Anti-FOLR1 antibody, preparation therefor and use thereof

Abstract

Provided are an anti-FOLR1 antibody, and preparation therefor and the use thereof. The anti-FOLR1 antibody comprises a heavy chain variable region and a light chain variable region, or the anti-FOLR1 antibody comprises a heavy chain variable region; and both the heavy chain variable region and the light chain variable region comprise three complementarity determining regions. The provided anti-FOLR1 antibody is a high-affinity antibody capable of targeting FOLR1 on the surface of tumor cells, has binding affinity to FOLR1 on the surface of tumor cells similar as that of a control antibody, does not bind to other members of the FOLR family, and only specifically binds to FOLR1. Therefore, the anti-FOLR1 antibody can provide a new way for the development of products and technical means related to the prevention, treatment and detection of tumors such as ovarian cancer.

Description

Anti-FOLR1 antibodies, their preparation and application

[0001] Priority Declaration

[0002] This application claims priority to Chinese Patent Application No. 202411861999.9, filed on December 17, 2024, entitled "Anti-FOLR1 Antibody and its Preparation and Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biomedical technology, and in particular to an anti-FOLR1 antibody and its preparation and application. Background Technology

[0004] Folic acid is a water-soluble B vitamin composed of pteridine, para-aminobenzoic acid, and glutamic acid residues. Also known as vitamin B12 or vitamin M, it is essential for cell growth and reproduction, aids in protein metabolism, and, together with vitamin B12, promotes the production and maturation of red blood cells. It is indispensable for red blood cell production. In the body, folic acid functions as tetrahydrofolate, which participates in the synthesis and conversion of purine and pyrimidine nucleotides. It plays a crucial role in the production of nucleic acids (ribonucleic acid and deoxyribonucleic acid) and is essential for the body's utilization of sugars and amino acids.

[0005] Folate receptors FOLR1 (FRα), FOLR2 (FRβ), and FOLR3 (FRγ) are cysteine-rich cell surface glycoproteins that bind to folate with high affinity to mediate cellular folate uptake. Folate receptors, especially FOLR1, are expressed at very low levels in most normal tissues, but at high levels in many cancer cells or tissues to meet the folate requirements of rapidly dividing cells under low folate conditions. Folate dependence in many tumors has been utilized in treatment and diagnosis through the administration of high-affinity antifolate agents, folate-based imaging agents, and folate-binding drugs and toxins. Beyond this, targeted therapies for cancer have also been developed to maximize tumor killing while minimizing toxicity. Monoclonal antibodies targeting FOLR1 on the surface of tumor cells can mediate specific antitumor activity by blocking cell signaling or by participating in effector cell or complement-induced immune-mediated cell killing. FRs are among the earliest confirmed cancer targets because they are expressed in a subset of malignant cells and a limited number of normal tissues, making them tumor-associated antigens (TAAs) and antibody-drug conjugates (ADCs), which are important target antigens for the development of ADC drugs. Therefore, FOLR1 is a very promising target in both tumor immunology and ADC drug development.

[0006] Currently, there are several antibodies and related ADC drugs targeting FOLR1 on the market, such as farletuzumab from Morphotek and mirvetuximab from IMMUNOGEN. Among them, mirvetuximab has seen relatively rapid progress, with the FDA approving its related antibody-drug conjugate, Mirvetuximab soravtansine-gyynx (MIRV), for the treatment of FOLR1-positive, platinum-resistant epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer. MIRV is a conjugate of a FOLR1-targeting antibody and a mirvetuximab microtubule inhibitor. Mirvetuximab targets FOLR1, binding to FOLR1 expressed on cells, leading to internalization of the receptor-ADC complex and lysosomal degradation, thereby releasing the DM4 payload from the antibody, resulting in cell cycle arrest and cell death. In vivo, MIRV has shown antitumor activity in a mouse xenograft model of FOLR1-positive ovarian cancer.

[0007] Therefore, developing antibodies targeting FOLR1 is of great significance for further research and development of FOLR1-targeting ADC drugs and for the treatment of related tumors. Developing new antibodies targeting FOLR1 would open new avenues for the treatment of related tumors. However, in actual research and development, it is very difficult to develop new antibodies targeting FOLR1 with high affinity, high selectivity, and high bioactivity. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an anti-FOLR1 antibody and its preparation and application. The anti-FOLR1 antibody is a high-affinity antibody that can target FOLR1 on the surface of tumor cells, and its binding affinity to FOLR1 on the surface of tumor cells is similar to that of the control antibody. It has good specificity and can provide a new approach for the development of products and technologies related to the prevention, treatment and detection of tumors such as ovarian cancer.

[0009] To achieve the above and other related objectives, the first aspect of the present invention provides an anti-FOLR1 antibody, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region includes three complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the light chain variable region includes three complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequentially include, as shown in SEQ ID NO. 5–10, SEQ ID NO. 15–20, SEQ ID NO. 25–30, SEQ ID NO. 35–40, SEQ ID NO. 45–50, SEQ ID NO. 55–60, SEQ ID NO. 65–70, SEQ ID NO. 75–80, SEQ ID NO. 85–90, SEQ ID NO. 95–100, SEQ ID NO. 105–110, SEQ ID NO. ... The amino acid sequences shown in NO. 115–120, SEQ ID NO. 125–130, or SEQ ID NO. 135–140;

[0010] Alternatively, the anti-FOLR1 antibody may contain a heavy chain variable region comprising three complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 sequentially contain the amino acid sequences shown in SEQ ID NO. 143–145, SEQ ID NO. 148–150, SEQ ID NO. 153–155, SEQ ID NO. 158–160, or SEQ ID NO. 163–165.

[0011] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 5, 6, and 7, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 8, 9, and 10, respectively.

[0012] Furthermore, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO.15, 16, and 17, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO.18, 19, and 20, respectively.

[0013] Furthermore, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO.25, 26, and 27, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO.28, 29, and 30, respectively.

[0014] Furthermore, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO.35, 36, and 37, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO.38, 39, and 40, respectively.

[0015] Furthermore, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO.45, 46, and 47, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO.48, 49, and 50, respectively.

[0016] Furthermore, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 55, 56, and 57, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 58, 59, and 60, respectively.

[0017] Furthermore, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 65, 66, and 67, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 68, 69, and 70, respectively.

[0018] Furthermore, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO.75, 76, and 77, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO.78, 79, and 80, respectively.

[0019] Furthermore, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 85, 86, and 87, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 88, 89, and 90, respectively.

[0020] Furthermore, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 95, 96, and 97, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 98, 99, and 100, respectively.

[0021] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 105, 106, and 107, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 108, 109, and 110, respectively.

[0022] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 115, 116, and 117, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 118, 119, and 120, respectively.

[0023] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 125, 126, and 127, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 128, 129, and 130, respectively.

[0024] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 135, 136, and 137, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 138, 139, and 140, respectively.

[0025] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 143, 144, and 145, respectively.

[0026] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 148, 149, and 150, respectively.

[0027] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 153, 154, and 155, respectively.

[0028] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 158, 159, and 160, respectively.

[0029] Furthermore, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 163, 164, and 165, respectively.

[0030] Furthermore, the anti-FOLR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region sequentially comprise, as shown in SEQ ID NO.3-4, SEQ ID NO.13-14, SEQ ID NO.23-24, SEQ ID NO.33-34, SEQ ID NO.43-44, SEQ ID NO.53-54, SEQ ID NO.63-64, SEQ ID NO.73-74, SEQ ID NO.83-84, SEQ ID NO.93-94, SEQ ID NO.103-104, SEQ ID NO.113-114, SEQ ID NO.123-124, or SEQ ID NO.133-134, or the amino acid sequences shown in SEQ ID NO.3-4, SEQ ID NO.13-14, SEQ ID NO.23-24, SEQ ID NO.33-34, SEQ ID NO.43-44, SEQ ID NO.53-54, SEQ ID NO. Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity as the amino acid sequences shown in SEQ ID NO. 63–64, SEQ ID NO. 73–74, SEQ ID NO. 83–84, SEQ ID NO. 93–94, SEQ ID NO. 103–104, SEQ ID NO. 113–114, SEQ ID NO. 123–124, or SEQ ID NO. 133–134.

[0031] Furthermore, the anti-FOLR1 antibody comprises a heavy chain variable region, the amino acid sequence of which sequentially comprises the amino acid sequences shown in SEQ ID NO.142, SEQ ID NO.147, SEQ ID NO.152, SEQ ID NO.157 or SEQ ID NO.162, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequences shown in SEQ ID NO.142, SEQ ID NO.147, SEQ ID NO.152, SEQ ID NO.157 or SEQ ID NO.162.

[0032] Furthermore, the heavy chain also includes a heavy chain constant region, which is selected from any one of IgG1, IgG2, IgG3 and IgG4.

[0033] Furthermore, the light chain also includes a light chain constant region, which is selected from the κ chain or the λ chain.

[0034] Furthermore, the anti-FOLR1 antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, heavy chain antibody, or single-domain antibody, or a monoclonal antibody or polyclonal antibody prepared from the above antibodies.

[0035] Further, the anti-FOLR1 antibody comprises a heavy chain (H) and a light chain (L), the amino acid sequences of which sequentially include, as shown in SEQ ID NO.1–2, SEQ ID NO.11–12, SEQ ID NO.21–22, SEQ ID NO.31–32, SEQ ID NO.41–42, SEQ ID NO.51–52, SEQ ID NO.61–62, SEQ ID NO.71–72, SEQ ID NO.81–82, SEQ ID NO.91–92, SEQ ID NO.101–102, SEQ ID NO.111–112, SEQ ID NO.121–122, or SEQ ID NO.131–132, or the amino acid sequences shown in SEQ ID NO.1–2, SEQ ID NO.11–12, SEQ ID NO.21–22, SEQ ID NO.31–32, SEQ ID NO.41–42, SEQ ID NO. Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity as the amino acid sequences shown in SEQ ID NO.51–52, SEQ ID NO.61–62, SEQ ID NO.71–72, SEQ ID NO.81–82, SEQ ID NO.91–92, SEQ ID NO.101–102, SEQ ID NO.111–112, SEQ ID NO.121–122, or SEQ ID NO.131–132.

[0036] Furthermore, the anti-FOLR1 antibody comprises a heavy chain, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO.141, SEQ ID NO.146, SEQ ID NO.151, SEQ ID NO.156 or SEQ ID NO.161, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.141, SEQ ID NO.146, SEQ ID NO.151, SEQ ID NO.156 or SEQ ID NO.161.

[0037] A second aspect of the present invention provides an isolated nucleic acid encoding an anti-FOLR1 antibody as described in the first aspect.

[0038] A third aspect of the present invention provides an expression vector comprising isolated nucleic acids as described in the second aspect.

[0039] A fourth aspect of the present invention provides a transformant prepared by transforming an expression vector as described in the third aspect into a host cell, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

[0040] The fifth aspect of the present invention provides a method for preparing an anti-FOLR1 antibody, comprising: culturing a transformant as described in the fourth aspect, and obtaining the anti-FOLR1 antibody from the resulting culture.

[0041] The sixth aspect of the present invention provides the use of the anti-FOLR1 antibody as described in the first aspect, the isolated nucleic acid as described in the second aspect, the expression vector as described in the third aspect, the transformant as described in the fourth aspect, and / or the anti-FOLR1 antibody prepared by the method described in the fifth aspect in tumor prevention, and / or tumor treatment, and / or FOLR1 detection.

[0042] Furthermore, the application includes at least one of the following: preparing a drug for preventing tumors, preparing a drug for treating tumors, and preparing a reagent and / or kit for detecting FOLR1.

[0043] A seventh aspect of the present invention provides an antibody-drug conjugate comprising an antibody portion and a conjugation portion conjugated to the antibody portion; wherein the antibody portion is an anti-FOLR1 antibody as described in the first aspect.

[0044] An eighth aspect of the present invention provides a pharmaceutical composition comprising component one and component two;

[0045] Component one is selected from at least one of the following substances: anti-FOLR1 antibody as described in the first aspect, isolated nucleic acid as described in the second aspect, expression vector as described in the third aspect, transformant as described in the fourth aspect, anti-FOLR1 antibody prepared by the method described in the fifth aspect, and antibody-drug conjugate as described in the seventh aspect.

[0046] The second component is a pharmaceutically acceptable carrier.

[0047] The eighth aspect of the present invention provides a kit comprising reagents and instructions, wherein the reagents comprise at least one of the following substances: an anti-FOLR1 antibody as described in the first aspect, isolated nucleic acid as described in the second aspect, an expression vector as described in the third aspect, a transformant as described in the fourth aspect, and an anti-FOLR1 antibody prepared by the method described in the fifth aspect.

[0048] A ninth aspect of the present invention provides a drug delivery system comprising a drug and a drug delivery device for delivering the drug to a target, the drug comprising an antibody-drug conjugate as described in the seventh aspect or a drug composition as described in the eighth aspect.

[0049] As described above, the anti-FOLR1 antibody of the present invention, its preparation and application, have the following beneficial effects:

[0050] This invention utilizes Harbour's proprietary human mouse platform to develop a series of novel anti-FOLR1 antibodies. Experiments show that these antibodies not only possess high affinity for FOLR1 on the surface of tumor cells, but their binding affinity to FOLR1 on tumor cells is also similar to that of control antibodies. Furthermore, they do not bind to other members of the FOLR family (such as FOLR2 and FOLR3), only specifically binding to FOLR1. In addition, these antibodies also exhibit good binding activity with cynomolgus monkey FOLR, demonstrating excellent cross-species activity. Therefore, this invention has significant clinical application value and extremely high market potential. Attached Figure Description

[0051] Figures 1-1, 1-2, and 1-3 show the binding curves of FOLR1 antibody, control antibodies 1-2, and hIgG1 to human FOLR1-ECD protein in Example 2 of the present invention.

[0052] Figures 2-1, 2-2, and 2-3 show the binding curves of FOLR1 antibody, control antibodies 1-2, and hIgG1 to cynomolgus monkey FOLR1-ECD protein in Example 2 of this invention.

[0053] Figure 3 shows the binding curves of FOLR1 antibody, control antibodies 1-2 and hIgG1 to mouse FOLR1-ECD protein in Example 2 of the present invention.

[0054] Figures 4-1, 4-2, and 4-3 show the binding curves of FOLR1 antibody, control antibodies 1-2, and hIgG1 to SKOV3 tumor cells endogenously expressing FOLR1 in Example 3 of the present invention.

[0055] Figures 5-1, 5-2, and 5-3 show the binding curves of FOLR1 antibody, control antibodies 1-2, and hIgG1 to cynomolgus monkey FOLR1 molecules expressed on the surface of CHO-K1 cells in Example 3 of the present invention. Detailed Implementation

[0056] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0057] In this invention, unless otherwise stated, the term "a plurality of" means two or more.

[0058] The character " / " indicates that the objects before and after it are in an "or" relationship. For example, A / B means: A or B.

[0059] The term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0060] The terms “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of…” does not include any unspecified elements, steps, or components. The expression “essentially composed of…” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “essentially composed of…” and “composed of…” are encompassed within the meaning of the expression “comprising.”

[0061] The term "antibody" refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen-binding site. Antibody encompasses antibody fragments. As used herein, the term "antibody" includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, fully humanized antibodies, heavy chain antibodies, nanobodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0062] The term "variable region" (i.e., "binding domain") allows binding molecules to selectively recognize and specifically bind to epitopes on antigens. That is, for example, the light chain variable region (VL) and heavy chain variable region (VH) domains of an antibody binding molecule, or combinations of these complementarity-determining region (CDR) subgroups, form a variable region that defines a three-dimensional antigen-binding site. More specifically, the antigen-binding site is defined by three CDRs on each VH and VL chain. These "complementarity-determining regions," or "CDRs," are discontinuous short sequences of amino acids that are specifically localized to form a binding domain as the antibody adopts its three-dimensional conformation in an aqueous environment. The remaining amino acids in the binding domain are called "framework (FR)" regions, exhibiting minor intermolecular differences. The binding domain formed by the localized CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its complementary epitope. The amino acids constituting the CDRs and framework regions of any given heavy or light chain variable region can be identified by conventional methods (see “Sequences of Proteins of Immunological Interest”, Kabat, E. et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their full text). In this paper, the CDRs (CDRL or LCDR) of the light chain variable region may be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region may be referred to as HCDR1, HCDR2, and HCDR3.

[0063] The terms “heavy-chain-only antibody” and “heavy-chain antibody (HCAb)” are used interchangeably and exist in their broadest sense, referring to an antibody that lacks the conventional antibody light chain and contains only a heavy chain variable region and a heavy chain constant region (e.g., Fc fragment) that does not contain CH1.

[0064] The term "single-domain antibody," also known as "nanobody," refers to a VHH structure cloned from a heavy chain antibody. It is the smallest known unit that can bind to a target antigen.

[0065] In this invention, the amino acid sequence of CDR is shown according to the Chothia definition rule (the sequence in the claims of this invention is also shown according to the Chothia definition rule). However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)) based on the antibody's three-dimensional structure and the topology of the CDR ring; Kabat (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) based on antibody sequence variability; and AbM (Martin, ACR and J. Allen (2007) "Bioinformatics tools for antibody engineering," in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH). Verlag, pp. 95–118), Contact (MacCallum, R.M et al., (1996) J.Mol.Biol. 262: 732-745), IMGT (Lefranc, M.-P., 2011(6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev.Comp.Immunol., 27, 55-77(2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. In this paper, multiple CDR numbering systems, such as Chothia, Abm, Kabat, Contact, and IMGT, can be used for the same variable region. Those skilled in the art should understand that although CDRs defined by different numbering systems may be different, CDRs corresponding to the same numbering system represent effective antigen-binding sites that can bind antigenic epitopes.Unless otherwise specified, the terms “CDR” and “complementation-determining region” for a given antibody or its region (e.g., variable region) should be understood to encompass complementation-determining regions defined as described in any of the known schemes described herein. While the scope of protection claimed in the claims of this invention is based on the sequence shown in the Chothia definition rule, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0066] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0067] In this invention, the term "percentage (%) sequence identity" or "sequence identity" has a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a known algorithm). This can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0068] One embodiment of the present invention provides an anti-FOLR1 antibody, comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region includes three complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the light chain variable region includes three complementarity determining regions LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequentially include, for example, SEQ ID NO. 5–10, SEQ ID NO. 15–20, SEQ ID NO. 25–30, SEQ ID NO. 35–40, SEQ ID NO. 45–50, SEQ ID NO. 55–60, SEQ ID NO. 65–70, SEQ ID NO. 75–80, SEQ ID NO. 85–90, SEQ ID NO. 95–100, SEQ ID NO. 105–110, SEQ ID NO. 115–12 ... The amino acid sequence shown in NO.125–130 or SEQ ID NO.135–140.

[0069] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 5, 6, and 7, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 8, 9, and 10, respectively.

[0070] In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.15, 16 and 17, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.18, 19 and 20, respectively.

[0071] In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.25, 26 and 27, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.28, 29 and 30, respectively.

[0072] In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.35, 36 and 37, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.38, 39 and 40, respectively.

[0073] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO.45, 46, and 47, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO.48, 49, and 50, respectively.

[0074] In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 55, 56 and 57, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 58, 59 and 60, respectively.

[0075] In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 65, 66, and 67, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 68, 69, and 70, respectively.

[0076] In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.75, 76 and 77, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.78, 79 and 80, respectively.

[0077] In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 85, 86 and 87, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 88, 89 and 90, respectively.

[0078] In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 95, 96 and 97, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 98, 99 and 100, respectively.

[0079] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 105, 106, and 107, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 108, 109, and 110, respectively.

[0080] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 115, 116, and 117, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 118, 119, and 120, respectively.

[0081] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 125, 126, and 127, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 128, 129, and 130, respectively.

[0082] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 135, 136, and 137, respectively; and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 138, 139, and 140, respectively.

[0083] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region sequentially comprise, as shown in SEQ ID NO. 3-4, SEQ ID NO. 13-14, SEQ ID NO. 23-24, SEQ ID NO. 33-34, SEQ ID NO. 43-44, SEQ ID NO. 53-54, SEQ ID NO. 63-64, SEQ ID NO. 73-74, SEQ ID NO. 83-84, SEQ ID NO. 93-94, SEQ ID NO. 103-104, SEQ ID NO. 113-114, SEQ ID NO. 123-124, or SEQ ID NO. 133-134, or the amino acid sequences shown in SEQ ID NO. 3-4, SEQ ID NO. 13-14, SEQ ID NO. 23-24, SEQ ID NO. 33-34, SEQ ID NO. 43-44, SEQ ID NO. 53-54, SEQ ID NO. 63-64, SEQ ID NO. 73-74, SEQ ID NO. 133-134, or SEQ ID NO. 133-134, or the amino acid sequences shown in SEQ ID NO. 13 ...34, or the amino acid sequences shown in SEQ ID NO. 133-14, SEQ ID NO. 23-24, SEQ ID NO. Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity as the amino acid sequences shown in SEQ ID NO. 83–84, SEQ ID NO. 93–94, SEQ ID NO. 103–104, SEQ ID NO. 113–114, SEQ ID NO. 123–124, or SEQ ID NO. 133–134.

[0084] In some embodiments, the heavy chain further includes a heavy chain constant region selected from any one of IgG1, IgG2, IgG3 and IgG4, preferably IgG1, and more preferably human IgG1 (hIgG1).

[0085] In some embodiments, the light chain further includes a light chain constant region, which is selected from the κ chain or the λ chain, preferably the κ chain of a human antibody.

[0086] In some embodiments, the anti-FOLR1 antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, or multispecific antibody, or a monoclonal antibody or polyclonal antibody prepared from the above antibodies.

[0087] In some embodiments, the anti-FOLR1 antibody comprises a heavy chain (H) and a light chain (L), wherein the amino acid sequences of the heavy and light chains sequentially comprise, as shown in SEQ ID NO.1–2, SEQ ID NO.11–12, SEQ ID NO.21–22, SEQ ID NO.31–32, SEQ ID NO.41–42, SEQ ID NO.51–52, SEQ ID NO.61–62, SEQ ID NO.71–72, SEQ ID NO.81–82, SEQ ID NO.91–92, SEQ ID NO.101–102, SEQ ID NO.111–112, SEQ ID NO.121–122, or SEQ ID NO.131–132, or the amino acid sequences shown in SEQ ID NO.1–2, SEQ ID NO.11–12, SEQ ID NO.21–22, SEQ ID NO.31–32, SEQ ID NO.41–42, or SEQ ID NO. Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity as the amino acid sequences shown in SEQ ID NO.51–52, SEQ ID NO.61–62, SEQ ID NO.71–72, SEQ ID NO.81–82, SEQ ID NO.91–92, SEQ ID NO.101–102, SEQ ID NO.111–112, SEQ ID NO.121–122, or SEQ ID NO.131–132.

[0088] Another embodiment of the present invention provides an anti-FOLR1 antibody comprising a heavy chain variable region, wherein the heavy chain variable region includes three complementarity-determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 sequentially comprise the amino acid sequences shown in SEQ ID NO. 143-145, SEQ ID NO. 148-150, SEQ ID NO. 153-155, SEQ ID NO. 158-160 or SEQ ID NO. 163-165.

[0089] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 143, 144, and 145, respectively.

[0090] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 148, 149, and 150, respectively.

[0091] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 153, 154, and 155, respectively.

[0092] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 158, 159, and 160, respectively.

[0093] In one specific embodiment, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 163, 164, and 165, respectively.

[0094] In some embodiments, the anti-FOLR1 antibody comprises a heavy chain variable region, the amino acid sequence of which sequentially comprises the amino acid sequence shown in SEQ ID NO.142, SEQ ID NO.147, SEQ ID NO.152, SEQ ID NO.157 or SEQ ID NO.162, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.142, SEQ ID NO.147, SEQ ID NO.152, SEQ ID NO.157 or SEQ ID NO.162.

[0095] In some embodiments, the anti-FOLR1 antibody is a heavy chain antibody, a single-domain antibody, a bispecific antibody, or a multispecific antibody, or a monoclonal antibody or polyclonal antibody prepared from the above antibodies.

[0096] In some embodiments, the anti-FOLR1 antibody comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO. 141, SEQ ID NO. 146, SEQ ID NO. 151, SEQ ID NO. 156, or SEQ ID NO. 161, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 141, SEQ ID NO. 146, SEQ ID NO. 151, SEQ ID NO. 156, or SEQ ID NO. 161.

[0097] Another embodiment of the present invention provides an isolated nucleic acid encoding an anti-FOLR1 antibody as described in the above embodiments / examples.

[0098] Furthermore, the method for preparing the isolated nucleic acid is a conventional method in the art. In some embodiments, the method for preparing the isolated nucleic acid includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned antibody through gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned antibody through artificial full-sequence synthesis. Other technical details can be found in the conventional methods in the art, and will not be repeated here.

[0099] Another embodiment of the present invention provides an expression vector comprising the isolated nucleic acid as described in the above embodiments / examples. The expression vector can be any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecules, including but not limited to plasmids, granules, bacteriophages, and viral vectors. The viral vector can be, for example, a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0100] Another embodiment of the present invention provides a transformant, obtained by transforming the expression vector as described in the above embodiments / examples into a host cell, wherein the host cell is a prokaryotic cell or a eukaryotic cell. The host cell can be any conventional host cell in the art, as long as it allows the expression vector to stably replicate spontaneously and the carried nucleic acid to be effectively expressed. In some embodiments, the host cell is E. coli TG1 or BL21 cells (expressing single-chain antibodies or Fab antibodies), or CHOK1 cells (expressing full-length IgG antibodies). The transformation method is a conventional transformation method in the art, such as chemical transformation, heat shock, or electroporation.

[0101] Another embodiment of the present invention provides a method for preparing an anti-FOLR1 antibody, comprising: culturing a transformant as described in the above embodiments / examples, and obtaining the anti-FOLR1 antibody from the resulting culture. The methods for culturing the transformant and obtaining the antibody from the resulting culture can be performed using conventional methods in the art, and will not be described in detail here.

[0102] Another embodiment of the present invention provides the application of the anti-FOLR1 antibody as described in the above embodiments / examples, the isolated nucleic acid as described in the above embodiments / examples, the expression vector as described in the above embodiments / examples, the transformant as described in the above embodiments / examples, and / or the anti-FOLR1 antibody prepared by the method described in the above embodiments / examples in tumor prevention, and / or tumor treatment, and / or FOLR1 detection.

[0103] In some embodiments, the application includes at least one of the following: preparing a drug for preventing tumors, preparing a drug for treating tumors, and preparing a reagent and / or kit for detecting FOLR1.

[0104] In some embodiments, the reagent for treating tumors is an antibody-drug conjugate.

[0105] In some embodiments, the tumor includes, but is not limited to, ovarian cancer, fallopian tube cancer, peritoneal cancer, breast cancer, uterine cancer, endometrial cancer, pancreatic cancer, brain cancer, lung cancer, kidney cancer, liver cancer, head and neck cancer, stomach cancer, colorectal cancer, esophageal cancer, and metastatic lesions of the above cancers.

[0106] Another embodiment of the present invention provides an antibody-drug conjugate, comprising an antibody portion and a conjugation portion conjugated to the antibody portion; the antibody portion is an anti-FOLR1 antibody as described in the above embodiments / examples.

[0107] In some embodiments, the coupling portion includes, but is not limited to, detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, etc.

[0108] In some embodiments, the antibody portion and the conjugation portion are conjugated by chemical bonds or adapters.

[0109] In the above embodiments / examples, the antibody-drug conjugate further includes a drug portion, which is linked to the antibody portion via the conjugation portion. The drug portion includes an antitumor drug, which includes, but is not limited to, known antitumor drugs or products obtained by chemical modification of known antitumor drugs. The purpose of the chemical modification is to optimize its physical properties and drug-like characteristics.

[0110] Another embodiment of the present invention provides a pharmaceutical composition comprising component one and component two;

[0111] Component 1 is an active ingredient, which is selected from at least one of the following substances: anti-FOLR1 antibody as described in the above embodiments / examples, isolated nucleic acid as described in the above embodiments / examples, expression vector as described in the above embodiments / examples, transformant as described in the above embodiments / examples, and anti-FOLR1 antibody prepared by the method described in the above embodiments / examples;

[0112] The second component is a pharmaceutically acceptable carrier.

[0113] In some embodiments, the pharmaceutical composition further includes a third component that has a synergistic effect with the first component, the third component being selected from at least one of the following substances: known antibodies targeting FOLR1, and known drugs for the prevention and / or treatment of tumors.

[0114] In the above embodiments / examples, components one and three are both active ingredients, playing a promoting and synergistic role in tumor prevention / treatment. The pharmaceutically acceptable carrier can be a conventional carrier in the art, such as any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, including, but not limited to, pharmaceutically acceptable excipients, fillers, or diluents. Furthermore, the content of each component in the pharmaceutical composition, the route of administration, and the dosage can be selected and determined according to conventional methods and actual conditions in the art, and will not be elaborated here.

[0115] Another embodiment of the present invention provides a kit comprising reagents and instructions. The reagents include at least one of the following substances: an anti-FOLR1 antibody as described in the above embodiments / examples; isolated nucleic acid as described in the above embodiments / examples; an expression vector as described in the above embodiments / examples; a transformant as described in the above embodiments / examples; and an anti-FOLR1 antibody prepared by the method described in the above embodiments / examples. The instructions may include information such as the method of use, shelf life, and manufacturer of the reagents.

[0116] In some embodiments, the kit may further include, but is not limited to, buffer solutions, standards, controls, tools, and containers for holding the reagents. The buffer solution is generally used for dilution and / or pH adjustment; the standards or controls are used to calibrate experimental instruments, verify experimental methods, or serve as a reference for experimental results; the tools include, but are not limited to, pipettes, centrifuge tubes, petri dishes, and test strips, and are used for sample processing, reaction operations, and result observation during the experiment. The specific composition of the kit can be selected and adjusted according to actual application needs (such as the experimental purpose and the target being detected), which will not be elaborated further here.

[0117] Another embodiment of the present invention provides a drug delivery system, including a drug and a drug delivery device for delivering the drug to a target, wherein the drug includes an antibody-drug conjugate as described in the above embodiments / examples or a drug composition as described in the above embodiments / examples.

[0118] In some embodiments, the drug delivery system may further include a drug monitoring device, which is used to detect the type of drug, monitor the dosage and drug delivery rate, etc., but is not limited thereto.

[0119] Another embodiment of the present invention provides a FOLR1 detection method, comprising detecting FOLR1 in the test subject using an anti-FOLR1 antibody as described in the above embodiments / examples and / or an anti-FOLR1 antibody prepared by the method described in the above embodiments / examples.

[0120] In some embodiments, the FOLR1 detection method is used to perform qualitative and / or quantitative detection of FOLR1 in the test object.

[0121] The following specific examples illustrate the present invention in detail. It should also be understood that the following examples are only for specific illustrative purposes and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0122] Unless otherwise specified, all reagents and raw materials used in the following examples are commercially available.

[0123] Example 1

[0124] Preparation and screening of high-affinity antibodies targeting FOLR1 on the surface of tumor cells

[0125] I. Immunization of Harbour transgenic mice

[0126] 1.1 Immunogen:

[0127] The protein immunogen was recombinant human FOLR1-ECD-Fc protein purchased from ACRO, catalog number FO1-H5253, which was dissolved, aliquoted, and frozen at -80°C; the cell immunogen was a 293T cell line overexpressing FOLR1, purchased from Kyinno Bio Cat, KC-1235, 293T-FOLR1 cell line.

[0128] 1.2. Immunization with Harbour HCAb and H2L2 mouse proteins:

[0129] huFOLR1-ECD-Fc protein was used to immunize Harbour HCAb transgenic mice (which produced antibodies without light chains, only half the size of conventional IgG antibodies, but with similar pharmacokinetic properties and Fc-mediated effector functions) for 6-8 weeks and Harbour H2L2 transgenic mice (Harbour Antibodies BV, a transgenic mouse carrying a human immunoglobulin repertoire, whose antibodies have complete human antibody variable domains and rat constant domains). All transgenic mice were housed in an SPF-grade environment. For primary immunization, 50 μg of huFOLR1-ECD-Fc protein was emulsified with Freund's complete adjuvant (Sigma, F5881) and injected intraperitoneally into mice. For subsequent booster immunizations, 25 μg of huFOLR1-ECD-Fc protein was mixed with RIBI adjuvant (Sigma, S6322) and injected intraperitoneally into mice. The interval between each immunization was 2 weeks. Seven days after immunization, blood samples were collected and serum antibody titers were detected by ELISA. Mice with high serum titers were selected for subsequent single B cell screening experiments.

[0130] 1.3. Harbour HCAb, H2L2 mouse cellular immunity:

[0131] For the initial immunization, administer 1×10⁻⁶ doses per mouse. 7 250 μL of 293T-FOLR1 cells were injected intraperitoneally into mice. For subsequent booster immunizations, the cell seeding volume was the same as the initial injection. The interval between each immunization was 3 weeks. Seven days post-immunization, blood samples were collected for ELISA and FACS analysis to determine serum antibody titers. Mice with high serum titers were selected for subsequent single B-cell selection experiments.

[0132] II. Based on Single B-cell screening in the Optofluidic system

[0133] Optofluidic system uses optical-electronic positioning (OEP) TM Beacon technology is used to move individual cells. The Beacon system is an automated biological instrument that allows for simultaneous biological function assays, experimental analysis, positive clone selection, and other operations under cell culture conditions. The Beacon platform can perform these tasks in massive parallel and automated manner on thousands of cells.

[0134] This embodiment uses a plasma cell discovery workflow, screening up to 14,000 individual plasma cells per experiment to select antigen-specific plasma cells that secrete antibodies. These antibody-secreting plasma cells are then exported to 96-well plates containing cell lysis buffer for subsequent single-B cell sequencing to identify the heavy and light chain sequences of antibodies produced by individual B cells (monoclonals).

[0135] III. Single B-cell sequencing

[0136] This invention utilizes single-cell B-cell sequencing to recover antibody heavy and light chain sequences from individual plasma cells. Single-cell B-cell sequencing has become a powerful tool for obtaining antibody sequences. The general procedure includes purifying RNA from single-cell lysates, reverse transcription synthesis of cDNA, amplification and purification of cDNA, amplification of heavy and light chains, cloning and transfection, and Sanger sequencing. Uniqueness and clustering analysis are performed on the obtained sequences, and then the paired heavy and light chain DNA sequences are used for plasmid synthesis.

[0137] After obtaining the light and heavy chain variable domain sequences encoding the antibody molecule, conventional recombinant DNA technology can be used to fuse the light and heavy chain variable domain sequences with the corresponding human antibody light and heavy chain constant domain sequences to obtain recombinant antibody molecules. Specifically, in this embodiment, the antibody heavy chain variable domain sequence (VH) is synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG1 antibody heavy chain constant domain sequence to encode the full-length heavy chain that produces the IgG1 antibody. The antibody light chain variable domain sequence (VL) is synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody Igκ light chain constant domain sequence to encode the full-length light chain that produces the antibody. Simultaneously, since the variable domain sequences of the anti-FOLR1 monoclonal antibody molecules obtained from immunized Harbour HCAb and H2L2 mice are human antibody sequences, this embodiment also yields a fully human anti-FOLR1 recombinant IgG1 antibody. The heavy chain and light chain complementary region (CDR) sequences, heavy chain and light chain variable region (V) sequences of human FORL1 H2L2 antibodies 1-14 screened in this embodiment are shown in Tables 1-1 to 1-14 below. The CDR and VH sequences of human FORL1 HCAb antibodies 1-5 screened in this embodiment are shown in Tables 1-15 to 1-19 below. The heavy chain and light chain complementary region (CDR) sequences, heavy chain and light chain variable region (V) sequences of control antibody 1 (PR001640, derived from patent US20170184603A1, applicant Morphotek) and control antibody 2 (PR001641, derived from patent US20210032327A1, applicant IMMUNOGEN) used in the following embodiments are shown in Tables 1-20 to 1-21 below.

[0138] Table 1-1 Sequence listing of FOR1 H2L2 antibody 1

[0139] Table 1-2 FOLR1 H2L2 antibody sequence listing

[0140] Table 1-3 FOLR1 H2L2 antibody 3 sequence listing

[0141] Table 1-4 FOLR1 H2L2 antibody 4 sequence listing

[0142] Table 1-5 FOLR1 H2L2 antibody sequence listing

[0143] Table 1-6 FOLR1 H2L2 antibody 6 sequence listing

[0144] Table 1-7 FOLR1 H2L2 antibody 7 sequence listing

[0145] Table 1-8 FOLR1 H2L2 antibody sequence listing

[0146] Table 1-9 FOLR1 H2L2 antibody sequence listing

[0147] Table 1-10 FOLR1 H2L2 antibody 10 sequence listing

[0148] Table 1-11 FOLR1 H2L2 antibody 11 sequence listing

[0149] Table 1-12 FOLR1 H2L2 antibody 12 sequence listing

[0150] Table 1-13 FOLR1 H2L2 antibody 13 sequence listing

[0151] Table 1-14 FOLR1 H2L2 antibody 14 sequence listing

[0152] Table 1-15 FOLR1 HCAb antibody 1 sequence listing

[0153] Table 1-16 FOLR1 HCAb antibody 2 sequence listing

[0154] Table 1-17 FOLR1 HCAb antibody 3 sequence listing

[0155] Table 1-18 FOLR1 HCAb antibody 4 sequence listing

[0156] Table 1-19 FOLR1 HCAb antibody 5 sequence listing

[0157] Table 1-20 Sequence Listing of Control Antibody 1

[0158] Table 1-21 Sequence listing of control antibody 2

[0159] IV. Expression and Purification of Recombinant Antibodies

[0160] Uniqueness and clustering analysis were performed on the obtained sequences, and then plasmids were synthesized from the paired heavy and light chain DNA sequences. Expi293F cells were used for antibody expression. The cell density was adjusted to 3 × 10e6 cells / mL. The plasmids of heavy and light chain antibodies were added to the tube at a ratio of 2:3, followed by the addition of transfection reagent PEI (PEI to DNA ratio of 4:1). After mixing, the mixture was incubated at room temperature for 15 min. The above mixture was then added to the cells, and the transfected cells were cultured in a 37°C, 5% CO2 incubator with shaking at 125 rpm. 24 h after transfection, MetaCell™ 293 TransFeed High-Glucose (Sipeng, catalog number L2009-1000) was added to a final concentration of 3%. After 5 days of cell culture, when the cell viability dropped below 70%, the supernatant was collected. The transfection supernatant was purified using a Protein G column.

[0161] Example 2

[0162] Detection of antibody binding ability to FOLR1-ECD at the protein level

[0163] The binding affinity of the antibodies prepared and screened in Example 1, control antibodies 1-2, and hIgG1 to the FOLR1-ECD protein was detected using an ELISA assay. The binding affinity was determined by comparing the binding curves of different antibodies to the FOLR1-ECD protein. The specific experimental procedure is as follows:

[0164] First, human FOLR1-ECD (ACRO, FO1-H52H1), mouse FOLR1-ECD (ACRO, FO1-M5225), and cynomolgus monkey (Cyno) FOLR1-ECD (ACRO, FO1-C52H8) proteins were sequentially diluted to a concentration of 1 μg / mL and added to 96-well plates, 100 μL per well, and incubated overnight at 4°C. After washing the 96-well plates three times with PBST solution, 2% BSA in PBS solution was added and incubated at 37°C for 1 h. The test antibody was then serially diluted (100 nM, 10-fold dilution) and added to the 96-well plates, incubated at 37°C for 1 h. After washing three times with PBST solution, anti-human IgG Fc-HRP secondary antibody (5000× dilution) was added and incubated at 37°C for 30–60 min. After washing three times with PBST solution, add TMB colorimetric solution for 5-15 minutes, and then add stop solution to stop the color development.

[0165] Table 2. Results of the binding ability of different antibodies to FOLR1-ECD at the protein level.

[0166] The detection results are shown in Table 2 and Figures 1-1, 1-2, 1-3, 2-1, 2-2, 2-3 and Figure 3. Antibodies PR306163, PR306164, PR306167, PR306346, PR306398, PR306403, PR306415, PR306422, PR306411, PR306496, PR306503, PR306504 and PR306510 all have the ability to bind to human FOLR1-ECD and monkey FOLR1-ECD proteins, and are comparable to the control antibodies PR001640 (Morphotek: US20170184603A1) and PR001641 (IMMUNOGEN: US20210032327A1). Among them, the PR306143 and PR306346 antibodies also have the ability to bind to the mouse FORL1-ECD protein.

[0167] Example 3

[0168] Detection of antibody binding ability to FOLR1 at the cellular level

[0169] The binding affinity of the FOLR1 antibody, control antibodies 1-2, and hIgG1 prepared and screened in Example 1 to FOLR1 molecules expressed on the surface of CHO-K1 cells, as well as the binding affinity of SKOV3 tumor cells endogenously expressing FOLR1, was detected by flow cytometry. The binding affinity was determined by comparing the binding curves of different antibodies to FOLR1 molecules expressed on the cell surface. The specific experimental procedure is as follows:

[0170] (1) The cells were incubated with different concentrations of the test antibody at 4°C for 60 min. After washing with PBS three times, the cells were incubated with Alex-647 labeled goat anti-human secondary antibody at 4°C for 50 min. After washing with PBS three times again, the cells were resuspended in 30 μL FACS buffer.

[0171] (2) The median fluorescence value of one channel was determined using flow cytometry. The EC50 was compared by plotting the logarithm of antibody concentration to base 10 on the x-axis and the median fluorescence value of one channel on the y-axis.

[0172] Table 3. Results of FOLR1 binding ability of different antibodies at the cellular level.

[0173] The test results are shown in Table 3 and Figures 4-1, 4-2, 4-3, 5-1, 5-2 and 5-3. The antibodies PR306163, PR306167, PR306398, PR306415, PR306413, PR306420, PR306411, PR306484, PR306496, PR306502, PR306503, PR306504, PR306510 and PR306517 showed strong binding ability to SKOV3 cells. Except for PR306143, the binding ability of the other antibodies to CHOK1-cynoFOLR1 cells was comparable to that of the control antibodies PR001640 (Morphotek: US20170184603A1) and PR001641 (IMMUNOGEN: US20210032327A1).

[0174] Example 4

[0175] Cross-reactivity of antibodies with other FOLR family proteins

[0176] The cross-reactivity of the FOLR1 antibody prepared and screened in Example 1, control antibodies 1-2, and hIgG1 with other FOLR1 family proteins (FOLR2, FOLR3) was tested to compare the binding ability of different antibodies with other FOLR1 family proteins (FOLR2, FOLR3), thus verifying their ability to specifically bind to FOLR1. The specific experimental procedure is as follows:

[0177] 100 μL of 1 μg / mL human FOLR2 (ACRO, Cat:FO2-H5223) and FOLR3 (ACRO, Cat:FO3-H52H3) protein was used for ELISA coating and incubated overnight at 4°C. The plates were washed three times with PBST. 200 μL of a solution of PBST (PBS + 0.05% Tween 20) + 2% BSA was used for blocking and incubated at 37°C for 1 hour. The plates were then washed three times with PBST. 100 μL of 100 nM FOLR1 antibody was added to each well of the ELISA plate and incubated at 37°C for 1 hour. The plates were washed three times with PBST. 100 μL of Goat Anti-Human IgG, H+L-HRP (1:5000, Jackson, Cat:109-035-088) was added and incubated at 37°C for 1 hour. The plates were washed three times with PBST. Add 100 μL of TMB substrate (Biopanda, Cat: TMB-S-003) and incubate at room temperature for 5 minutes. Then add 50 μL of stop buffer (Solarbio, Cat#: C1058) to terminate the reaction. Record OD using a microplate reader (Molecular Devices, Spectra max 384plus). 450nm Read value.

[0178] Table 4. Cross-reactivity results of different antibodies with other FOR1 family proteins.

[0179] The test results are shown in Table 4, indicating that the FOLR1 antibody prepared and screened in Example 1 did not bind to other FOLR family members FOLR2 and FOLR3, but only specifically bound to FOLR1.

[0180] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

An anti-FOLRl antibody characterized in that, The anti-FOLR1 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes three complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region includes three complementarity-determining regions LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequentially contain the amino acid sequences shown in SEQ ID NO. 5–10, SEQ ID NO. 15–20, SEQ ID NO. 25–30, SEQ ID NO. 35–40, SEQ ID NO. 45–50, SEQ ID NO. 55–60, SEQ ID NO. 65–70, SEQ ID NO. 75–80, SEQ ID NO. 85–90, SEQ ID NO. 95–100, SEQ ID NO. 105–110, SEQ ID NO. 115–120, SEQ ID NO. 125–130, or SEQ ID NO. 135–140. Alternatively, the anti-FOLR1 antibody may contain a heavy chain variable region comprising three complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 sequentially contain the amino acid sequences shown in SEQ ID NO. 143–145, SEQ ID NO. 148–150, SEQ ID NO. 153–155, SEQ ID NO. 158–160, or SEQ ID NO. 163–165. The anti-FOLR1 antibody according to claim 1 is characterized in that, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 5, 6, and 7, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 8, 9, and 10, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 15, 16, and 17, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 18, 19, and 20, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 25, 26, and 27, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 28, 29, and 30, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 35, 36, and 37, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 38, 39, and 40, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 45, 46, and 47, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 48, 49, and 50, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 55, 56, and 57, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 58, 59, and 60, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 65, 66, and 67, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 68, 69, and 70, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 75, 76, and 77, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 78, 79, and 80, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 85, 86, and 87, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 88, 89, and 90, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 95, 96, and 97, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 98, 99, and 100, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 105, 106, and 107, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 108, 109, and 110, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 115, 116, and 117, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 118, 119, and 120, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 125, 126, and 127, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 128, 129, and 130, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 135, 136, and 137, respectively; the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 138, 139, and 140, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 143, 144, and 145, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 148, 149, and 150, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 153, 154, and 155, respectively; or, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 158, 159, and 160, respectively; or, The heavy chain variable region contains amino acid sequences HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 163, 164 and 165, respectively. The anti-FOLRl antibody according to claim 1 or 2, characterized in that: The anti-FOLR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region sequentially comprise, as shown in SEQ ID NO.3-4, SEQ ID NO.13-14, SEQ ID NO.23-24, SEQ ID NO.33-34, SEQ ID NO.43-44, SEQ ID NO.53-54, SEQ ID NO.63-64, SEQ ID NO.73-74, SEQ ID NO.83-84, SEQ ID NO.93-94, SEQ ID NO.103-104, SEQ ID NO.113-114, SEQ ID NO.123-124, or SEQ ID NO.133-134, or the amino acid sequences shown in SEQ ID NO.3-4, SEQ ID NO.13-14, SEQ ID NO.23-24, SEQ ID NO.33-34, SEQ ID NO.43-44, SEQ ID NO.53-54, SEQ ID NO. Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequences shown in SEQ ID NO. 63–64, SEQ ID NO. 73–74, SEQ ID NO. 83–84, SEQ ID NO. 93–94, SEQ ID NO. 103–104, SEQ ID NO. 113–114, SEQ ID NO. 123–124, or SEQ ID NO. 133–134; Alternatively, the anti-FOLR1 antibody comprises a heavy chain variable region, the amino acid sequence of which sequentially comprises the amino acid sequence shown in SEQ ID NO.142, SEQ ID NO.147, SEQ ID NO.152, SEQ ID NO.157 or SEQ ID NO.162, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.142, SEQ ID NO.147, SEQ ID NO.152, SEQ ID NO.157 or SEQ ID NO.

162. The anti-FOLRl antibody of claim 1, wherein: The heavy chain further includes a heavy chain constant region, which is selected from any one of IgG1, IgG2, IgG3 and IgG4; And / or, the light chain further includes a light chain constant region, which is selected from the κ chain or the λ chain. The anti-FOLRl antibody of claim 1, wherein: The anti-FOLR1 antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, heavy chain antibody, or single-domain antibody, or a monoclonal antibody or polyclonal antibody prepared from the above antibodies. The anti-FOLRl antibody according to claim 5, characterized in that: The anti-FOLR1 antibody comprises a heavy chain and a light chain, the amino acid sequences of which sequentially include, as shown in SEQ ID NO.1–2, SEQ ID NO.11–12, SEQ ID NO.21–22, SEQ ID NO.31–32, SEQ ID NO.41–42, SEQ ID NO.51–52, SEQ ID NO.61–62, SEQ ID NO.71–72, SEQ ID NO.81–82, SEQ ID NO.91–92, SEQ ID NO.101–102, SEQ ID NO.111–112, SEQ ID NO.121–122, or SEQ ID NO.131–132, or the amino acid sequences shown in SEQ ID NO.1–2, SEQ ID NO.11–12, SEQ ID NO.21–22, SEQ ID NO.31–32, SEQ ID NO.41–42, SEQ ID NO.51–52, SEQ ID NO.61–62, SEQ ID NO. Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequences shown in SEQ ID NO. 71–72, SEQ ID NO. 81–82, SEQ ID NO. 91–92, SEQ ID NO. 101–102, SEQ ID NO. 111–112, SEQ ID NO. 121–122, or SEQ ID NO. 131–132; Alternatively, the anti-FOLR1 antibody comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO.141, SEQ ID NO.146, SEQ ID NO.151, SEQ ID NO.156, or SEQ ID NO.161, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.141, SEQ ID NO.146, SEQ ID NO.151, SEQ ID NO.156, or SEQ ID NO.

161. An isolated nucleic acid, characterized in that: The antibody is encoded as described in any one of claims 1 to 6. An expression vector, characterized in that, Includes the isolated nucleic acid as described in claim 7. A transformant characterized by comprising: It is prepared by transforming the expression vector as described in claim 8 into host cells. A method of making an anti-FOLR1 antibody, characterized in that, include: The transformant as described in claim 9 is cultured to obtain the anti-FOLR1 antibody from the resulting culture. The use of the anti-FOLR1 antibody as described in any one of claims 1 to 6, the isolated nucleic acid as described in claim 7, the expression vector as described in claim 8, the transformant as described in claim 9, and / or the anti-FOLR1 antibody prepared by the method described in claim 10 in tumor prevention, and / or tumor treatment, and / or FOLR1 detection, wherein the use is for purposes other than disease diagnosis or treatment. The use according to claim 11, characterized in that The applications include at least one of the following: preparing drugs for preventing tumors, preparing drugs for treating tumors, and preparing reagents and / or kits for detecting FOLR1. An antibody-drug conjugate characterized in that, It includes an antibody portion and a coupling portion conjugated to the antibody portion; the antibody portion is an anti-FOLR1 antibody as described in any one of claims 1 to 6. A pharmaceutical composition, characterized by Includes component one and component two; Component one is selected from at least one of the following substances: the anti-FOLR1 antibody as described in any one of claims 1 to 6, the isolated nucleic acid as described in claim 7, the expression vector as described in claim 8, the transformant as described in claim 9, the anti-FOLR1 antibody prepared by the method described in claim 10, and the antibody-drug conjugate as described in claim 13. The second component is a pharmaceutically acceptable carrier. A kit characterized in that, Includes reagents and instructions, wherein the reagents include at least one of the following substances: the anti-FOLR1 antibody as described in any one of claims 1 to 6, the isolated nucleic acid as described in claim 7, the expression vector as described in claim 8, the transformant as described in claim 9, and the anti-FOLR1 antibody prepared by the method described in claim 10. A drug delivery system characterized in that, The invention includes a drug and a drug delivery device for delivering the drug to a target, the drug comprising an antibody-drug conjugate as claimed in claim 13 or a drug composition as claimed in claim 14.

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