual, and can be a clinical intervention performed for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or slow the progression of symptoms.

[0061] A "therapeuticly effective amount" of a pharmaceutical agent, such as a pharmaceutical composition, refers to the amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dosage and time period. A therapeutically effective amount of a pharmaceutical agent may eliminate, reduce, delay, minimize, or prevent adverse effects of disease.

[0062] I, FORL1

[0063] In some embodiments, the recombinant human FOLR1 protein bound by the anti-FOLR1 chimeric antibody disclosed herein is recombinant human FOLR1 with a linker having the nucleic acid sequence sequence GGGGGTGGAGGCTCT added to its C-terminus and an HA tag having the nucleic acid sequence sequence TATCCTTACGACGTGCCTGACTACGCC added. In some embodiments, the recombinant human FOLR1 with the added linker and HA tag comprises an amino acid sequence having at least 95% (e.g., at least 91%, 92%, 93%, 94%, or 95%) sequence identity with the amino acid sequence shown in SEQ ID NO: 23, encoded by a nucleic acid having at least 95% (e.g., at least 91%, 92%, 93%, 94%, or 95%) sequence identity with the polynucleotide sequence shown in SEQ ID NO: 24.

[0064] MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTN

[0065] TSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKED

[0066] CEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLSGGGGSYPYDVPDYA (SEQ ID NO: 23).

[0067] ATGGCTCAGCGGATGACAACACAGCTGCTGCTCCTTCTAGTGTGGGTGGCTGTAGTAGGGGAGGCTCAG

[0068] ACAAGGATTGCATGGGCCAGGACTGAGCTTCTCAATGTCTGCATGAACGCCAAGCACCACAAGGAAAG

[0069] CCAGGCCCCGAGGACAAGTTGCATGAGCAGTGTCGACCCTGGAGGAAGAATGCCTGCTGTTCTACCAAC

[0070] ACCAGCCAGGAAGCCCATAAGGATGTTTCCTACCTATATAGATTCAACTGGAACCACTGTGGAGAGATG

[0071] GCACCTGCCTGCAAACGGCATTTCATCCAGGACACCTGCCTCTACGAGTGCTCCCCCAACTTGGGGCCC

[0072] TGGATCCAGCAGGTGGATCAGAGCTGGCGCAAAGAGCGGGTACTGAACGTGCCCCTGTGCAAAGAGGAC

[0073] TGTGAGCAATGGTGGGAAGATTGTCGCACCTCCTACACCTGCAAGAGCAACTGGCACAAGGGCTGGAAC

[0074] TGGACTTCAGGGTTTAACAAGTGCGCAGTGGGAGCTGCCTGCCAACCTTTCCATTTCTACTTCCCCACA

[0075] CCCACTGTTCTGTGCAATGAAATCTGGACTCACTCCTACAAGGTCAGCAACTACAGCCGAGGGAGTGGC

[0076] CGCTGCATCCAGATGTGTTTCGACCCAGCCCAGGGCAACCCCAATGAGGAGGTGGCGAGGTTCTATGCT

[0077] GCAGCCATGAGTGGGGCTGGGCCCTGGGCAGCCTGGCCTTTCCTGCTTAGCCTGGCCCTAATGCTGCTGTGGCTGCTCAGCGGGGGTGGAGGCTCTTATCCTTACGACGTGCCTGACTACGCCTAA (SEQ ID NO: 24).

[0078] II. Anti-FOLR1 antibody

[0079] In one aspect, the present invention provides an anti-FOLR1 antibody or an antigen-binding fragment thereof.

[0080] In some embodiments, the anti-FOLR1 antibody is a monoclonal antibody.

[0081] In other embodiments, the monoclonal antibody includes murine antibodies and murine chimeric antibodies.

[0082] In some embodiments, the anti-FOLR1 antibody of the present invention is a murine chimeric antibody.

[0083] 1. CDR

[0084] Through in-depth research, the inventors obtained a murine antibody against folate receptor 1 (i.e., Folate Receptor α or FOLR1) using the hybridoma method, which can specifically bind to FOLR1.

[0085] In one exemplary embodiment, the anti-FOLR1 murine antibody comprises heavy chain variable regions (VH) of VHCDR1, VHCDR2, and VHCDR2 having amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and light chain variable regions (VL) of VLCDR1, VLCDR2, and VLCDR2 having amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, and is exemplaryly named 5G12.

[0086] In one exemplary embodiment, the anti-FOLR1 murine antibody comprises heavy chain variable regions (VH) of VHCDR1, VHCDR2, and VHCDR2 having amino acid sequences as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and light chain variable regions (VL) of VLCDR1, VLCDR2, and VLCDR2 having amino acid sequences as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, exemplarily named 14H4.

[0087] Exemplary VHCDR and VLCDR of antiFOLR1 murine antibodies are shown in Table 1 and Table 2, respectively.

[0088] Table 1. Amino acid sequence and SEQ ID NO of VHCDR of anti-FOLR1 murine antibody:

[0089]

[0090] Table 2. Amino acid sequence and SEQ ID NO: of VLCDR of antiFOLR1 murine antibody.

[0091]

[0092] 2. Variable area

[0093] In one exemplary embodiment, the anti-FOLR1 murine antibody comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:13 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:14, and is exemplary named 5G12.

[0094] In one exemplary embodiment, the anti-FOLR1 murine antibody comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:15 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:16, exemplarily named 14H4.

[0095] Exemplary murine antibodies contain heavy chain variable regions (VH) and light chain variable regions (VL) as shown in Table 3.

[0096] Table 3. Mouse anti-variable region sequence binding to FOLR1

[0097]

[0098] In some embodiments, the murine chimeric anti-FOLR1 antibody of the present invention contains an amino acid sequence of the sequence shown in SEQ ID NO:13 or 15 in the heavy chain variable region.

[0099] In some embodiments, the murine chimeric anti-FOLR1 antibody of the present invention contains a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 14 or 16.

[0100] In some exemplary embodiments, the murine chimeric anti-FOLR1 antibody of the present invention contains amino acid sequences of the sequences shown in SEQ ID NO:13 and 14, respectively, such as the chimeric antibody named ch5G12.

[0101] In some exemplary embodiments, the murine chimeric anti-FOLR1 antibody of the present invention contains amino acid sequences of the sequences shown in SEQ ID NO:15 and 16, respectively, such as the chimeric antibody named ch14H4.

[0102] Preferably, the amino acids corresponding to the sites listed in Table 3 in the heavy chain variable region / light chain variable region of the chimeric antibody are conserved.

[0103] 3. Constant Region

[0104] In some embodiments, the C-terminus of the anti-FOLR1 antibody VH and VL are respectively connected to the N-terminus of the heavy chain constant region (CH) and the light chain constant region (CL) to form a full-length heavy chain (HC) and a full-length light chain (LC).

[0105] In another preferred embodiment, the constant region of the anti-FOLR1 antibody is derived from the heavy chain constant region of IgG.

[0106] In another preferred embodiment, the constant region of the anti-FOLR1 antibody is derived from the heavy chain constant region of hIgG.

[0107] In another preferred embodiment, the heavy chain constant region source of the anti-FOLR1 antibody is one of the heavy chain constant regions of hIgG1, hIgG2, hIgG3, or hIgG4.

[0108] In another preferred embodiment, the constant region of the anti-FOLR1 antibody is the heavy chain constant region of hIgG1.

[0109] In another preferred embodiment, the light chain constant region of the anti-FOLR1 antibody is one of the hκ and hλ type light chain constant regions.

[0110] In another preferred embodiment, the constant region of the anti-FOLR1 antibody is the hκ type light chain constant region.

[0111] In some common embodiments in the art, modifications to the constant regions compatible with the present invention may include the addition, deletion, or substitution of one or more amino acids in one or more regions.

[0112] Those skilled in the art will understand that the modified anti-FOLR1 antibody of the present invention may be an antibody (e.g., a full-length antibody or its immunoreactive fragment) containing at least a small portion of one or more constant regions that have been deleted or otherwise altered to provide desired biochemical properties, such as increased tumor locality or reduced serum half-life when compared with antibodies containing native or unaltered constant regions of approximately the same immunogenicity.

[0113] In addition, in some common embodiments in this field, the modified chimeric anti-FOLR1 antibody may include more alterations or modifications to the three portions (CH1, CH2, or CH3) of the heavy chain constant region and / or to the light chain constant region (CL).

[0114] In some commonly used chimeric anti-FOLR1 antibody embodiments in the art, it is conceivable that one or more regions are modified constant regions that have been partially or completely deleted. In some commonly used embodiments in the art, the modified antibody will comprise a construct or variant with the deleted region, wherein the entire CH2 region has been removed (ΔCH2 construct).

[0115] In some commonly used chimeric anti-FOLR1 antibody embodiments in the art, the omitted constant region is replaced by a short amino acid interval (e.g., 10 residues), which provides some of the molecular flexibility typically given by the absence of the constant region.

[0116] In addition to their conformation, constant regions are known in the art to mediate several effector functions. For example, the C1 component of complement binds to antibodies to activate the complement system. Complement activation is important for opsonization and cytopathogenic lysis. Complement activation also stimulates inflammatory responses and can be associated with autoimmune hypersensitivity. Furthermore, antibodies bind to cells via Fc regions to bind to Fc receptors (FcRs) on the cell surface at Fc receptor sites on the antibody Fc region. Several Fc receptors are specific to different classes of antibodies, including IgG (γ receptor), IgE (ε receptor), IgA (α receptor), and IgM (μ receptor). Antibody binding to Fc receptors on the cell surface triggers several important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[0117] In some embodiments of the art, constant region modifications of chimeric FOLR1-binding antibodies can provide altered effector functions, such as enhancement or weakening, which in turn affect the biological function of the added antibody. For example, deletion or deactivation of the constant region (through point mutation or other methods) can reduce the binding of circulating modified antibody to the Fc receptor, thereby increasing tumor localization. In other examples, constant region modifications consistent with the present invention may weaken complement binding and thus reduce serum half-life. In some embodiments of the art, constant region modifications can be used to remove disulfide bonds or oligosaccharide motifs, which result in increased localization due to increased antigen specificity or antibody flexibility. Similarly, constant region modifications consistent with the present invention can be readily fabricated using well-known biochemical or molecular engineering techniques, within the understanding of those skilled in the art.

[0118] 4. Full-length heavy chain and full-length light chain

[0119] In some embodiments, the antiFOLR1 antibody of the present invention is a chimeric antibody.

[0120] In some embodiments, the chimeric anti-FOLR1 antibody of the present invention comprises a full-length heavy chain (HC) and a full-length light chain (LC).

[0121] The heavy and light chains of exemplary chimeric anti-FOLR1 antibodies are shown in Table 4.

[0122] Table 4. Heavy chain (HC) and light chain (LC) sequences of chimeric antibodies binding to FOLR1

[0123]

[0124]

[0125] In some embodiments, the HC and LC of the chimeric anti-FOLR1 antibody of the present invention comprise the amino acid sequences shown in SEQ ID NO:17 and SEQ ID NO:18, respectively, and the exemplary antibody is named ch5G12.

[0126] In some embodiments, the HC and LC of the chimeric anti-FOLR1 antibody of the present invention respectively contain the amino acid sequences shown in SEQ ID NO:19 and SEQ ID NO:20, and the exemplary antibody is named ch14H4.

[0127] III. Functionally Conserved Variants

[0128] In some embodiments, the murine chimeric anti-FOLR1 antibody of the present invention also includes functionally conserved variants of VHCDR1, VHCDR2, and VHCDR3, each having an amino acid sequence having at least 95% identity with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0129] In some embodiments, the murine chimeric anti-FOLR1 antibody of the present invention also includes functionally conserved variants of VLCDR1, VLCDR2, and VLCDR3, each having an amino acid sequence having at least 95% identity with the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0130] In some embodiments, the anti-FOLR1 murine chimeric antibody of the present invention also includes functionally conserved variants of anti-FOLR1 antibodies comprising VHCDR1, VHCDR2, and VHCDR3 having amino acid sequences that are at least 95% identical to the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

[0131] In some embodiments, the antiFOLR1 murine chimeric antibody of the present invention also includes functionally conserved variants of antiFOLR1 antibodies comprising VLCDR1, VLCDR2, and VLCDR3 having amino acid sequences that are at least 95% identical to the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively.

[0132] In some embodiments, the anti-FOLR1 chimeric antibody of the present invention comprises a derived sequence of any one of the above-mentioned amino acid sequences, which has been added, deleted, modified and / or substituted at least one (e.g., 1-8, 1-5, or 1-3) amino acids and is capable of retaining the ability to specifically bind to FOLR1, or an amino acid sequence with homology or sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0133] The antigen-binding properties of an antibody are typically determined by three complementarity-determining regions (CDRs). In the variable region, the CDRs and FRs are arranged in an orderly fashion, with the FRs not directly participating in the binding reaction. These CDRs form a ring structure, spatially close to each other through β-sheets formed by the FRs, constituting the antigen-binding site of the antibody. The CDR region is the sequence of the protein that elicits an immunologically specific response.

[0134] In some embodiments, the murine chimeric anti-FOLR1 antibody of the present invention contains an amino acid sequence that is 85% or more (e.g., 86%, 87%, 88%, 90%, 91%, 92%, 93%, 95%, 97%, or 99% or more) identical to the sequence shown in SEQ ID NO:13, and has the same function as the antibody described in the embodiments of the present invention.

[0135] In some embodiments, the mouse-derived chimeric anti-FOLR1 antibody of the present invention contains an amino acid sequence that is 85% or more (e.g., 86%, 87%, 88%, 90%, 91%, 92%, 93%, 95%, 97%, or 99% or more) identical to the sequence shown in SEQ ID NO:15, and has the same function as the antibody described in the embodiments of the present invention.

[0136] In some embodiments, the murine chimeric anti-FOLR1 antibody of the present invention contains an amino acid sequence that is 85% or more (e.g., 86%, 87%, 88%, 90%, 91%, 92%, 93%, 95%, 97%, or 99% or more) identical to the sequence shown in SEQ ID NO:14, and has the same function as the antibody described in the embodiments of the present invention.

[0137] In some embodiments, the murine chimeric anti-FOLR1 antibody of the present invention contains an amino acid sequence that is 85% or more (e.g., 86%, 87%, 88%, 90%, 91%, 92%, 93%, 95%, 97%, or 99% or more) identical to the sequence shown in SEQ ID NO:16, and has the same function as the antibody described in the embodiments of the present invention.

[0138] It should be understood that modified antibodies can contain any type of variant region that enables the antibody to bind to human FOLR1. In this respect, the variant region can be contained in or derived from any type of mammal that can induce a humoral response and generate immunoglobulins against FOLR1. Therefore, the variant region of a modified antibody can be, for example, human, rodent, or non-human primate (e.g., cynomolgus monkey, rhesus macaque, etc.).

[0139] Using the aforementioned CDR region sequence as the starting sequence, ordinary technicians in the antibody field can perform various sequence modifications on the CDR region and the framework region.

[0140] In some well-known embodiments in the field of antibody engineering, both the variant and constant regions of the modified immunoglobulin can be human. For example, the variant regions of compatible antibodies (often derived from non-human sources) can be designed or specifically modified to improve binding properties or reduce the immunogenicity of the molecule. In this regard, the variant regions useful in this invention can be humanized or otherwise altered by inserting replacement amino acid sequences.

[0141] For example, variations in the heavy and light chains can be altered by at least partial substitution of one or more CDRs, and, if necessary, by partial backbone substitution and sequence changes. CDRs can be derived from antibodies of the same class or even subclass as those derived from the backbone region, or from antibodies of different classes, or from antibodies of different species. It may not be necessary to replace all CDRs of the receptor antibody with all CDRs from the variant region to convert the antigen-binding capacity of the receptor variant region into the antigen-binding capacity of the donor CDRs; it may only be necessary to transfer the residues necessary to maintain the activity of the antigen-binding site. This will be readily understood and mastered by those skilled in the art, whether through routine experiments or trial-and-error testing, to obtain functional antibodies with reduced immunogenicity.

[0142] In some implementations, the framework region (FR) in the monoclonal antibody, as described above, can be or replaced with a humanized FR, thereby obtaining, for example, only the CDR is retained, while the others are humanized variable regions of a humanized structure.

[0143] Based on the VH and VL encoding nucleic acids of the chimeric anti-FOLR1 antibody described above in this invention, humanized anti-FOLR1 antibody VH and VL can be further obtained through humanization modification techniques such as CDR transplantation and reverse mutation, and then further combined to obtain humanized anti-FOLR1 antibody.

[0144] In some embodiments, the present invention provides a functionally conserved variant of the chimeric anti-FOLR1 antibody described above, which has the same specific FOLR1 binding function as the antibody described in the embodiments of the present invention.

[0145] In some embodiments, in variants of the chimeric anti-FOLR1 antibody of the present invention, the heavy chain comprises an amino acid sequence having more than 85% (e.g., more than 88%, 90%, 93%, 95%, 97%, or 99%) the same identity as the sequence shown in SEQ ID NO:17 or 19, and has the same function as the antibody described in the embodiments of the present invention.

[0146] In some embodiments, variants of the chimeric anti-FOLR1 antibody of the present invention have an amino acid sequence that is 85% or more (e.g., 88%, 90%, 93%, 95%, 97%, or 99% or more) identical to the sequence shown in SEQ ID NO: 18 or 20. Furthermore, they possess the same function as the antibodies described in the embodiments of the present invention.

[0147] Among them, any of the above amino acid sequences also includes a derivative sequence which has been optionally added, deleted, modified and / or substituted at least one (e.g., 1-8, 1-5 or 1-3) amino acids and retains the ability to specifically bind to FOLR1.

[0148] IV. Antigen-binding fragments

[0149] In some embodiments, the present invention provides an antigen-binding fragment that specifically binds to FOLR1.

[0150] "Antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding affinity to the target. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. The antigen-binding fragments of the present invention can be recombinant peptides, natural peptides, or synthetic peptides comprising an antibody against human FOLR1 or a fragment thereof. Those skilled in the art will recognize that some amino acid sequences of the present invention can be altered without significantly affecting the structure or function of the protein. Therefore, the present invention further includes variants of antigen-binding fragments that exhibit substantial activity against human folate receptor proteins or that comprise regions of antibodies or fragments thereof. Such mutations include deletions, insertions, inversions, duplications, and type substitutions.

[0151] This article includes not only complete monoclonal antibodies, but also antibody fragments (antigen-binding fragments) with immune activity, such as Fab, Fab', F(ab')2, Fd, or scFv, disulfide-linked Fv, V-NAR domain, F(ab')3, tetraantibodies, triantibodies, bispecific antibodies, single-domain antibodies, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc, etc.

[0152] The “Fab fragment” is a heterodimer formed by the heavy chain Fd and the intact light chain linked by disulfide bonds, containing only one antigen-binding site. The heavy chain Fd refers to approximately half of the H chain portion of Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0153] The “Fab′ fragment” contains a light chain and a heavy chain portion containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby enabling the formation of interchain disulfide bonds between the two heavy chains of the two Fab′ fragments to form the F(ab′)2 molecule.

[0154] The “F(ab′)2 segment” contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by disulfide bonds between the two heavy chains.

[0155] "Single-chain antibody (ScFv)" refers to an antibody that uses an appropriate oligonucleotide linker to connect the light chain and heavy chain variable region genes, enabling the expression of a single polypeptide chain. The polypeptide chain can spontaneously fold into its native conformation, maintaining the specificity and affinity of the Fv.

[0156] "Nanobodies (single-domain antibodies)" refer to antibodies containing only the VH fragment obtained by expressing the V region of the antibody heavy chain through genetic engineering methods. The ability of single-domain antibodies to bind to antigens and their stability are basically the same as those of complete antibodies.

[0157] This invention further includes variants and equivalents that are substantially homologous to the chimeric, humanized, and human antibodies or antibody fragments thereof described herein. These may include, for example, conserved substitution mutations, i.e., the substitution of one or more amino acids with similar amino acids. For example, conserved substitution means the substitution of an amino acid with another amino acid in the same general class; for example, an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. The intent of conserved amino acid substitution is well known in the art.

[0158] In another preferred embodiment, the antibody or its antigen-binding fragment may include a monoclonal antibody, a bivalent antibody, and / or a multivalent antibody.

[0159] Recombinant protein

[0160] In a second aspect of the invention, a recombinant protein is provided, said recombinant protein having:

[0161] (i) an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and

[0162] (ii) Optional tag sequences to assist in expression and / or purification.

[0163] In another preferred embodiment, the tag sequence includes Fc tag, HA tag, GGGS sequence, FLAG tag, Myc tag, 6His tag, or a combination thereof.

[0164] In another preferred embodiment, the recombinant protein specifically binds to FOLR1.

[0165] In some embodiments in this art, the recombinant protein (or polypeptide) includes a fusion protein.

[0166] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0167] In another preferred embodiment, the bivalent antibody may also be a bispecific antibody.

[0168] In another preferred embodiment, the multivalent antibody may also be a multispecific antibody.

[0169] V. Biomaterials

[0170] 1. Nucleic acid

[0171] In a third aspect of the invention, a nucleic acid molecule is provided, the nucleic acid molecule encoding a protein selected from the group consisting of: an anti-FOLR1 antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, or a recombinant protein as described in the second aspect of the invention.

[0172] In another preferred embodiment, the nucleic acid of the present invention may be RNA, DNA or cDNA.

[0173] In another preferred embodiment, the nucleic acid molecule comprises: a polynucleotide sequence encoding VH as shown in SEQ ID NO:13 and a polynucleotide sequence encoding VL as shown in SEQ ID NO:14, wherein the exemplary antiFOLR1 antibody encoded is a chimeric antibody ch5G12; in another preferred embodiment, the nucleic acid molecule comprises: a polynucleotide sequence encoding VH as shown in SEQ ID NO:15 and a polynucleotide sequence encoding VL as shown in SEQ ID NO:16, wherein the exemplary antiFOLR1 antibody encoded is a chimeric antibody ch14H4.

[0174] In another preferred embodiment, the nucleic acid molecule comprises: a polynucleotide sequence having at least 80% sequence homology to the polynucleotide sequence encoding VH as shown in SEQ ID NO:13 and a polynucleotide sequence having at least 80% sequence homology to the polynucleotide sequence encoding VL as shown in SEQ ID NO:14; in another preferred embodiment, the nucleic acid molecule comprises a polynucleotide sequence having at least 80% sequence homology to the polynucleotide sequence encoding VH as shown in SEQ ID NO:15 and a polynucleotide sequence having at least 80% sequence homology to the polynucleotide sequence encoding VL as shown in SEQ ID NO:16.

[0175] The sequences of these nucleic acid molecules can be obtained using conventional techniques, such as PCR amplification to fuse the coding sequences of VH and CH together to form a full-length heavy chain (HC) coding nucleic acid, or PCR amplification to fuse the coding sequences of VL and CL together to form a full-length light chain (LC) coding nucleic acid.

[0176] The sequences of these nucleic acid molecules can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0177] 2. Expression vehicle

[0178] In a fourth aspect of the invention, an expression vector is provided, the expression vector containing the nucleotide molecule described in the third aspect of the invention.

[0179] The vectors described herein are well known to those skilled in the art and include, but are not limited to, DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector includes viral vectors, such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0180] In another preferred embodiment, the expression vector includes, but is not limited to, pTomo lentiviral vector or plasmid vectors such as plenti, pLVTH, and pHr.

[0181] In another preferred embodiment, the expression vector is a pHr plasmid vector.

[0182] In another preferred embodiment, the expression vector further includes expression elements selected from the group consisting of promoters, transcriptional enhancement elements (WPREs), long terminal repeat sequences (LTRs), and signal peptides.

[0185] 3. Host cells

[0186] In a fifth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the fourth aspect of the invention, or having the nucleotide molecules described in the third aspect of the invention integrated into its genome.

[0187] "Cell" and "cell line" are used interchangeably, and all such names include their descendants.

[0188] In some embodiments, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0189] In some embodiments, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0190] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

[0191] Mammalian cell lines are commonly used as host cells for expressing eukaryotic cell-derived peptides. Preferred mammalian cell lines are many commercially available, indefinitely proliferating cell lines. These include, but are not limited to, Chinese hamster ovary (CHO) cells, Vero cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (such as Hep G2), and many other cell lines. They provide post-translational modifications for protein molecules, including proper folding, proper disulfide bond formation, and glycosylation at the correct sites.

[0192] In some embodiments, the host cell is, for example, a CHO cell or a COS cell.

[0193] In some embodiments, the host cell is a CHO cell.

[0194] In some embodiments, the host cell is an Expi CHO-S cell.

[0195] VI. Antibody Preparation

[0196] In a sixth aspect, the present invention provides a method for preparing a chimeric anti-FOLR1 antibody.

[0197] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector, then transfected into host cells, and the transfected host cells are cultured under specific conditions to express the antibodies of the present invention.

[0198] Variants of the antibodies described in this invention, exhibiting improved affinity and / or titer, can be obtained using methods known in the art and are included within the scope of this invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can also be used to improve translation efficiency in expression systems used to generate antibodies. Furthermore, polynucleotides comprising sequences whose antibody specificity or neutralizing activity is optimized by applying directed evolution to any nucleic acid sequence of this invention are also within the scope of this invention.

[0199] Any method suitable for producing monoclonal antibodies can be used to produce the FOLR1 antibody of this invention. For example, animals can be immunized with recombinant or naturally occurring FOLR1 protein or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, or one or more routes.

[0200] The murine monoclonal antibody of the present invention can be prepared by a variety of techniques, such as hybridoma technology, recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al. Nature 352: 624-628, 1991, or Marks et al. J. Mol. Biol. 222: 581-597, 1991).

[0201] For example, the murine anti-FOLR1 monoclonal antibody of the present invention can be prepared using the following hybridoma technique. First, mice, such as BalB / C mice or other suitable host animals, are immunized with a human FOLR1 protein immunogen fused with a 6×His tag at the C-terminus (with adjuvants added if necessary). Following immunization, the animals will produce lymphocytes that secrete antibodies specifically binding to the immunogen. Alternatively, lymphocytes can be obtained through in vitro immunization. After the serum titer of the immunized mice reaches the fusion standard, the spleen is collected to extract the target lymphocytes, which are then fused with myeloma cells, such as SP20 cells, by electrofusion to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The hybridoma cells prepared above can be seeded into a suitable culture medium for growth, wherein the culture medium containing hybridoma cells is used to detect the production of monoclonal antibodies against the specific antigen. Methods for determining the binding specificity of the monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). For example, the affinity of monoclonal antibodies can be determined using the BLI assay. Once the specificity, affinity, and reactivity of the hybridoma-produced antibody are determined, the target cell line can be subcloned using the standard limiting dilution method described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). A suitable culture medium may be IMDM medium, etc. Alternatively, hybridoma cells can also grow in animals in the form of ascites tumors. Further limiting dilution methods can be used to isolate monoclonal antibodies with FOLR1 binding activity, which contain VHCDRs and VLCDRs of the exemplary anti-FOLR1 antibody of the first aspect of this invention.

[0202] Traditional immunoglobulin purification methods, such as protein A agarose gel chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, can be used to separate monoclonal antibodies secreted by subclonal cells from cell culture medium, ascites, or serum.

[0203] Monoclonal antibodies can also be obtained through genetic engineering recombination technology. By using nucleic acid primers that specifically bind to the heavy and light chain genes of monoclonal antibodies for PCR amplification, DNA molecules encoding the heavy and light chain genes of monoclonal antibodies can be isolated from hybridoma cells.

[0204] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This usually involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods, such as VH, VL, HC, and LC.

[0205] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0206] Currently, the nucleic acid sequence encoding the monoclonal antibody (or a fragment thereof, or a derivative thereof) described herein can be obtained entirely through chemical synthesis. This sequence can then be introduced into various existing molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence described herein through chemical synthesis.

[0207] The obtained DNA molecule is inserted into an expression vector, then transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins), and cultured under appropriate conditions to obtain recombinantly expressed target antibodies.

[0208] In some implementations, the obtained light and heavy chain expression vectors are co-transfected into host mammalian cells, such as Expi CHO-S cells.

[0209] There are many methods for transforming host cells using expression vectors, and the transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene (1,5-dimethyl-1,5-diazadecylmethylene polymethyl bromide)-mediated transfection, protoplast fusion, electroporation, liposome-mediated transfection, and direct microinjection of DNA into the cell nucleus.

[0210] In some implementations, a preferred method is liposome-mediated transfection, etc.

[0211] In some implementations, a preferred method is to use a cationic transfection reagent.

[0212] In some implementations, electroporation is a preferred method.

[0213] The transformed host cells were cultured under conditions suitable for the expression of the monoclonal antibody described in this paper. The monoclonal antibody was then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well-known to those skilled in the art.

[0214] Antibodies can be purified using known techniques, such as affinity chromatography using protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and purified by immunoaffinity chromatography to purify the immunospecific antibody.

[0215] In some embodiments, as described above, the V region of the monoclonal antibody can be linked to the C region of the human antibody, and expression in a suitable host cell can yield a human-mouse chimeric antibody.

[0216] Based on the VH and VL encoding nucleic acids of the chimeric anti-FOLR1 antibody of the present invention, those skilled in the art of antibodies can further design humanized anti-FOLR1 antibody encoding nucleic acids through humanization techniques such as CDR transplantation and reverse mutation.

[0217] In some implementations, the framework region (FR) in the monoclonal antibody, as described above, may be or replaced with a humanized FR, for example, retaining only the CDR while the others are humanized structures.

[0218] The chimeric antibody of the present invention can be prepared according to the sequence of the mouse monoclonal antibody prepared above. The DNA encoding the heavy chain variable region and the light chain variable region can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to produce an immunoglobulin sequence containing non-mouse (e.g., human) components.

[0219] To prepare chimeric antibodies, variable regions of mouse immunoglobulins can be ligated to constant regions of human immunoglobulins using methods known in the art. For example, DNA encoding VH can be operatively ligated to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene, or DNA encoding VL can be operatively ligated to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and the Fab light chain gene). The sequences of the human heavy chain constant region and light chain constant region genes are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification.

[0220] The heavy chain constant region can be the human IgG constant region. The heavy chain constant region can be hIgG1 (e.g., uniprot IDP01857), hIgG2 (e.g., uniprot ID P01859), hIgG3 (e.g., uniprot ID P01860), or hIgG4 (e.g., uniprot ID P01861).

[0221] In a preferred embodiment, the heavy chain constant region is typically an IgG1 or IgG4 constant region.

[0222] In one exemplary embodiment, the heavy chain constant region is the human IgG1 constant region.

[0223] The light chain constant region can be either κ or λ constant region, but is usually preferred to be hκ constant region.

[0224] VII. Function and activity of chimeric anti-FOLR1 antibodies

[0225] The chimeric anti-FOLR1 antibody provided by this invention can bind specifically to human FOLR1 with high efficiency and high affinity.

[0226] The chimeric antibody of the present invention can bind to the recombinantly expressed FOLR1 protein.

[0227] In some embodiments, the EC50 of the chimeric antibodies ch5G12 and ch14H4 of the present invention binding to recombinant human FOLR1 protein, as determined by ELISA, is about 0.0960 (nM), preferably about 0.0820 (nM), more preferably about 0.0650 (nM), or most preferably about 0.050 (nM), which is close to or better than the reference antibody Mirvetuximab.

[0228] The chimeric antibody of the present invention can bind to the FOLR1 protein on the cell surface.

[0229] In some embodiments, the chimeric antibody of the present invention can bind to the FOLR1 protein on the surface of CAL51 cells.

[0230] In some embodiments, the EC50 of the chimeric antibodies ch5G12 and ch14H4 of the present invention binding to the hFOLR1 protein on the surface of CAL51 cells, as determined by FACS, is about no more than 0.960 (nM), or preferably about no more than 0.820 (nM), or more preferably about no more than 0.650 (nM), or more preferably about no more than 0.50 (nM), or even more preferably about no more than 0.41 (nM), which is close to that of the reference antibody Mirvetuximab.

[0231] In some embodiments, the chimeric antibody of the present invention can bind to the FOLR1 protein on the surface of CHO-hFOLR1 cells.

[0232] In some embodiments, the EC50 of the chimeric antibodies ch5G12 and ch14H4 of the present invention binding to the FOLR1 protein on the surface of CHO-hFOL1 cells, as determined by FACS, is about 1.32 (nM), preferably about 1.48 (nM), more preferably about 1.65 (nM), more preferably about 1.78 (nM), or even more preferably about 0.196 (nM), which is close to that of the reference antibody Mirvetuximab.

[0233] The chimeric antibodies ch5G12 and ch14H of the present invention can bind to the recombinantly expressed cynomolgus monkey FOLR1 protein.

[0234] In some embodiments, the EC50 of the chimeric antibodies ch5G12 and ch14H4 of the present invention binding to recombinant cynomolgus monkey FOLR1 protein, as measured by ELISA, is approximately no higher than 0.960 (nM), or preferably no higher than 0.820 (nM), or more preferably no higher than 0.650 (nM), or more preferably no higher than 0.50 (nM), or more preferably no higher than 0.41 (nM), or more preferably no higher than 0.25 (nM), or more preferably no higher than 0.124 (nM), or more preferably no higher than 0.099 (nM), or more preferably no higher than 0.076 (nM), or more preferably no higher than 0.058 (nM), or most preferably no higher than 0.039 (nM), which is close to the reference antibody Mirvetuximab.

[0235] The chimeric antibody of this invention does not bind to other proteins in the same family as the FORL1 protein.

[0236] In some embodiments, ELISA showed that the chimeric antibodies ch5G12 and ch14H4 of the present invention did not bind to the recombinant proteins FOLR2, FOLR3 and FOLR4 of the FOLR1 family.

[0237] In some embodiments, the equilibrium dissociation constant (KD) of the chimeric antibodies ch5G12 and ch14H4 binding to the recombinant human FOLR1 protein is determined using biofilm thin-layer interferometry (BLI).

[0238] In some embodiments, the equilibrium dissociation constant (KD) of the chimeric antibodies ch5G12 and ch14H4 of the present invention binding to recombinant human FOLR1 protein, as determined by the BLI method, is approximately no higher than 1.5E-08, or approximately no higher than 3.5E-08, or approximately no higher than 5.6E-08, or approximately no higher than 8.6E-08, or approximately no higher than 1.02E-09, or approximately no higher than 2.6E-09, or approximately no higher than 3.2E-09, or approximately no higher than 4.8E-09, or approximately no higher than 6.9E-09.

[0239] The FOLR1 chimeric antibody of the present invention has the activity of promoting tumor cell endocytosis.

[0240] In some embodiments, flow cytometry analysis showed that the average fluorescence signal intensity of PE in tumor cells after treatment with the FOLR1 chimeric antibody of the present invention was higher than 100,000, which was higher than the average fluorescence signal intensity of PE in cells after treatment with the reference antibody Mirvetuximab.

[0241] In some embodiments, the FOLR1 chimeric antibody of the present invention exhibits stronger endocytic activity than the reference antibody Mirvetuximab.

[0242] VIII. Uses of chimeric anti-FOLR1 antibodies

[0243] The chimeric antibodies provided in this article can be used to develop humanized antibodies for the treatment of FORL1-positive tumors, or bispecific antibodies, or for use as CAR-T or ADC.

[0244] In some implementations, non-human monoclonal antibodies can be humanized through the following pathways: (1) homologous substitution, replacing the non-human corresponding part with a human FR; (2) surface remodeling, remodeling the amino acid residues on the surface of the non-human CDR and FR to make them resemble the outline of the human antibody CDR or the FR pattern; (3) compensatory changes, modifying the amino acid residues at key positions to compensate for CDR transplantation; (4) conserved positioning, humanizing the monoclonal antibody with the conserved FR sequence as a template, but retaining the key amino acid residues of the variable region of the non-human monoclonal antibody.

[0245] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art. Alternatively, transgenic animals that do not produce endogenous immunoglobulins after immunization and can generate a complete human antibody library can be used.

[0246] In some embodiments, the bispecific or multispecific antibody includes an antigen-binding arm that specifically binds to antigen targets other than FOLR1.

[0247] In some embodiments, the other antigens include, but are not limited to, immune checkpoint drugs, which include: PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR.

[0248] In some embodiments, the bispecific antibody comprises an antigen-binding arm that specifically binds to FOLR1 and an antigen-binding arm that specifically binds to the PD-1 target.

[0249] In some embodiments, the bispecific antibody comprises an antigen-binding arm that specifically binds to FOLR1 and an antigen-binding arm that specifically binds to the CTLA-4 target.

[0250] In some embodiments, the bispecific antibody comprises an antigen-binding arm that specifically binds to FOLR1 and an antigen-binding arm that specifically binds to the TIGIT target.

[0251] In some embodiments, the preparation method of the bispecific antibody or multispecific antibody includes constructing recombinant expression vectors of the FOLR1 binding arm and other specific binding arms by genetic engineering methods, then transiently transfecting host cells to generate recombinant expression host cells, and culturing and purifying the target bispecific antibody or multispecific antibody.

[0252] In some embodiments, the use of the aforementioned anti-FOLR1 antibody in the preparation of an immunoconjugate is provided, wherein the immunoconjugate comprises:

[0253] (a) An antibody portion, said antibody portion comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and

[0254] (b) A conjugate selected from the group consisting of: detectable markers, therapeutic agents, toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0255] In another preferred embodiment, the (a) portion is coupled to the coupling portion by a chemical bond or a connector.

[0256] In another preferred embodiment, the coupling portion may contain at least one therapeutic agent.

[0257] In another preferred embodiment, the coupling portion is a therapeutic agent.

[0258] In another preferred embodiment, the therapeutic agent is an immune checkpoint drug, the immune checkpoint including: PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR.

[0259] In another preferred embodiment, the therapeutic agent is a PD-1 antibody and / or a PD-L1 antibody.

[0260] In another preferred embodiment, the immune checkpoint drug is an inhibitor or activator of the immune checkpoint.

[0261] In another preferred embodiment, the therapeutic agent is a cytotoxic drug.

[0262] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0263] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, docarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0264] In another preferred embodiment, the coupling portion is a toxin.

[0265] In another preferred embodiment, the immunoconjugate contains: a multivalent (e.g., bivalent) antibody or an antigen-binding fragment thereof as described in the first aspect of the invention.

[0266] In another preferred embodiment, the term "multivalent" refers to the inclusion of multiple repeating identical or different antibodies or antigen-binding fragments thereof as described in the first aspect of the invention in the amino acid sequence of the immunoconjugate.

[0267] In some embodiments, the coupling is achieved through enzymatic coupling, chemical coupling, fusion, or other methods. The adapter can be a "cleavable adapter" that promotes the release of the coupling portion into the cell, such as an acid-labile adapter, a peptidase-sensitive adapter, a light-labile adapter, a dimethyl adapter, and a disulfide-containing adapter.

[0268] IX. Composition

[0269] In a ninth aspect of the invention, the use of the antiFOLR1 chimeric antibody of the invention in the preparation of pharmaceutical compositions for treating cancer, wherein the pharmaceutical compositions inhibit tumor growth and treat cancer in human patients are provided.

[0270] In a tenth aspect of the invention, a pharmaceutical composition comprising the anti-FOLR1 chimeric antibody of the present invention is provided, the pharmaceutical composition being used in human patients to inhibit tumor growth and treat cancer, the pharmaceutical composition comprising:

[0271] (i) an active ingredient selected from the group consisting of: antibodies or antigen-binding fragments thereof as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, engineered immune cells as described in the seventh aspect of the invention, or immunoconjugates as described in the ninth aspect of the invention, or combinations thereof; and

[0272] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0273] "Pharmaceutical acceptable" means that when the molecular bulk and the composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions. As used herein, "pharmaceutical acceptable carriers" should be compatible with the active substance described herein, meaning they can be miscible with it without significantly reducing the efficacy of the pharmaceutical composition under normal circumstances. These carriers are well known to those skilled in the art.

[0274] These carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof. Additionally, these carriers may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.

[0275] These substances are typically formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH usually around 5-8, preferably around 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0276] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections and lyophilized preparations.

[0277] In another preferred embodiment, the pharmaceutical composition may include 0.01 to 99.99% of an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, or a recombinant protein as described in the second aspect of the invention, or an immunoconjugate as described in the ninth aspect of the invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0278] Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions should preferably be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0279] In some embodiments, when using the pharmaceutical composition, a safe and effective amount of the immunoconjugate may be administered to mammals, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. The specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0280] In another preferred embodiment, the pharmaceutical composition further includes a second active ingredient.

[0281] In another preferred embodiment, the pharmaceutical composition comprises:

[0282] (a) A first active ingredient, such as the antiFOLR1 antibody or its antigen-binding fragment as described in the first aspect of the present invention;

[0283] (b) The second active ingredient, and

[0284] (c) Pharmaceutically acceptable carriers, diluents or excipients.

[0285] In another preferred embodiment, the second active ingredient is an antibody that binds to targets other than FOLR1.

[0286] In another preferred embodiment, the second active ingredient is an anti-PD1 antibody.

[0287] In another preferred embodiment, the ratio of the first active ingredient to the second active ingredient is 5:1 to 1:1.

[0288] In another preferred embodiment, the dosage of the active ingredient in the pharmaceutical composition is 5-60 mg / kg, preferably 10-30 mg / kg. Typically, the dosage of the first active ingredient in the pharmaceutical composition is 5-60 mg / kg, preferably 10-30 mg / kg; and the dosage of the PD-1 antibody is 5-20 mg / kg, preferably 10 mg / kg.

[0289] Combination therapy can provide “synergy” and is demonstrated to be “synergistic,” meaning that the efficacy achieved when the active ingredients are used together is greater than the sum of the effects of using the compounds individually. Synergistic effects are achieved when the active ingredients are: (1) co-formulated and administered or administered simultaneously in a combined, unit-dose formulation; (2) administered individually in alternation or parallel; or (3) administered through some other therapy. Synergistic effects are achieved when the compounds are administered or delivered continuously, for example, through different injections in individual syringes, during alternating therapy. Generally, during alternating therapy, the effective dose of each active ingredient is administered continuously, i.e., sequentially, while in combination therapy, the effective doses of two or more active ingredients are administered together.

[0290] X. Uses in the preparation of therapeutic drugs

[0291] In a tenth aspect of the invention, there is provided the use of an active ingredient selected from the group consisting of: an anti-FOLR1 antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, or a recombinant protein such as a bispecific antibody as described in the second aspect of the invention, or an immunoconjugate as described in the sixth aspect of the invention, or a combination thereof, wherein the active ingredient is used to prepare a medicament for the prevention and / or treatment of diseases or conditions associated with abnormal overexpression of FOLR1.

[0292] In some implementations, a bispecific antibody is an antibody that is capable of specifically recognizing and binding to at least one different antigen containing human folate receptor 1.

[0293] In some implementations, the FOLR1 chimeric antibody can specifically bind to FOLR1-overexpressing tumor cells, producing one or more of the following effects: inhibiting tumor cell proliferation, reducing tumor carcinogenicity by decreasing the frequency of cancer stem cells in the tumor, inhibiting tumor growth, increasing survival, triggering tumor cell death, differentiating carcinogenic cells into a non-carcinogenic state, or preventing tumor cell metastasis.

[0294] In some embodiments, the disease treated with anti-FOLR1 antibodies (e.g., antibodies or immunoconjugates) is cancer. In some embodiments, cancer is characterized by tumors expressing FOLR1 overexpression that is bound by a FOLR1 binder (e.g., an antibody).

[0295] In some embodiments, the cancer is selected from the group consisting of rectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer.

[0296] In some embodiments, the FRα-expressing cancer in the control subjects is ovarian cancer, endometrial cancer, colorectal cancer, breast cancer, thyroid cancer, fallopian tube cancer, or lung cancer such as adenocarcinoma. In some embodiments, the control subjects are known to have early-stage FRα-expressing cancer, such as stage I ovarian cancer, endometrial cancer, colorectal cancer, breast cancer, thyroid cancer, fallopian tube cancer, or lung cancer (e.g., adenocarcinoma). In some embodiments, the control subjects are known to have intermediate-stage FRα-expressing cancer, such as stage II ovarian cancer, endometrial cancer, colorectal cancer, breast cancer, thyroid cancer, fallopian tube cancer, or lung cancer (e.g., adenocarcinoma). In some embodiments, the control subjects are known to have late-stage FRα-expressing cancer, such as stage III or IV ovarian cancer, endometrial cancer, colorectal cancer, breast cancer, thyroid cancer, fallopian tube cancer, or lung cancer (e.g., adenocarcinoma).

[0297] In some embodiments, the cancer is ovarian cancer. In other embodiments, the cancer is lung cancer.

[0298] In some embodiments, the object is a human being.

[0299] The present invention further provides a method for inhibiting tumor growth using antibodies or other reagents described herein. In some embodiments, the method for inhibiting tumor growth includes contacting cells with a FOLR1 binding agent (e.g., an antibody) in vitro. For example, an immortal cell line or cancer cell line expressing FOLR1 is cultured in a culture medium containing an antibody or other reagent to inhibit tumor growth.

[0300] In some embodiments, the method of inhibiting tumor growth includes isolating tumor cells from a patient sample, such as, for example, a tissue biopsy, pleural effusion, or blood sample, and culturing them in a culture medium containing a FOLR1 binder to inhibit tumor growth.

[0301] In some embodiments, the illustrated method for inhibiting tumor growth includes administering a therapeutically effective amount of the FOLR1 binder to a subject. In some embodiments, the subject is a human. In some embodiments, the subject has a tumor or has a tumor that has already been removed.

[0302] In some embodiments, the tumor expresses a folate receptor that binds to or is bound to a FOLR1 binder. In some embodiments, the tumor overexpresses human FOLR1.

[0303] In some embodiments, the tumor is selected from the group consisting of brain cancer, rectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer.

[0304] In some embodiments, the tumor is ovarian cancer.

[0305] Furthermore, the present invention provides a method for reducing tumor carcinogenicity in a subject, comprising applying a therapeutically effective amount of a FOLR1 binder to the subject. In some embodiments, the tumor comprises cancer stem cells. In some embodiments, the frequency of cancer stem cells in the tumor is reduced by applying the agent.

[0306] Therefore, in some embodiments, the present invention provides a method for treating cancer using ch5G12 and ch14H4 antibodies.

[0307] The embodiments disclosed herein may be further defined by reference to the following non-limiting examples, which detail the preparation of specific antibodies disclosed herein and methods of using the antibodies disclosed herein. Many modifications to both the materials and methods may be made without departing from the scope of this disclosure and will be apparent to those skilled in the art.

[0308] Example

[0309] It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes thereof will be proposed by those skilled in the art and are included within the spirit and scope of this application.

[0310] Example 1: Construction of a human FOLR1 overexpressing cell line

[0311] Human FOLR1 overexpressing cell lines were constructed using liposome transfection and Lipofectamine was used. TMThe pCMV3 plasmid (Yiqiao Shenzhou, HG11241-CY) expressing human FOLR1 (amino acid sequence as shown in SEQ ID NO:23, nucleic acid encoding as shown in SEQ ID NO:24) was transfected into CHO-K1 cells (hamster ovary cell subline, ATCC#CCL-61) using a 3000 transfection reagent (Invitrogen, L3000008). The specific procedure was as follows: one day before transfection, 4 × 10⁻⁶ cells were transfected. 5 Cell / well quantity: CHO-K1 cells were seeded into 6-well plates and cultured overnight to allow cell adhesion. The next day, the procedure was performed according to the reagent manufacturer's instructions, with 2.5 μg of experimental DNA used per transfected cell. After transfection, the cells were placed in a CO2 incubator for 48 hours. The medium was then replaced with one containing Hygromycin B (Invitrogen, 10687010). After 10 days of antibiotic-resistant selection, a mixed cell pool expressing human FOLR1 was obtained. The mixed cell pool was then plated using the limiting dilution method to obtain monoclonal cell lines. The isolated monoclonal cell lines were identified using flow cytometry (FACS). Specifically, the monoclonal cells to be identified were digested and washed twice with FACS buffer (PBS + 1% BSA). 50 μL of anti-FOLR1 antibody Mirvetuximab (sequence reference patent WO2011106528A1) was added to each cell, with an antibody concentration of 10 μg / mL, and incubated at 4°C for 30 min. Centrifuge at 500g for 3 min and discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Add 50 μL of PE-labeled goat anti-mouse Fc secondary antibody (Jackson, 115-115-164) to each well and incubate at 4°C for 30 min. Centrifuge at 500g for 3 min and discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Resuspend the cells in 50 μL of FACS buffer to each well and use flow cytometry (Agilent, Novocyte) to identify the FOLR1 expression level in monoclonal cells.

[0312] MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTN

[0313] TSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKED

[0314] CEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLSGGGGSYPYDVPDYA(SEQ ID NO:23)。

[0315] ATGGCTCAGCGGATGACAACACAGCTGCTGCTCCTTCTAGTGTGGGTGGCTGTAGTAGGGGAGGCTCAG

[0316] ACAAGGATTGCATGGGCCAGGACTGAGCTTCTCAATGTCTGCATGAACGCCAAGCACCACAAGGAAAAG

[0317] CCAGGCCCCGAGGACAAGTTGCATGAGCAGTGTCGACCCTGGAGGAAGAATGCCTGCTGTTCTACCAAC

[0318] ACCAGCCAGGAAGCCCATAAGGATGTTTCCTACCTATATAGATTCAACTGGAACCACTGTGGAGAGATG

[0319] GCACCTGCCTGCAAACGGCATTTCATCCAGGACACCTGCCTCTACGAGTGCTCCCCCAACTTGGGGCCC

[0320] TGGATCCAGCAGGTGGATCAGAGCTGGCGCAAAGAGCGGGTACTGAACGTGCCCCTGTGCAAAGAGGAC

[0321] TGTGAGCAATGGTGGGAAGATTGTCGCACCTCCTACACCTGCAAGAGCAACTGGCACAAGGGCTGGAAC

[0322] TGGACTTCAGGGTTTAACAAGTGCGCAGTGGGAGCTGCCTGCCAACCTTTCCATTTCTACTTCCCCACA

[0323] CCCACTGTTCTGTGCAATGAAATCTGGACTCACTCCTACAAGGTCAGCAACTACAGCCGAGGGAGTGGC

[0324] CGCTGCATCCAGATGTGTTTCGACCCAGCCCAGGGCAACCCCAATGAGGAGGTGGCGAGGTTCTATGCT

[0325] GCAGCCATGAGTGGGGCTGGGCCCTGGGCAGCCTGGCCTTTCCTGCTTAGCCTGGCCCTAATGCTGCTGTGGCTGCTCAGCGGGGGTGGAGGCTCTTATCCTTACGACGTGCCTGACTACGCCTAA (SEQ ID NO: 24).

[0326] Figure 1 The results showed that the proportion of FOLR1-positive cells in the obtained FOLR1-expressing mixed cell pool reached 64.82%, and human FOLR1-high-expressing cell clone 1B6 was successfully obtained by limiting dilution method. The obtained single clone was named CHO-hFOLR1.

[0327] Example 2: Generation and Identification of Anti-FOLR1 Monoclonal Antibody

[0328] This invention employs hybridoma technology for screening anti-FOLR1 antibodies. Specifically, human FOLR1 protein (Bepsys, FO1-H52H1) fused with a 6×His tag at its C-terminus was used as an immunogen, emulsified with a rapid adjuvant (Bio-Lon, KX0210041), and then immunized with BalB / C mice (purchased from Spiefol Biotechnology Co., Ltd.). Serum titers of the immunized mice were detected using ELISA. Mice that reached the fusion standard were euthanized, and spleens were collected and single-cell suspensions were prepared. Hybridomas were generated by electrofusion with SP20 myeloma cells (Nanjing Kebai, CBP60881). After hybridomas grew in 96-well cell culture plates for 10 days, the parent clones 5G12 and 14H4, which could bind human FOLR1, were screened by ELISA and FACS. Single clones with FOLR1 binding activity were isolated by limiting dilution and sequenced. The heavy chain variable region sequences and light chain variable region sequences of mouse clones 5G12 and 14H4 were obtained by sequencing. The variable region sequences are shown in Table 1.

[0329] Table 3. Mouse anti-variable region sequence binding to FOLR1

[0330]

[0331] Example 3 Construction, Expression and Purification of Recombinant FOLR1 Chimeric Antibody

[0332] The variable regions of the heavy and light chains of the murine antibody were placed at the N-terminus of the human IgG1 heavy chain constant region (Uniprot ID: P0DOX5) and the light chain kappa constant region (Uniprot ID: P01834). The fused gene fragment was then constructed into a pHr expression vector with an N-terminal signal peptide. The obtained light and heavy chain expression vectors were then co-transfected into Expi CHO-S cells (Gibco, A29129) using a transfection reagent (Gibco, A29133). Specifically, Expi CHO-S cells were passaged according to the required transfection volume. The cell density was adjusted the day before transfection to achieve a cell density of approximately 6 × 10⁶ cells on the day of transfection. 6 Cells / mL. Take 4% OptiPRO. TM SFM complexing medium (purchased from Gibco) was used as the transfection buffer. 0.8 μg of plasmid DNA was added to each milliliter of transfection buffer, mixed well, and then ExpiFectamine was added. TM Mix CHO reagent (purchased from Gibco) thoroughly. Gently pour the cationic transfection reagent / DNA mixture into the Expi CHO-S cell suspension. Gently mix and incubate overnight at 37°C with 5% CO2. After 18-22 hours of overnight incubation, add 24% of the transfected culture volume of ExpiCHO reagent to the culture flask. TM Excipients (purchased from Gibco) and 0.6% ExpiCHO by volume of the transfected culture. TM The cell culture supernatant was collected after gently mixing with an enhancer (purchased from Gibco) and continuous culturing until day 10 or when cell viability was below 70%. The target antibody was purified from the culture supernatant using a Protein A affinity chromatography column. Specifically, before purification, the collected cell supernatant was centrifuged at 10,000 rpm for 10 min and filtered through a 0.22 μm filter. The Protein A column was equilibrated with 5 column volumes of equilibration buffer (10 mM Pb, pH 7.0). The filtered supernatant was added to the purification column and equilibrated with 10 column volumes of equilibration buffer. 5 mL of elution buffer (100 mM Gly-NaCl, pH 3.8) was added, and the eluent was collected. The antibody concentration was detected using the A280 method, and the antibody purity was determined by SEC-HPLC. Recombinant chimeric antibodies with a purity greater than 95% were obtained and named ch5G12 and ch14H4, respectively. After ultrafiltration concentration and concentration determination, they were used for subsequent experiments.

[0333] The heavy and light chain amino acid sequences of ch5G12 and ch14H4 are shown in Table 4.

[0334] Table 4. Heavy chain (HC) and light chain (LC) sequences of chimeric antibodies binding to FOLR1

[0335]

[0336]

[0337] Example 4: ELISA Binding Activity Analysis of FOLR1 Chimeric Antibody

[0338] This embodiment detects the binding activity of the obtained chimeric antibodies ch5G12 and ch14H4 with the recombinantly expressed FOLR1 protein. The specific procedure is as follows: Human FOLR1-His (Bepsys, FO1-H52H1) was diluted to 2 μg / mL with coating buffer (a mixture of 8 mL of 0.2 mol / L Na2CO3 and 17 mL of 0.2 mol / L NaHCO3, then 75 mL of distilled water was added to adjust the pH to 9.6). 50 μL of this solution was added to each well of a microplate (Corning, 3590) and incubated at 37°C for 2 hours. After discarding the liquid in the wells, 200 μL of washing buffer (PBS + 0.5‰ Tween-20) was added to each well, and the plate was washed three times. Then, 200 μL of blocking buffer (PBS + 0.5‰ Tween-20 + 5% skim milk powder) was added to each well, and the plate was incubated at 37°C for 2 hours. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the plate was washed three times. 50 μL of serially diluted chimeric antibodies ch5G12 and ch14H4, and the reference antibody Mirvetuximab, were added to the wells of an ELISA plate. The initial antibody concentration was 100 nM, and each well was diluted 5-fold, resulting in 11 concentration points. The plates were incubated at 37°C for 60 min. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the plates were washed 3 times. 50 μL of HRP-labeled goat anti-human Fc secondary antibody (Jackson, 109-035-170) was added to each well, and the plates were incubated at 37°C for 60 min. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the plates were washed 3 times. 50 μL of chromogenic reagent (Sigma, T4444) was added to each well, and the plates were incubated at room temperature for 2 min. Then, 50 μL of 2 mol / L H2SO4 was added to terminate the reaction. The OD values ​​were read using a ThermoScientific Microplate Reader (MμLtiskan FC, 8D-030A). 450 Numerical value. Based on OD 450 Plot the numerical values ​​against antibody concentrations.

[0339] Figure 2 The chimeric antibodies ch5G12 and ch14H4 were shown to bind to recombinant human FOLR1 protein, and their EC50 values ​​were close to those of the reference antibody Mirvetuximab.

[0340] Example 5: FACS Binding Activity Analysis of FOLR1 Chimeric Antibody

[0341] This embodiment tested the binding activity of the obtained chimeric antibodies ch5G12 and ch14H4 to the FOLR1 protein on the cell surface. Specifically, the CHO-hFOLR1 cell line obtained in Example 1 and the purchased CAL51 tumor cell line (Nanjing Kebai, CBP60360) were digested into single-cell suspensions using trypsin. The cells were washed twice with FACS buffer, and then the density was adjusted to 2 × 10⁻⁶ cells / mL. 6 Cells were added to each well at a rate of 100 μL / well in a 96-well U-bottom plate (BIOFIL, 002096). After centrifugation at 500g for 3 min, the supernatant was discarded. 50 μL of serially diluted chimeric antibodies ch5G12, ch14H4, and the reference antibody Mirvetuximab were added to each well. The initial antibody concentration was 100 nM, and each well was diluted 5-fold for a total of 10 concentration points. Cells were incubated at 4°C for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded. 200 μL of FACS buffer was added to each well, and the cells were washed twice. 50 μL of PE-labeled goat anti-human Fc secondary antibody (Jackson, 109-115-098) was added to each well, and the cells were incubated at 4°C for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded. 200 μL of FACS buffer was added to each well, and the cells were washed twice. 50 μL of FACS buffer was added to each well to resuspend the cells, and the fluorescence intensity of the cells was detected by flow cytometry. Plot the antibody concentration on the x-axis and the PE fluorescence area on the y-axis.

[0342] Figure 3 The chimeric antibodies ch5G12 and ch14H4 were shown to be able to bind to FOLR1 on the cell membrane.

[0343] Example 6: Species Cross-Binding Activity Analysis of FOLR1 Chimeric Antibody

[0344] This embodiment tested the binding activity of the obtained chimeric antibodies ch5G12 and ch14H4 with recombinantly expressed FOLR1 proteins from different species. Specifically, the recombinant expressed cynomolgus monkey FOLR1-His protein (Bepsys, FO1-C52H8) and mouse FOLR1-His protein (Bepsys, FO1-M5225) were diluted to 2 μg / mL with coating buffer and added to the wells of an ELISA plate at 50 μL / well, and incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing 3 times. 200 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing 3 times. 50 μL of serially diluted chimeric antibodies ch5G12 and ch14H4, along with the control antibody Mirvetuximab, were added to the wells of an ELISA plate. The initial antibody concentration was 100 nM, and the plates were serially diluted 5-fold, resulting in 11 concentration points. The plates were incubated at 37°C for 60 min. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the plates were washed 3 times. 50 μL of HRP-labeled goat anti-human Fc secondary antibody was added to each well, and the plates were incubated at 37°C for 60 min. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the plates were washed 3 times. 50 μL of chromogenic buffer was added to each well, and the plates were incubated at room temperature for 2 min. Then, 50 μL of 2 mol / L H2SO4 was added to terminate the reaction, and the OD450 values ​​were read on a microplate reader. The OD450 values ​​were plotted on the x-axis as antibody concentration. 450 Plot the values ​​on the ordinate.

[0345] like Figure 4 As shown, both chimeric antibodies ch5G12 and ch14H4 can bind to recombinant cynomolgus monkey FOLR1 protein, but have no binding activity to mouse FOLR1 protein.

[0346] Figure 4 This demonstrates the ability of ELISA to detect the binding activity of antibodies to FORR1 proteins from other species.

[0347] Example 7: Analysis of the cross-binding activity of FOLR1 chimeric antibody with its family of proteins

[0348] This embodiment tested the binding activity of the obtained chimeric antibodies ch5G12 and ch14H4 with recombinant FOLR2, FOLR3, and FOLR4 proteins, which are related to the FOLR1 family. Specifically, the recombinant human FOLR2 protein (Bepsys, FO2-H5223), human FOLR3 protein (Bepsys, FO3-H52H3), and human FOLR4 protein (Bepsys, FO4-H52H3) were diluted to 2 μg / mL with coating buffer and added to the wells of an ELISA plate at 50 μL / well, and incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing 3 times. 200 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing 3 times. Add 50 μL of serially diluted chimeric antibodies ch5G12 and ch14H4, and the reference antibody Mirvetuximab, to the wells of an ELISA plate. The initial antibody concentration was 100 nM, and each well was diluted 5-fold, resulting in 11 concentration points. Incubate at 37°C for 60 min. After discarding the liquid in the wells, add 200 μL of washing buffer to each well and wash 3 times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. After discarding the liquid in the wells, add 200 μL of washing buffer to each well and wash 3 times. Add 50 μL of chromogenic buffer to each well and incubate at room temperature for 2 min. Then, add 50 μL of 2 mol / L H2SO4 to stop the reaction and read the OD on a microplate reader. 450 Numerical values. OD is plotted on the x-axis as antibody concentration. 450 Plot the values ​​on the ordinate.

[0349] Figure 5 The results showed that the chimeric antibodies ch5G12 and ch14H4 could not bind to other proteins in the FORL1 family.

[0350] Example 8: Affinity assay of FOLR1 chimeric antibody

[0351] The equilibrium dissociation constant (KD) of the chimeric antibodies ch5G12 and ch14H4 binding to recombinant human FOLR1 protein was determined using biofilm thin-layer interferometry (BLI). The specific procedures were as follows: One hour before the experiment, the Protein A (Sartorius, 18-5010) sensor was immersed in PBST solution (10 mM PBS + 0.1% BSA + 0.2‰ Tween-20, pH 7.4). The recombinant human FOLR1 protein was serially diluted with PBST, starting at 100 nM and then sequentially diluted 2-fold, for a total of 7 concentration points. A zero-concentration control well was also included. The antibody to be tested was diluted to 5 μg / mL. The Fortebio molecular interaction analyzer (Sartorius, Octet R8) was set to the following operating conditions: temperature 30℃, Shake speed 1000 rpm. Antibody was captured using a pre-coated Protein A probe for 180 s; it was then bound to serially diluted antibody samples for 120 s; dissociation was performed for 300 s; and regeneration was repeated three times with regeneration buffer (10 mM glycine, pH 1.7) for 30 s each time. Detection was performed using ForteBio's Octet System. After obtaining the sensor data, the binding constant (k) was analyzed using Octet BLI Analysis software. on ) and dissociation constant (k off By fitting an ideal binding-dissociation curve, the equilibrium dissociation constant KD(k) between the antibody and antigen is calculated. off / k on (See results) Figure 6 According to Table 5, the affinity of the chimeric antibodies obtained through screening was lower than that of the reference antibody Mirvetuximab.

[0352] Table 5. Results of antibody affinity detection by BLI method

[0353] <![CDATA[k on (1 / Ms)]]> <![CDATA[k dis (1 / s)]]> KD(M) Mirvetuximab 3.08E+05 3.28E-04 1.07E-09 ch5G12 3.33E+05 4.79E-03 1.44E-08 ch14H4 3.54E+05 2.44E-03 6.88E-09

[0354] Example 9: Detection of FOLR1 chimeric antibody endocytosis activity

[0355] In this embodiment, the endpoint method was used to detect the endocytic activity of the chimeric antibodies ch5G12 and ch14H4. Specifically, the density of digested CAL51 cells (Nanjing Kebai, CBP60360) was adjusted to 1×10⁻⁶ cells one day before the experiment. 5The antibody was seeded at a rate of 50 μL / well into 96-well cell culture plates and cultured overnight at 37°C with 5% CO2 to allow cell adhesion. The next day, the antibody was diluted to 24 μg / mL, and the goat anti-human Fc secondary antibody (SSA015) labeled with pHAb Amine and Thiol Reactive Dyes (Promega, G9841) was diluted to 72 μg / mL. 25 μL of the antibody and 25 μL of the labeled goat anti-human Fc secondary antibody were mixed and incubated at room temperature in the dark for 30 min before being added to the corresponding cell wells. After culturing at 37°C with 5% CO2 for 24 hours, the cells were digested with trypsin and washed twice with FACS buffer. The mean fluorescence signal intensity of the PE cells was detected by flow cytometry and plotted. The results are shown below. Figure 7 The endocytic activity of ch5G12 and ch14H4 was stronger than that of the reference antibody Mirvetuximab.

[0356] Figure 7 Results of antibody endocytosis activity assay.

Claims

1. An antifolate receptor 1 (FOLR1) antibody or its antigen-binding fragment, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises complementarity-determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL comprises complementarity-determining regions VLCDR1, VLCDR2, and VLCDR3, characterized in that: The VHCDR1, VHCDR2, and VHCDR3 each contain the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or amino acid sequences having at least 95% sequence identity with them; the VLCDR1, VLCDR2, and VLCDR3 each contain the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or amino acid sequences having at least 95% sequence identity with them.

2. The anti-FOLR1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The VH contains an amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 80% sequence identity with it.

3. The anti-FOLR1 antibody or its antigen-binding fragment according to claim 1, characterized in that: The VL contains an amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 80% sequence identity with it.

4. The anti-FOLR1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The VH and VL respectively contain amino acid sequences as shown in SEQ ID NO:15 and SEQ ID NO:

16.

5. The anti-FOLR1 antibody or its antigen-binding fragment as described in claim 2, characterized in that: The anti-FOLR1 antibody is a chimeric antibody, wherein, The C-terminus of the VH and the N-terminus of the heavy chain constant region (CH) are connected to form the heavy chain (HC), and the HC has an amino acid sequence as shown in SEQ ID NO:19 or an amino acid sequence having at least 80% sequence identity with it. The C-terminus of the VL and the N-terminus of the light chain constant region (CL) are connected to form a light chain (LC), the LC having an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 80% sequence identity with it.

6. The anti-FOLR1 antibody or its antigen-binding fragment according to any one of claims 1-5, characterized in that, The HC and the LC respectively contain amino acid sequences as shown in SEQ ID NO:19 and SEQ ID NO:

20.

7. The use of the antiFOLR1 antibody or its antigen-binding fragment as described in any one of claims 1-6 in the preparation of humanized antibodies, bispecific antibodies or antibody-drug conjugates (ADCs).

8. A pharmaceutical composition comprising the anti-FOLR1 antibody or its antigen-binding fragment as described in any one of claims 1-6, characterized in that: It consists of the anti-FOLR1 antibody and a pharmaceutical carrier.

9. Use of the anti-FOLR1 antibody or its antigen-binding fragment according to any one of claims 1-6, or the pharmaceutical composition according to claim 9, in the preparation of a medicament for the treatment or prevention of FOL1-positive malignant tumors.

10. The use according to claim 9, wherein, The FOL1-positive malignant tumors include choriocarcinoma, ovarian cancer, fallopian tube cancer, endometrial cancer, breast cancer, thyroid cancer, colorectal cancer, primary peritoneal cancer, lung cancer, pancreatic cancer, and kidney cancer; or the FOL1-positive malignant tumors are selected from one of choriocarcinoma, ovarian cancer, fallopian tube cancer, endometrial cancer, breast cancer, thyroid cancer, colorectal cancer, primary peritoneal cancer, lung cancer, pancreatic cancer, and kidney cancer.

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