Antibody binding to FOLR1 protein, antibody-drug conjugate, and use

Abstract

The present application relates to an antibody specifically binding to an FOLR1 protein or an antigen-binding fragment thereof, and an antibody-drug conjugate comprising the antibody or the antigen-binding fragment thereof. The present application also relates to a nucleic acid which codes the antibody specifically binding to the FOLR1 protein or the antigen-binding fragment thereof, a cell and pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof or the nucleic acid or the antibody-drug conjugate, and a use of the antibody or the antigen-binding fragment thereof or the antibody-drug conjugate in the prevention and / or treatment of FOLR1-mediated diseases.

Description

Antibodies binding to FOLR1 protein, antibody-drug conjugates and uses thereof

[0001] This application claims priority to Chinese invention patent application No. 2024103385743, filed on March 22, 2024, entitled “Antibodies that bind to FOLR1 protein, antibody-drug conjugates, and uses thereof,” and Chinese invention patent application No. 202411103842.X, filed on August 12, 2024, entitled “Antibodies that bind to FOLR1 protein, antibody-drug conjugates, and uses thereof.” The contents of both applications are incorporated herein by reference. Technical Field

[0002] The present application relates to the field of biomedicine or biopharmaceuticals, and more specifically to anti-FOLR1 antibodies, antibody-drug conjugates (ADCs) prepared from the antibodies, and applications thereof. Background Art

[0003] Folate receptor α (FR-α), also known as FOLR1 or folate-binding protein, is a glycoprotein anchored to the cell membrane via glycosylated phosphatidylinositol (GPI). It has a high affinity for folate and can transport folate via receptor-mediated endocytosis. FOLR1 expression is limited in non-malignant tissues, but it is overexpressed in malignant tissues of certain cancers, making it a potential target for a variety of anticancer drugs, such as antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, or chimeric antigen receptor (CAR) T cells.

[0004] FOLR1 is known to be expressed on the surface of tumor cells in a variety of cancers, including ovarian cancer, fallopian tube cancer, triple-negative breast cancer, endometrial cancer, mesothelioma, primary peritoneal cancer, and lung cancer. Currently, antibodies or ADCs targeting FOLR1 are relatively well-researched in ovarian cancer. Elahere, a FR-α-targeting ADC developed by ImmunoGen, received accelerated approval from the FDA in November 2022 for the treatment of adult patients with FR-α-positive, platinum-resistant epithelial ovarian, fallopian tube, or primary peritoneal cancer who have received one to three prior systemic therapies. Elahere utilizes a cleavable sulfo-SPDB linker to conjugate the anti-FR-α humanized monoclonal antibody M9346A to DM4. This cleavable linker maintains stability in the bloodstream and targets FR-α-positive tumor cells. However, Elahere has significant drawbacks: its limited patient population is limited, with efficacy limited to patients with high FR-α expression. However, this type of patients only accounts for 35-40% of the ovarian cancer population. Elahere is helpless for the majority of patients with medium and low expression of FR-α; secondly, there is the safety issue of eye toxicity.

[0005] MORAb-202, Eisai's first ADC, is a FR-α-targeted ADC composed of the anti-FR-α humanized IgG1 monoclonal antibody farletuzumab (MORAb-003) and the anticancer agent eribulin, linked via an enzymatically cleavable linker. Its DAR is 4.0. Upon entry of MORAb-202 into target FR-α-positive cancer cells, the linker undergoes enzymatic cleavage, releasing eribulin from the antibody and initiating anti-tumor activity.

[0006] Currently, there is still a need to develop new antibodies and ADCs targeting FOLR1 for the treatment of various FOLR1-positive cancers. Summary of the Invention

[0007] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to FOLR1 protein, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0008] (1) the VH comprises the heavy chain complementary determining region (HCDR) 1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 49; and the VL comprises the light chain complementary determining region (LCDR) 1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 50;

[0009] (2) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 51; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 52;

[0010] (3) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 53; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 54;

[0011] (4) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 7; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 8;

[0012] (5) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 9; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 10; or

[0013] (6) The VH comprises the heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO: 11; and the VL comprises the LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO: 12.

[0014] In some embodiments, the antibodies of the invention comprise a VH and a VL, wherein

[0015] (1) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 62, 21, and 22, respectively;

[0016] (2) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 16, and 15, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 62, 21, and 22, respectively;

[0017] (3) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 17, 18, and 19, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively;

[0018] (4) the VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22, respectively; or

[0019] (5) The VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 16 and 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22, respectively.

[0020] In some embodiments, the antibodies of the invention comprise a VH and a VL, wherein

[0021] (1) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50;

[0022] (2) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52;

[0023] (3) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 53, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 54;

[0024] (4) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 7, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 8;

[0025] (5) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 10; or

[0026] (6) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12.

[0027] In some embodiments, the antibodies of the present invention are selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0028] In some embodiments, the antibodies of the present invention are of an isotype selected from IgG, IgA, IgM, IgE, and IgD. In some preferred embodiments, the antibodies are of the IgG isotype.

[0029] In some embodiments, the antibodies of the present invention are of a subtype selected from IgG1, IgG2, IgG3, and IgG4. In some preferred embodiments, the antibodies are of the IgG1 subtype.

[0030] In some embodiments, the antibodies of the present invention comprise a heavy chain (HC) and a light chain (LC), wherein

[0031] (1) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:43, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:44;

[0032] (2) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 45, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 46;

[0033] (3) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 47, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 48;

[0034] (4) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2;

[0035] (5) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:3, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:4; or

[0036] (6) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:5, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:6.

[0037] In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv.

[0038] In some embodiments, the antibody is a monovalent, bivalent, or multivalent antibody.

[0039] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0040] In some embodiments, the antibody is a bispecific antibody, and the bispecific antibody further comprises a second antigen binding region that binds to a second antigen. In some preferred embodiments, the second antigen is selected from a cancer-associated antigen, an immune cell antigen, and an immune checkpoint molecule.

[0041] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0042] In a third aspect, the present invention provides a vector comprising a nucleic acid molecule according to the second aspect of the invention.

[0043] In a fourth aspect, the present invention provides a cell comprising a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention.

[0044] In a fifth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the method comprising:

[0045] (1) culturing the cell of the fourth aspect of the present invention under conditions suitable for expression of the antibody or antigen-binding fragment thereof;

[0046] (2) isolating the antibody or antigen-binding fragment thereof from the culture medium and / or culture supernatant of the cell.

[0047] In a sixth aspect, the present invention provides a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0048] In a seventh aspect, the present invention provides a genetically modified cell comprising a chimeric antigen receptor according to the sixth aspect of the present invention. In some preferred embodiments, the genetically modified cell is an immune cell, such as a T cell or a NK cell.

[0049] In an eighth aspect, the present invention provides an antibody conjugate comprising an antibody or antigen-binding fragment thereof according to the first aspect of the present invention and a chemical moiety coupled to the antibody or antigen-binding fragment thereof. In some preferred embodiments, the chemical moiety is selected from a cytotoxic drug, an immunostimulatory molecule, and a detectable label.

[0050] In a ninth aspect, the present invention provides an antibody drug conjugate (ADC), wherein the antibody drug conjugate has the structure of the following formula (I):

[0051] Ab-(L-(D) v ) w (I)

[0052] wherein Ab is an antibody or antigen-binding fragment thereof according to the first aspect of the present invention,

[0053] L is a linker,

[0054] D is a drug group,

[0055] v is 1, 2 or 3, preferably 3;

[0056] w ranges from about 1 to about 20, preferably 1 to 8, and more preferably w is 2 or 4.

[0057] In a tenth aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a chimeric antigen receptor according to the sixth aspect of the invention, a genetically modified cell according to the seventh aspect of the invention, an antibody conjugate according to the eighth aspect of the invention or an antibody-drug conjugate according to the ninth aspect of the invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0058] In the eleventh aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof according to the first aspect of the invention, the nucleic acid molecule according to the second aspect of the invention, the vector according to the third aspect of the invention, the chimeric antigen receptor according to the sixth aspect of the invention, the genetically modified cell according to the seventh aspect of the invention, the antibody conjugate according to the eighth aspect of the invention, the antibody-drug conjugate according to the ninth aspect of the invention, or the pharmaceutical composition according to the tenth aspect of the invention; optionally, the kit further comprises instructions for use and / or an administration device.

[0059] In a twelfth aspect, the present invention provides a method for treating, alleviating and / or preventing a FOLR1-mediated disease in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, the chimeric antigen receptor according to the sixth aspect of the present invention, the genetically modified cell according to the seventh aspect of the present invention, the antibody conjugate according to the eighth aspect of the present invention, the antibody drug conjugate according to the ninth aspect of the present invention, or the pharmaceutical composition according to the tenth aspect of the present invention.

[0060] In a thirteenth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, the chimeric antigen receptor according to the sixth aspect of the present invention, the genetically modified cell according to the seventh aspect of the present invention, the antibody conjugate according to the eighth aspect of the present invention, the antibody drug conjugate according to the ninth aspect of the present invention, or the pharmaceutical composition according to the tenth aspect of the present invention in the preparation of a medicament for treating, alleviating and / or preventing a FOLR1-mediated disease in a subject.

[0061] In a fourteenth aspect, the present invention provides an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a chimeric antigen receptor according to the sixth aspect of the present invention, a genetically modified cell according to the seventh aspect of the present invention, an antibody conjugate according to the eighth aspect of the present invention, an antibody-drug conjugate according to the ninth aspect of the present invention, a pharmaceutical composition according to the tenth aspect of the present invention, or a kit according to the eleventh aspect of the present invention, for use in treating, alleviating and / or preventing a FOLR1-mediated disease in a subject.

[0062] In some embodiments, the FOLR1-mediated disease of the twelfth, thirteenth, and / or fourteenth aspects of the present invention includes a cancer in which FOLR1 is highly expressed. In some preferred embodiments, the cancer is selected from lung cancer, such as non-small cell lung cancer, ovarian cancer, mesothelioma, breast cancer, such as triple-negative breast cancer, endometrial cancer, fallopian tube cancer, or primary peritoneal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 shows the results of the chimeric antibodies and ADCs of the present application binding to cells expressing human FOLR1, wherein W3922-BMK1 (MORAB-003) was used as a positive control antibody and hIgG1 isotype antibody was used as a negative control antibody.

[0064] FIG2 shows the internalization of the chimeric antibody of the present application on NCI-H2110 cells at 37° C. (upper panel) and 4° C. (lower panel).

[0065] Figure 3 shows the killing effect of the chimeric antibody ADC of the present application on NCI-H2110 cells, wherein WBP3922-BMK1-HG012 (i.e., MORAB-202) is used as a positive control ADC.

[0066] FIG4 shows the detection results of the binding of the humanized antibodies and ADCs of the present application to cells expressing human FOLR1.

[0067] FIG5 shows the internalization of the humanized antibodies and ADCs of the present application on NCI-H2110 cells.

[0068] FIG6 shows the in vitro killing effect of the humanized antibody ADC of the present application on NCI-H2110 cells.

[0069] FIG7 shows the bystander killing effect of the humanized antibody ADC of the present application.

[0070] FIG8 shows the ADCC killing effect mediated by the humanized antibody and ADC of the present application.

[0071] FIG9 shows the stability test results of the humanized antibody of the present application in plasma.

[0072] Figure 10 shows the changes in body weight of lung cancer model mice after administration of the humanized antibody ADC of the present application. The black arrows represent the administration time, n=8.

[0073] Figure 11 shows the changes in tumor volume after administration of the humanized antibody ADC of the present application to lung cancer model mice. The black arrows represent the administration time, n=8.

[0074] Figure 12 shows the binding curves of the humanized antibody ADC of the present application and the control on OV-90 cells (first round), wherein MORAb-202 and IMGN-853 were used as positive controls, and Isotype ctrl was used as a negative control.

[0075] Figure 13 shows the binding analysis of the humanized antibody ADC of the present application and the control on OV-90 cells (second round), wherein IMGN-853 was used as a positive control and Isotype ctrl was used as a negative control.

[0076] FIG14 shows the proliferation inhibition curves of Eribulin, MORAb-202, HFRAO34-HG004-W2, HFRA34-HG004-W2, HFRA34-HG004-D2 and IMGN853 on the human ovarian cancer cell line OV-90.

[0077] Figure 15 shows the changes in body weight of mice bearing OV-90 xenograft tumors after administration of ADC in different treatment groups. The data points represent the mean body weight within the group, and the error bars represent the standard error (SEM).

[0078] Figure 16 shows the relative weight changes (%) of OV-90 xenograft tumor-bearing mice in different treatment groups after administration of ADC. The data points represent the mean weight change percentage within the group, and the error bars represent the standard error (SEM).

[0079] Figure 17 shows the tumor growth curves of OV-90 xenograft tumor-bearing mice in different treatment groups after administration of ADC. The data points represent the mean tumor volume within the group, and the error bars represent the standard error (SEM). DETAILED DESCRIPTION

[0080] The above features and advantages of the present invention and additional features and advantages will be more clearly understood from the following detailed description of embodiments taken in conjunction with the accompanying drawings.

[0081] Throughout this application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those commonly used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0082] Definition of terms

[0083] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies and in some embodiments, reference to "an antibody" includes a plurality of antibodies, and so forth.

[0084] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items. For example, a composition comprising A and / or B can be interpreted as a composition comprising A, a composition comprising B, or a composition comprising A and B.

[0085] Unless otherwise stated or defined, the term “comprise” and variations such as “includes”, “comprising” and “having” will be understood to imply the inclusion of stated elements or steps or groups of elements or steps but not the exclusion of any other elements or steps or groups of elements or steps.

[0086] All numbers, such as pH, temperature, time, concentration and molecular weight, including ranges, are approximate values. It should be understood that all numbers are preceded by the term "about". It should also be understood that the reagents described herein are only exemplary, and their equivalents are known in the art. When relating to measurable values ​​such as amount or concentration, the term "about" generally refers to changes in the range of 0.5%-10% above or below the specified value, such as changes in the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

[0087] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically comprise a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical pathway of complement activation). Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

[0088] A "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of a "framework" region separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs that specifically bind to an antigenic epitope. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0089] The amino acid arrangement of each VL and VH domain is consistent with any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of the Chothia Kabat CDRs, in which CDR-H1 is a composite of the Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition by Martin et al. (world wide web bioinfo.org.uk / abs).

[0090] In the present application, the amino acid sequences of the CDRs are all shown in accordance with the Kabat definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in this area by a variety of methods. In the technical solution of the present invention, the Combined definition rule comprising the Kabat definition and the Chothia definition can also be used to determine the amino acid residues in the variable domain sequence. The Combined definition rule combines the scope of the Kabat definition and the Chothia definition, and takes a larger scope based on this. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or its region (such as a variable region) should be understood to cover the complementary determining region defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection claimed in the present invention is based on the sequence shown in the Kabat definition rule, the amino acid sequence corresponding to the definition rules of other CDRs should also be within the scope of protection of the present invention.

[0091] The term "antibody" as used herein should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit the desired biological activity.

[0092] As used herein, the term "monoclonal antibody" generally refers to the antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies in the cluster are identical, except for a small amount of natural mutations that may exist. Monoclonal antibodies are generally highly specific for a single antigenic site. And, different from conventional polyclonal antibody preparations (generally having different antibodies for different determinants), each monoclonal antibody is for a single determinant on the antigen. Except for their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture, and are not polluted by other immunoglobulins. The modifier "monoclonal" represents the feature of the antibody obtained from a substantially homogeneous antibody population, and is not interpreted as needing to produce antibodies by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared in hybridoma cell, or can be prepared by recombinant DNA method.

[0093] The term "bispecific antibody" should be understood in the context of the present invention as an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a target. The term "bispecific antibody" as used herein should be understood in its broadest sense, including full-length bispecific antibodies and antigen-binding fragments thereof. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing the antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0094] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is less likely to induce an adverse immune response in a human individual than the parent (e.g., mouse-derived) antibody.

[0095] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulin. In the CDR region, small additions, deletions, insertions, replacements, or modifications of amino acids may also be permitted, as long as they still retain the ability of the antibody to bind to a specific antigen. Humanized antibodies may optionally comprise at least a portion of a human immunoglobulin constant region. "Humanized antibodies" retain antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally comprise a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, the CDR region residues in the human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) with desired properties, affinity, and / or ability. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity.

[0096] As used herein, the term "fully human antibody" generally refers to antibodies expressed by animals by transferring human antibody-encoding genes into genetically engineered antibody gene-deficient animals. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Methods for obtaining fully human antibodies in this area include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

[0097] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., FOLR1 of the present invention). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0098] Examples of the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a partial hinge region; (iv) an Fd fragment, which consists of the VH and CH1 domains; (v) an Fd' fragment, which has the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of the VH domain; (viii) a single complementarity determining region (CDR); and (ix) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by synthetic linkers using recombinant methods so that they can form a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv)). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes "straight-chain antibodies" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptides and any modified forms of the aforementioned fragments that retain antigen-binding activity form an antigen-binding region.

[0099] These antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0100] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Avidity is represented by the equilibrium constant (KD) for the dissociation of antigen and antibody, and is a measure of the binding strength between an antigenic determinant and the antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD. Avidity is a measure of the binding strength between an antibody and the associated antigen. Avidity relates to the affinity between an antigenic determinant and the antigen binding site of an antibody and the number of associated binding sites present on the antibody.

[0101] Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and various variations thereof known in the art.

[0102] In the present invention, the term "KD" generally refers to the equilibrium dissociation constant, which is the ratio of the dissociation rate constant (kdis, also known as "off-rate (koff)" or "kd") to the association rate constant (kon, also known as "binding rate (kon)" or "ka"). The association rate constant (kon), dissociation rate constant (kdis) and equilibrium dissociation constant (KD) can be used to represent the binding affinity of an antigen-binding protein (e.g., an antibody) to an antigen. Methods for determining association and dissociation rate constants are well known in the art and include, but are not limited to, biomembrane interferometry (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (Co-IP), and protein chip technology. The affinity of a particular protein-protein interaction measured may be different if measured under different conditions (e.g., salt concentration, pH).

[0103] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or double-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting of, or consisting essentially of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0104] The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably and in their broadest sense refer to a polymeric form of two or more amino acid subunits, amino acid analogs, or peptidomimetics. "Protein," "peptide," "polypeptide," and "amino acid sequence" contain at least two amino acids, and there is no limitation on the maximum number of amino acids. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including D and L optical isomers and amino acid analogs.

[0105] As used herein, "expression" refers to the process by which a nucleic acid sequence is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, amino acid sequence, or protein. If the nucleic acid sequence is from genomic DNA, expression may include splicing of mRNA in a eukaryotic cell.

[0106] The term "encoding" when applied to a nucleic acid sequence refers to a nucleic acid sequence that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA and / or translated to produce a polypeptide. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0107] The term "sequence identity" refers to the sequence similarity between two polypeptides or between two nucleic acid sequences. The percent identity can be determined by comparing positions in each sequence that can be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The degree of identity between sequences depends on the number of shared matching positions. "Unrelated" or "non-homologous" sequences share less than 40% identity, less than 25% identity with one of the sequences of the present invention. The alignment and percent sequence identity of the nucleic acid or amino acid sequences provided herein can be determined by importing the nucleic acid or amino acid sequence into ClustalW (available from https: / / genome.jp / tools-bin / clustalw / ) and using ClustalW.

[0108] When referring to specific molecules, biological materials or cellular substances, the terms "equivalent" or "functional variant" can be used interchangeably and refer to those with minimal homology while still retaining the desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with a reference polypeptide (e.g., an antibody or antigen-binding fragment thereof described herein); or polypeptides encoded by polynucleotides having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with a reference polynucleotide (e.g., a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein).

[0109] Compared to the antibodies or antigen-binding fragments thereof described in the present invention, equivalents with one or more amino acid modifications are also encompassed within the scope of the present invention, provided that the one or more amino acid modifications do not affect or substantially do not affect the ability of the antibody or antigen-binding fragment thereof to specifically bind to the antigen. As used herein, amino acid modifications can be amino acid substitutions, amino acid deletions, or amino acid insertions. Amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions. Conservative substitutions (also referred to as conservative mutations, conservative substitutions, or conservative variations) are amino acid substitutions in proteins that change a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, or size). As used herein, "conservative substitutions" refer to amino acid residues that are replaced by another biologically similar residue. Examples of conservative substitutions include one hydrophobic residue such as isoleucine, valine, leucine, or methionine replacing another; or one charged or polar residue replacing another, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, etc. Other illustrative examples of conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine or glutamic acid; phenylalanine to tyrosine, serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and the like.

[0110] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression by transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0111] As used herein, the term "cell" generally refers to a plasmid or vector that may or has contained a nucleic acid molecule according to the present invention, or an individual cell, cell line, or cell culture that can express an antibody or antigen-binding fragment thereof according to the present invention. The cell may include progeny of a single host cell. Due to natural, accidental, or deliberate mutations, the progeny cells may not necessarily be identical in morphology or genome to the original parent cell, but may be able to express the antibody or antigen-binding fragment thereof described herein. The cell may be obtained by in vitro transfection of cells using the vectors described herein. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell, or a myeloma cell). In some cases, the cell may be a mammalian cell. As used herein, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a specific cell, but also the offspring of these cells.

[0112] In this article, the term "subject" generally refers to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). The term "primate" generally refers to monkey and ape species, and includes monkey species, such as monkeys from the genus Macaca (such as, cynomolgus monkeys (Macaca fascicularis) and / or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), and marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), and ape species, such as chimpanzees (Pan troglodytes), and also includes Homo sapiens.

[0113] The term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and that contains no additional ingredients that are unacceptably toxic to the subject to which the composition is administered. The composition is sterile. A "sterile" composition is sterile, or free of all living microorganisms and their spores.

[0114] As used herein, the term "administering" is intended to mean delivering a substance to a subject, such as an animal or a human. Administration can be performed in a single dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with factors such as the composition used for treatment, the purpose of treatment, and the age, health, or sex of the subject being treated. In some embodiments, single or multiple administrations may be performed, with the dosage level and pattern being selected by a physician, or in the case of pets and other animals, by a veterinarian.

[0115] As used herein, "treatment" includes limiting, slowing down or stopping the progression or severity of a patient's existing symptoms, condition, disease or illness. As understood in the art, "treatment" is a method for obtaining a beneficial or desired result, including a clinical result. For the purposes of the present invention, a beneficial or desired result may include alleviating the extent of one or more diseases or conditions, delaying or slowing the progression of a disease or condition, improving or alleviating the state of a disease or condition (whether partial or complete). The term "prevention" means prophylactically administering an antibody or antigen-binding fragment thereof or ADC of the present invention to an asymptomatic patient or a patient who does not exhibit obvious symptoms to prevent the onset or progression of a disease.

[0116] In this application, the term "tumor" generally refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. In this application, tumors can include solid tumors and / or non-solid tumors (e.g., hematologic tumors, lymphomas).

[0117] As used herein, the term "cancer-associated antigen" or "tumor-associated antigen" refers to an antigen that is differentially expressed in cancer cells or tumor cells compared to normal cells, and thus can be used to target cancer cells or tumor cells.

[0118] In this application, the term "antibody conjugate" generally refers to a substance that an antibody is connected to another chemical moiety. The chemical moiety can be a cytotoxic drug, an immunostimulatory molecule, and a detectable marker. The drug can be, for example, a microtubule inhibitor, an antibiotic, a DNA synthesis inhibitor, a topoisomerase inhibitor, an RNA polymerase II inhibitor, and an RNA spliceosome inhibitor. The terms "antibody-drug conjugate," "antibody conjugate," and "ADC" can be used interchangeably.

[0119] The terms "therapeutic agent," "drug," or "drug moiety" refer to an agent that modulates a biological process and / or possesses biological activity.

[0120] The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with the expression activity and / or function of the cell. Examples of cytotoxic agents include, but are not limited to, antimitotic agents such as eribulin and auristatins (e.g., monomethyl auristatin E (MMAE)).

[0121] In this application, the term "FOLR1" may also be referred to as "folate receptor alpha, FR-alpha, or folate binding protein," which can transport folate via receptor-mediated endocytosis. The term "FOLR1" encompasses any native FOLR1 from any vertebrate source, including mammals, such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). The term "FOLR1" also encompasses "full-length," unprocessed FOLR1, as well as any form of FOLR1 produced by processing in cells. The term also encompasses naturally occurring variants of FOLR1, such as splice variants or allelic variants.

[0122] "Stereoisomers" are compounds that have identical chemical constitutions but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric (cis / trans) isomers, atropisomers, and the like.

[0123] "Chiral" refers to a molecule that is non-superimposable on its mirror image; "achiral" refers to a molecule that is superimposable on its mirror image.

[0124] "Enantiomers" refer to two non-superimposable isomers of a compound that are mirror images of each other.

[0125] "Diastereoisomers" refers to stereoisomers that have two or more chiral neutral groups whose molecules are not mirror images of each other. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Diastereomeric mixtures can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0126] Stereochemical definitions and conventions used herein generally follow SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994.

[0127] Many organic compounds exist in optically active forms, meaning they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are the symbols used to designate the rotation of plane-polarized light caused by the compound, where (-) or l indicates that the compound is left-handed. A compound prefixed with (+) or d is right-handed. A specific stereoisomer is an enantiomer, and a mixture of such isomers is called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.

[0128] Any asymmetric atom (e.g., carbon, etc.) of the compounds disclosed herein can exist in a racemic or enantiomerically enriched form, such as in the (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in terms of the (R)- or (S)-configuration.

[0129] Depending on the choice of starting materials and process, the compounds of the present invention may exist as one of the possible isomers or a mixture thereof, such as a racemate or a mixture of diastereoisomers (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituents may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be in the cis or trans configuration.

[0130] Any resulting mixture of stereoisomers can be separated into the pure or substantially pure geometric isomers, enantiomers, and diastereomers on the basis of the differences in the constituent physicochemical properties, for example, by chromatography and / or fractional crystallization.

[0131] Any racemate of the resulting final product or intermediate can be separated into its optical antipodes by methods familiar to those skilled in the art using known methods, such as by separating the diastereomeric salts obtained. The racemic products can also be separated by chiral chromatography, such as high performance liquid chromatography (HPLC) using a chiral adsorbent. In particular, enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2 nd Ed.Robert E.Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, ELStereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, SHTables of Resolving Agents and Optical Resolutions p.268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G.Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0132] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be converted into each other through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of the tautomers can be reached. For example, proton tautomers (also known as prototropic tautomers) include interconversions performed by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions performed by the reorganization of some bonding electrons. A specific example of keto-enol tautomerism is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-ene-2-one tautomers. Another example of tautomerism is phenol-keto tautomerism. A specific example of phenol-keto tautomerism is the interconversion of pyridine-4-ol and pyridine-4(1H)-one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0133] As described herein, the compounds of the present invention may be optionally substituted with one or more substituents, such as the compounds of the above general formula, or as specifically exemplified in the Examples, and a class of compounds encompassed by the present invention.

[0134] "Pharmaceutically acceptable" means suitable, within the scope of sound medical judgment, for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio.

[0135] As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in S.M. Berge et al., "Describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19." Pharmaceutically acceptable salts formed from non-toxic acids include, but are not limited to, inorganic acid salts formed by reaction with amino groups, such as hydrochlorides, hydrobromides, phosphates, sulfates, and perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, and malonates, or salts obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C1-C4 alkyl) 4 salts. The present invention also contemplates quaternary ammonium salts formed by any compound containing a N group. Water-soluble or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include appropriate, non-toxic ammonium, quaternary ammonium salts and amine cations formed by counter ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 Sulfonates and aromatic sulfonates.

[0136] Certain embodiments of the present invention will now be described in more detail, examples of which are illustrated by the accompanying structural formulas and chemical formulae. The present invention is intended to encompass all substitutions, modifications, and equivalent technical solutions, which are all included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the combined documents, patents, and similar materials differ from or contradict the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0137] It will be further appreciated that certain features of the invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0138] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0139] Unless otherwise indicated, the definitions used herein shall apply. For purposes of the present invention, the chemical elements are consistent with the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994. In addition, general principles of organic chemistry can be found in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0140] Detailed Description of the Invention

[0141] Anti-FOLR1 antibodies

[0142] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to FOLR1 protein, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0143] (1) the VH comprises the heavy chain complementary determining region (HCDR) 1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 49; and the VL comprises the light chain complementary determining region (LCDR) 1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 50;

[0144] (2) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 51; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 52;

[0145] (3) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 53; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 54;

[0146] (4) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 7; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 8;

[0147] (5) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 9; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 10; or

[0148] (6) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 11; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 12;

[0149] The amino acid sequences of HCDRs and LCDRs are defined according to the Kabat, IMGT or Chothia numbering systems or other antibody numbering systems known in the art.

[0150] In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein, VH and VL each comprise the following CDR sequences:

[0151] (1) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 62, 21, and 22, respectively;

[0152] (2) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 16, and 15, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 62, 21, and 22, respectively;

[0153] (3) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 17, 18, and 19, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively;

[0154] (4) the VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22, respectively; or

[0155] (5) The VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 16 and 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22, respectively.

[0156] In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein, VH and VL each comprise the following amino acid sequence:

[0157] (1) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50;

[0158] (2) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52;

[0159] (3) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 53, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 54;

[0160] (4) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 7, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 8;

[0161] (5) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 10; or

[0162] (6) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12.

[0163] In some embodiments, the VH and VL of an anti-FOLR1 antibody include functional variants formed by insertion, deletion and / or substitution of one or more amino acids therein compared to the VH and VL of a reference antibody (e.g., an anti-FOLR1 antibody comprising the VH and VL set forth herein), provided that the functional variant retains the ability to bind to FOLR1.

[0164] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the reference antibody.

[0165] In the context of functional variants, the number of inserted, deleted and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the reference antibody amino acid sequence, more preferably does not exceed 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, and more preferably 15% to 20%. For example, the number of inserted, deleted and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0166] In some embodiments, insertions, deletions and / or substitutions may be made in the framework (FR) regions, eg, in FR1, FR2, FR3 and / or FR4.

[0167] In some embodiments, insertions, deletions and / or substitutions can be in the CDR regions, e.g., CDR1, CDR2 and / or CDR3, provided that the insertions, deletions and / or substitutions do not affect or do not substantially affect the ability of the antibody or antigen-binding fragment thereof to bind to FOLR1.

[0168] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention specifically bind to human FOLR1 protein. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention specifically bind to monkey FOLR1 protein. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention specifically bind to both human FOLR1 protein and monkey FOLR1 protein.

[0169] In some preferred embodiments, the VH and VL of the antibodies disclosed herein respectively comprise the following amino acid sequences:

[0170] (1) the VH comprises the amino acid sequence shown in SEQ ID NO: 49, and the VL comprises the amino acid sequence shown in SEQ ID NO: 50;

[0171] (2) the VH comprises the amino acid sequence shown in SEQ ID NO: 51, and the VL comprises the amino acid sequence shown in SEQ ID NO: 52;

[0172] (3) the VH comprises the amino acid sequence shown in SEQ ID NO: 53, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54;

[0173] (4) the VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO: 8;

[0174] (5) the VH comprises the amino acid sequence shown in SEQ ID NO: 9, and the VL comprises the amino acid sequence shown in SEQ ID NO: 10; or

[0175] (6) The VH comprises the amino acid sequence shown in SEQ ID NO: 11, and the VL comprises the amino acid sequence shown in SEQ ID NO: 12.

[0176] In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0177] Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of the antibody can be divided into lambda (λ) chain and kappa (κ) chain. The antibodies disclosed herein can be any of the above classes or subtypes.

[0178] In some embodiments, the antibody is an antibody isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is an antibody subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0179] In some embodiments, the antibodies of the present invention comprise an Fc region. The Fc region can be of any isotype or subtype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can comprise one or more substitutions or modifications. In some embodiments, the Fc region is an IgG1 Fc region or is derived therefrom, optionally comprising one or more substitutions or modifications. In some embodiments, the Fc region comprises one or more amino acid substitutions that extend the half-life of the antibody. In preferred embodiments, the Fc region comprises the L234A and L235A amino acid substitutions.

[0180] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions include substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another; or substitution of one charged or polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, etc. Preferred exemplary conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine or glutamic acid; phenylalanine to tyrosine, serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; etc.

[0181] In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein, the antibody comprises a heavy chain (HC) and a light chain (LC), wherein

[0182] (1) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:43, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:44;

[0183] (2) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 45, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 46;

[0184] (3) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 47, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 48;

[0185] (4) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2;

[0186] (5) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:3, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:4; or

[0187] (6) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:5, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:6.

[0188] In some embodiments, the HC and LC of an anti-FOLR1 antibody include functional variants formed by insertion, deletion and / or substitution of one or more amino acids therein compared to the HC and LC of a reference antibody, provided that the functional variant retains the ability to bind to FOLR1.

[0189] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the reference antibody.

[0190] In some embodiments, the number of inserted, deleted and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the reference antibody amino acid sequence, more preferably no more than 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, and more preferably 15% to 20%. For example, the number of inserted, deleted and / or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0191] In some embodiments, insertions, deletions and / or substitutions may be made in the framework (FR) regions, e.g., FR1, FR2, FR3 and / or FR4; and / or constant regions, e.g., CL, CH1, CH2 and / or CH3.

[0192] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0193] In some preferred embodiments, the heavy and light chains of the antibodies disclosed herein respectively comprise the following amino acid sequences:

[0194] (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 43, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 44;

[0195] (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:45, and the light chain comprises the amino acid sequence shown in SEQ ID NO:46;

[0196] (3) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 47, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 48;

[0197] (4) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 2;

[0198] (5) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 3, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; or

[0199] (6) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 5, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 6.

[0200] The antibodies disclosed herein may be intact antibodies or antigen-binding fragments thereof. An antigen-binding fragment may be any fragment of an antibody that retains the ability to specifically bind to FOLR1. Examples of antigen-binding fragments include, but are not limited to, Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; individual complementarity determining regions (CDRs); nanobodies; linear antibodies consisting of a pair of tandem Fd fragments (VH-CH1-VH-CH1), and modified forms of any of the foregoing fragments that retain antigen-binding activity.

[0201] In some embodiments, the antibody is a monovalent, bivalent, or multivalent antibody.

[0202] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0203] In some embodiments, the antibody is a bispecific antibody. The bispecific antibody comprises an antibody or antigen-binding fragment thereof that binds to FOLR1 and a second antigen-binding domain or region. The second antigen can be selected from a cancer-associated antigen, an immune cell antigen, and an immune checkpoint molecule.

[0204] Many cancer-associated antigens relevant to specific cancers have been identified in this area. In some embodiments, cancer-associated antigens are antigens that can stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as antigens that are usually silent (i.e., not expressed) in normal cells, antigens that are only expressed at certain stages of differentiation, and antigens expressed over time such as embryonic and fetal antigens. Other cancer cell antigens are encoded by mutant cell genes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., P53 mutants), and fusion proteins produced by internal deletions or chromosomal translocations. Other cancer antigens can be encoded by genes carried by viral genes such as RNA and DNA tumor viruses. Many other cancer-associated antigens and antibodies against them are known and / or commercially available, and can also be prepared by those skilled in the art.

[0205] Examples of cancer associated antigens include, but are not limited to, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, and combinations thereof.

[0206] In some embodiments, the second antigen is an immune cell antigen, such as a T cell antigen. In some embodiments, the T cell antigen is selected from T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) and NKG2D or any combination thereof. In some embodiments, the T cell antigen is CD3, and the second antigen binding region is combined with any one of the gamma chains, delta chains, epsilon chains, zeta chains and n chains of CD3.

[0207] In some embodiments, the second antigen is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule can be selected from PD-1, PD-L1, CTLA-4, CD4, CD40, CD80, CD86, B7-H3, LAG3, TIM-3, IDO1, etc.

[0208] Nucleic acid molecules

[0209] In yet another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof disclosed herein.

[0210] For example, the present invention provides nucleic acid molecules encoding the CDR sequences in any one of the VH and / or VL disclosed herein. In some embodiments, the present invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid molecules encoding the CDR sequences in any one of the VH and / or VL disclosed herein.

[0211] For another example, the present invention provides nucleic acid molecules encoding any one of the VH and / or VL disclosed herein. In some embodiments, the present invention also provides nucleic acid molecules encoding any one of the VH and / or VL disclosed herein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0212] For another example, the present invention provides nucleic acid molecules encoding any one of the heavy and / or light chains of the antibodies disclosed herein. In some embodiments, the present invention also provides nucleic acid molecules encoding any one of the heavy and / or light chains of the antibodies disclosed herein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0213] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an antibody disclosed herein.

[0214] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of the cell.

[0215] In some embodiments, the ribonucleic acid (RNA) can be introduced into human cells in vivo.In some embodiments, the ribonucleic acid (RNA) of the present invention is contained in a vector or delivery agent.

[0216] carrier

[0217] In another aspect, the present invention provides a vector comprising a nucleic acid molecule disclosed herein, which comprises a nucleotide sequence encoding an anti-FOLR1 antibody or antigen-binding fragment thereof disclosed herein.

[0218] In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising the heavy chain or light chain variable region of an antibody.For example, the present invention provides an expression vector comprising any of the above-mentioned nucleic acid molecules.

[0219] Any vector can be suitable for use in the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus (AAV) vector, a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pBY, pGAR, pBABE-puro, pBABE-neo largeT cDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0220] The vector can be any suitable recombinant expression vector. Suitable vectors include vectors designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, vectors can be selected from pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript sequence (Stratagene, LaJolla, Calif.), pET sequence (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors that can be used for the present invention include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the present invention include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0221] Recombinant expression vectors can be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. The replication system can be derived, for example, from ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0222] For example, the vector can be an adenovirus vector comprising a nucleotide sequence encoding an antibody disclosed herein. The vector can be administered to a subject and then enter the subject's cells, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the genome of the cell, which then expresses the antibody disclosed herein.

[0223] cell

[0224] In another aspect, the present invention provides a cell comprising a nucleic acid molecule disclosed herein or a vector disclosed herein.

[0225] Any cell can be used as the host cell of the nucleic acid molecule of the present invention or vector. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include but are not limited to true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae (Enterobactehaceae), such as Escherichia (Escherichia), such as Escherichia coli (E.coli); Enterobacter (Enterobacter); Erwinia (Erwinia); Klebsiella (Klebsiella); Proteus (Proteus); Salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); Serratia (Serratia), such as Serratia marcescens (Serratia marcescens). marcescans and Shigella; Bacilli, such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0226] The cells of the present invention are prepared by introducing the vectors disclosed herein or the nucleic acid molecules disclosed herein in vitro or ex vivo. The cells of the present invention can be administered to a subject, and the cells express the antibodies or antigen-binding fragments thereof disclosed herein in vivo.

[0227] Preparation method

[0228] In another aspect, the present invention provides a method for preparing the antibodies or antigen-binding fragments thereof disclosed herein, comprising culturing the cells provided by the present invention under conditions suitable for expression of the antibodies or antigen-binding fragments thereof disclosed herein; and isolating the antibodies or antigen-binding fragments thereof from the culture and / or culture supernatant of the cells.

[0229] Any method suitable for producing antibodies can be used to produce the antibodies of the present invention. For example, antibodies can be prepared using a hybridoma method, such as the method described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0230] Immunizing agents typically include protein antigens, fragments thereof, or fusion proteins thereof. In this context, any suitable form of FOLR1 can be used as an immunizing agent (antigen) to generate antibodies specific for FOLR1 and screen for the biological activity of the antibodies. For example, antigens suitable for use in the present invention include, but are not limited to, full-length human FOLR1 protein, full-length monkey FOLR1 protein, fragments thereof, or derivatives thereof.

[0231] The immunizing agent can be used alone or in combination with one or more immunogenicity enhancers known in the art. Typically, if human cells are needed, peripheral blood lymphocytes are used; if non-human mammalian sources are needed, spleen cells or lymph node cells are used. Lymphocytes are then fused with immortalized cell lines using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103). Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically will contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0232] Then can determine whether there is monoclonal antibody for antigen in the culture medium of culture hybridoma cell.Preferably, the binding specificity of the monoclonal antibody produced by hybridoma cell is measured by immunoprecipitation or by in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such technology and determination are known in the art.The binding affinity of monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal.Biochem., 107:220 (1980).In addition, in the therapeutic application of monoclonal antibody, it is important to identify antibodies with high specificity and high binding affinity to the target antigen.

[0233] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103.) Suitable culture media for this purpose include, for example, DMEM and RPMI-1640. Alternatively, hybridoma cells can be grown in vivo as ascites in mammalian fluid. The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0234] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). The hybridoma cells of the present invention are used as a preferred source of such DNA. The isolated DNA can be placed in an expression vector and then transfected into a host cell, such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain antibodies in recombinant host cells that synthesize monoclonal antibodies. The DNA can also be modified, for example, by replacing the homologous mouse sequence with the coding sequence of the human heavy and light chain constant domains (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0235] The sequences of the antibodies of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

[0236] Once the relevant sequence is obtained, the relevant antibody can be obtained in large quantities using recombinant methods. This typically involves cloning the sequence into a vector, transferring it into cells, and then isolating the relevant antibody from the propagated host cells using conventional methods. Alternatively, synthetic methods can be used to synthesize the relevant sequence, especially when the fragment length is relatively short. Typically, very long fragments of sequence can be obtained by first synthesizing multiple small fragments and then ligating them. The nucleic acid molecule can then be introduced into various existing vectors known in the art, which can be used to transform appropriate host cells to enable protein expression, thereby obtaining the antibodies of the present invention.

[0237] Chimeric antigen receptor and cell containing the same

[0238] In another aspect, the present invention provides a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof disclosed herein.

[0239] The classic chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that connects the extracellular antigen binding domain to the intracellular signaling domain. The antigen binding domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other forms of antibody-like antigen binding sites or antigen binding domains derived from the natural ligands of the antigen. A hinge domain is usually required to separate the antigen binding domain from the membrane and allow it to be properly oriented. The common hinge domain used is the Fc of IgG1. Depending on the antigen, a more compact spacer can meet the conditions, such as the stem from CD8α, and even just a single IgG1 hinge. The transmembrane domain anchors the protein in the cell membrane and connects the hinge domain to the intracellular domain (intracellular domain).

[0240] According to at least one non-limiting viewpoint, there are at least three "generations" of CAR molecules. In the first generation of CAR, it is designed to have an intracellular domain with an intracellular portion of the γ chain or CD3ζ derived from FcεR1. Therefore, these first generation CARs transmit immune signal 1, which is sufficient to trigger T cell killing of homologous target cells, but cannot fully activate T cell proliferation and survival. In order to overcome this limitation, a second generation of CAR has been constructed, which has a composite intracellular domain produced by fusion of the intracellular portion of the T cell costimulatory molecule with the intracellular portion of CD3ζ, so that activation signals and costimulatory signals can be transmitted simultaneously after antigen recognition. The most commonly used costimulatory domain is the costimulatory domain of CD28. This provides the most powerful costimulatory signal, i.e., immune signal 2, which triggers T cell proliferation. Some CARs have also been described, which include TNF receptor family intracellular domains, such as the closely related OX40 and 41BB that transmit survival signals. Even more powerful third generation CARs have now been described, which have intracellular domains capable of transmitting activation, proliferation and survival signals.

[0241] Thus, a CAR typically comprises: (i) an antigen binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain comprising a signaling domain and one or more costimulatory domains.

[0242] In the context of referring to CAR, "antigen binding domain" refers to the part of the chimeric antigen receptor that recognizes the antigen. In classical CAR, the antigen binding domain includes: a single-chain variable fragment (scFv) derived from a monoclonal antibody. CAR has also been produced using domain antibodies (dAbs), VHH antigen binding domains or antigen binding domains derived from natural ligands of antigens.

[0243] In some embodiments, the antigen binding domain is derived from an anti-FOLR1 antibody of the invention. In some embodiments, the antigen binding domain comprises a scFv derived from an anti-FOLR1 antibody of the invention.

[0244] "Hinge region" refers to the extracellular domain of the CAR molecule that is located between the antigen binding domain and the transmembrane domain and spatially separates the antigen binding domain from the intracellular domain. The hinge region may also be referred to as a "hinge domain" or "spacer". The hinge may contribute to receptor expression, activity and / or stability. The hinge may also provide flexibility in binding to the target antigen to allow the antigen binding domain to be oriented in different directions to facilitate binding.

[0245] The hinge region can be derived from a natural source or from a synthetic source. In some embodiments, the hinge region can comprise a hinge or fragment thereof selected from CD8, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. Preferably, the hinge region can be a CD8 hinge.

[0246] "Transmembrane domain" refers to a domain that has the property of being present in a membrane when present in a molecule at the cell surface or cell membrane (e.g., spanning part or all of the cell membrane). A transmembrane domain can be any protein structure that is thermodynamically stable in the membrane. This is typically an alpha helix containing several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of a chimeric receptor. The presence and span of a transmembrane domain of a protein can be determined by a person skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Alternatively, an artificially designed TM domain can be used.

[0247] The type of membrane-spanning domain included in the CAR of the present invention is not limited to any type. In some embodiments, the membrane-spanning domain naturally associated with the antigen-binding domain and / or the intracellular domain is selected. In some cases, the membrane-spanning domain includes one or more amino acid modifications (such as deletions, insertions and / or substitutions), such as to avoid such domains from being combined with the membrane-spanning domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The membrane-spanning domain can be derived from natural origin or from synthetic sources. When the source is natural origin, the domain can be derived from any membrane-bound protein or transmembrane protein. In some embodiments, the transmembrane domain can include a transmembrane domain selected from the group consisting of the α chain of a T cell receptor (TCR), the β chain of a TCR, the ζ chain of a TCR, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD19, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (41BB), CD152, CD154, and PD1, or any combination thereof. Preferably, the transmembrane domain is the transmembrane domain of CD8.

[0248] The intracellular domain (intracellular domain) is the signal transmission part of the chimeric antigen receptor. It includes a signal transduction domain and one or more costimulatory domains. After antigen recognition, the receptor cluster natural CD45 and CD148 are excluded from the synapse, and the signal is transmitted to the cell, thereby activating one or more immune cell effector functions (such as innate immune cell effector functions). The most commonly used intracellular domain component is the intracellular domain component of CD3ζ containing 3 ITAMs. After antigen binding, it transmits activation signals to T cells. CD3ζ may not provide completely sufficient activation signals, and may require additional costimulatory signal conduction. Costimulatory signals promote T cell proliferation and survival. There are two main types of costimulatory signals: costimulatory signals belonging to the Ig family (CD28, ICOS) and the TNF family (OX40, 41BB, CD27, GITR, etc.).

[0249] The signaling domain of the intracellular domain mediates the activation of at least one normal effector function of an immune cell. For example, the effector function of a T cell can be a cytolytic activity or auxiliary activity comprising cytokine secretion. In some embodiments, the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival and / or other T cell functions. The intracellular domain can include a signaling domain (intracellular signaling domain) as an activation domain. The intracellular domain can also include a signaling domain (intracellular costimulatory domain) as a costimulatory signaling domain. In some embodiments, the intracellular signaling domain can include a signaling region selected from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD5, CD22, CD79a, CD79b, ICOS (CD278) and CD66d, or any combination thereof. In some embodiments, the intracellular signaling domain includes a signaling region of CD3 ζ.

[0250] In some embodiments, the intracellular costimulatory domain can be selected from 4-1BB (CD137), OX40 (CD134), ICOS (CD278), 2B4, HVEM, LAG3, DAP10, DAP12, CD27, CD28, CD30, CD40, glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), MyD88, CD2, CD4, CD7, LIGHT, NKG2C and B7-H3 intracellular signaling region or its any combination.In some embodiments, the intracellular costimulatory domain is the intracellular signaling region of 41BB.

[0251] In another aspect, the present invention provides a genetically modified cell comprising a chimeric antigen receptor disclosed herein. In some embodiments, the genetically modified cell may express a chimeric antigen receptor disclosed herein.

[0252] In some embodiments, the genetically modified cells are eukaryotic cells. In preferred embodiments, the genetically modified cells are isolated human cells. In some embodiments, the genetically modified cells are selected from lymphocytes (e.g., T cells, NK cells) and monocytes (e.g., PBMCs). In some embodiments, the genetically modified cells are T cells. T cells can be any T cells, such as cultured T cells, such as primary T cells or T cells from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or liquids. T cells can also be enriched or purified. Preferably, T cells are human T cells. More preferably, T cells are T cells separated from people. T cell can be any type of T cell and can be the T cell of any developmental stage, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells, such as Th1 and Th2 cells, CD4+T cells, CD8+T cells (for example, cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (for example, central memory T cells and effector memory T cells), initial T cells etc. In some embodiments, the cell of genetic modification is an immune cell, preferably T cell, NK cell or macrophage. In certain embodiments, the cell of genetic modification is stem cell, preferably pluripotent stem cell, induced pluripotent stem cell (iPSC), mesenchymal stem cell, hematopoietic stem cell or lymphoid progenitor cell. In preferred embodiments, the cell of genetic modification is T cell or NK cell.

[0253] Antibody conjugates and antibody-drug conjugates

[0254] In another aspect, the present invention provides an antibody conjugate comprising an antibody or antigen-binding fragment thereof disclosed herein and a chemical moiety conjugated to the antibody or antigen-binding fragment thereof.

[0255] In the context of the present disclosure, an "antibody conjugate" or "antibody conjugate" is an antibody or antibody fragment (e.g., an antigen binding fragment) covalently linked to a chemical moiety. The chemical moiety can be selected from a cytotoxic drug, an immunostimulatory molecule, and a detectable marker, such as a drug, a toxin, a therapeutic agent, a detectable marker, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. When an antibody conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody drug conjugate" or "ADC."

[0256] The terms "coupled," "conjugated," or "linked" may refer to the making of two polypeptides into one continuous polypeptide molecule. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, the antibody linked to a chemical moiety is further linked to a lipid or other molecule to a protein or peptide to increase its half-life in vivo. The linking can be performed chemically or recombinantly. In one embodiment, the linking is chemical, wherein the reaction between the antibody moiety and the chemical moiety produces a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the chemical moiety.

[0257] Chemical moieties can be attached to the antibodies of the invention using any number of methods known to those skilled in the art. Herein, the number of chemical moieties attached to a single antibody of the invention can be represented by "DAR." For example, in some embodiments, "DAR" represents the number of linkers-payloads attached to a single antibody of the invention.

[0258] Covalent and non-covalent attachment modes can be used. The procedure for attaching the chemical moiety to the antibody varies according to the chemical structure of the chemical moiety. Polypeptides generally contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) moieties, which can be used to react with suitable functional groups on the antibody to cause the combination of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization can involve attaching any of many known linker molecules. The linker can be any molecule for connecting the antibody to the chemical moiety. The linker can form a covalent bond with both the antibody and the chemical moiety. Suitable linkers are well known to those skilled in the art, and include but are not limited to straight or branched carbon linkers, heterocyclic carbon linkers or peptide linkers. In the case where the antibody and the chemical moiety are polypeptides, the linker can be connected to the constituent amino acids (such as by disulfide bonds to cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acids through their side groups.

[0259] In some cases, it is desirable to release the chemical moiety from the antibody when the antibody conjugate reaches its target site. Therefore, in these cases, the antibody conjugate will contain a linkage that is cleavable near the target site.

[0260] Enzymatic activity or conditions to which the antibody conjugate is subjected within the target cell or near the target site may cause cleavage of the linker to release the chemical moiety from the antibody.

[0261] Given the multitude of methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one skilled in the art will be able to determine the appropriate method for attaching a given agent to an antibody or other polypeptide.

[0262] The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). Typically, the antibody or portion thereof is derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a bivalent antibody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0263] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same or different types). Suitable cross-linking agents include heterobifunctional cross-linkers having two distinct reactive moieties (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) separated by a suitable spacer, or homobifunctional cross-linkers (e.g., disuccinimidyl suberate). Such linkers are commercially available.

[0264] In some embodiments of the conjugates disclosed herein, the chemical moiety is selected from a therapeutic agent (eg, a cytotoxic drug), a detectable moiety, and an immunostimulatory molecule.

[0265] In some embodiments, therapeutic agents include but are not limited to immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins. In some embodiments, therapeutic agents can be selected from microtubule inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors. In some embodiments, therapeutic agents can be, for example, maytansine, geldanamycin, microtubule inhibitors such as microtubule binding agents (e.g., auristatins), or minor groove binding agents such as calicheamicin.

[0266] Other suitable therapeutic agents include, for example, small molecule cytotoxic agents, i.e., compounds with a molecular weight of less than 700 Daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals that can have cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decompose or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, razithromycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, metformin, auristatin E, vincristine, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes, and RNA enzymes; radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225, and astatine-213; chelating agents can be used to facilitate the binding of these radionuclides to molecules or polymers thereof.

[0267] In some embodiments, the detectable moiety can be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

[0268] The antibody can be coupled to a detectable label; for example, a detectable label that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optics, and laparoscopy. Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent labels such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) can also be used.

[0269] Antibodies or Fabs can also be coupled to enzymes that can be used for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When antibodies or Fabs are coupled to detectable enzymes, they can be detected by adding additional reagents that the enzyme is used to produce a recognizable reaction product. For example, when there is a horseradish peroxidase reagent, the addition of hydrogen peroxide and diaminobenzidine can result in a colored reaction product that can be detected visually. Antibodies or Fabs can also be coupled to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be coupled to an enzyme or a fluorescent label.

[0270] Antibodies can be fused to self-labeled protein tags (e.g., HaloTag). For example, protein tags can be cloned into the end of a constant region. HaloTag is a self-labeled protein tag derived from a bacterial enzyme (haloalkane dehalogenase) that is intended to be covalently bound to a synthetic ligand. In some cases, the synthetic ligand comprises a chloroalkane linker (Los et al. (2008) ACS Chem Biol. 3 (6): 373-82) attached to a fluorophore such as a near-infrared fluorophore.

[0271] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0272] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with a predetermined polypeptide epitope (such as a leucine zipper pair sequence, a binding site for a second antibody, a metal binding domain, an epitope tag) recognized by a second reporter gene. In some embodiments, the tag is attached by spacer arms of various lengths to reduce potential steric hindrance.

[0273] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for diagnostic and therapeutic purposes. For example, radiolabels can be used to detect expression of the target antigen by X-rays, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I.

[0274] In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule can be a cytokine such as IL-2 and IFN-γ, a chemokine such as IL-8, platelet factor 4, melanoma growth stimulating protein, a complement activator; a viral / bacterial protein domain, or a viral / bacterial peptide.

[0275] In some embodiments of the antibody drug conjugates disclosed herein, the antibody drug conjugate has the structure of Formula (I):

[0276] Ab-(L-(D) v ) w (I)

[0277] wherein Ab is an antibody or antigen-binding fragment thereof disclosed herein,

[0278] L is a linker,

[0279] D is a drug group,

[0280] v is 1, 2 or 3, preferably 3;

[0281] w ranges from about 1 to about 20, preferably 1 to 8, and more preferably w is 2 or 4.

[0282] In some embodiments, the -L-(D) v Selected from the compound of formula (II), or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0283] in,

[0284] Indicates -L-(D) v The site of attachment to Ab;

[0285] a, b, c, d, e, and f are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably 0, 1, 2, 3, 4, or 5; preferably 0, 1, 2, or 3; preferably 1 or 2;

[0286] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably 1, 2, 3, 4 or 5; preferably 1, 2 or 3; preferably 1;

[0287] m is 2 or 3;

[0288] X is C, N or Si;

[0289] A is selected from

[0290] R is

[0291] Where D is the drug group;

[0292] n2 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; preferably 7, 8, 9, 10 or 11;

[0293] Y is a bond or

[0294] wherein n1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably 1, 2, 3, 4 or 5; preferably 1, 2 or 3;

[0295] b1, c1, d1 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably 0, 1, 2, 3, 4 or 5; preferably 0, 1, 2 or 3; preferably 1 or 2;

[0296] When X is C or Si, m is 3;

[0297] When X is N, m is 2.

[0298] In some embodiments, the -L-(D) v A compound selected from formula (III), (III-1) or (III-2), or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof:

[0299] wherein R is as defined herein.

[0300] In some embodiments, R is

[0301] Preferably,

[0302] n2 is 7 or 11.

[0303] In some embodiments, the drug group in the antibody drug conjugate is derived from eribulin, methyl auristatin E, or SN-38, and eribulin, methyl auristatin E, and SN-38 have the following structures:

[0304] Preferably, the drug group is selected from: More preferably

[0305] In some embodiments, the -L-(D) v Selected from the following specific compounds or their tautomers, stereoisomers or pharmaceutically acceptable salts, which are represented by the structure shown in formula (IV)

[0306] Among them, Rx is

[0307] And n2, * and Payload are defined as follows:

[0308] The following compounds are particularly preferred:

[0309] Where Rx is:

[0310] In some preferred embodiments, the -L-(D) v The compound represented by formula (IV) or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0311] Among them, Rx is

[0312] In some preferred embodiments, the -L-(D) vThe compound represented by formula (IV) or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0313] Among them, Rx is

[0314] In some preferred embodiments, the antibody drug conjugate disclosed herein is selected from:

[0315] in,

[0316] a. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO:43, the light chain comprises the amino acid sequence set forth in SEQ ID NO:44, and w is 2;

[0317] b. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO:45, the light chain comprises the amino acid sequence set forth in SEQ ID NO:46, and w is 2;

[0318] c. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO:47, the light chain comprises the amino acid sequence set forth in SEQ ID NO:48, and w is 2;

[0319] d. The heavy chain of Ab comprises the amino acid sequence shown in SEQ ID NO: 1, the light chain comprises the amino acid sequence shown in SEQ ID NO: 2, and w is 2;

[0320] e. The heavy chain of Ab comprises the amino acid sequence shown in SEQ ID NO: 3, the light chain comprises the amino acid sequence shown in SEQ ID NO: 4, and w is 1, 2 or 3; or

[0321] f. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO: 5, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 6, and w is 2;

[0322] or

[0323] in,

[0324] a. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO:43, the light chain comprises the amino acid sequence set forth in SEQ ID NO:44, and w is 2;

[0325] b. The heavy chain of Ab comprises the amino acid sequence shown in SEQ ID NO:45, the light chain comprises the amino acid sequence shown in SEQ ID NO:46, and w is 2; or

[0326] c. The heavy chain of Ab comprises the amino acid sequence shown in SEQ ID NO: 47, the light chain comprises the amino acid sequence shown in SEQ ID NO: 48, and w is 2.

[0327] Pharmaceutical composition

[0328] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, a cell disclosed herein, a chimeric antigen receptor disclosed herein, a genetically modified cell disclosed herein, and / or an antibody conjugate or antibody drug conjugate disclosed herein, and optionally a pharmaceutically acceptable carrier.

[0329] In some embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0330] As used herein, the term "pharmaceutically acceptable" refers to a carrier or excipient that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0331] The exemplary carriers used in pharmaceutical compositions of the present invention include saline, buffered saline, glucose and water. The exemplary excipients used in compositions of the present invention include fillers, adhesives, disintegrants, coating agents, adsorbents, antiadherents, glidants, preservatives, antioxidants, flavorings, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tension regulators. Suitable excipients are known to those skilled in the art to prepare pharmaceutical compositions of the present invention. Usually, the selection of suitable excipients especially depends on the desired dosage form of employed activating agent, disease to be treated and compositions.

[0332] Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media for pharmaceutically active substances is well known in the art. Unless any conventional media is incompatible with the active compound, it is contemplated for use in the composition. Supplementary active compounds may also be incorporated into the composition.

[0333] The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended route of administration. The pharmaceutical compositions of the present invention are preferably administered parenterally, by injection, or orally. Injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The pharmaceutical compositions are in various dosage forms conventional in the art, preferably in solid, semisolid, or liquid form, i.e., aqueous, non-aqueous, or suspension solutions, more preferably tablets, capsules, granules, injections, or infusions. More preferably, they are administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the pharmaceutical compositions can also be administered as an aerosol or coarse spray, i.e., nasally; or intrathecally, intramedullary, or intraventricularly. More preferably, the pharmaceutical compositions can also be administered transdermally, percutaneously, topically, enterally, intravaginally, sublingually, or rectally. The pharmaceutical compositions of the present invention can be formulated into various dosage forms as needed, and a physician can determine the dosage that is beneficial to the patient based on factors such as the patient's type, age, weight, general condition, and route of administration. Administration may be by injection or other therapeutic methods.

[0334] The dosage level of the pharmaceutical composition of the present invention can be adjusted according to the amount of the composition reaching the required diagnosis or treatment result. The administration regimen can also be a single injection or multiple injections, or adjusted. The selected dosage level and regimen depend on the activity and stability (that is, half-life) of the pharmaceutical composition, preparation, route of administration, combination with other drugs or treatment, disease to be detected and / or treated or illness and the health status of the subject to be treated and various factors such as previous medical history and reasonably adjust.

[0335] Reagent test kit

[0336] In another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, a cell disclosed herein, a chimeric antigen receptor disclosed herein, a genetically modified cell disclosed herein, an antibody conjugate or antibody drug conjugate disclosed herein, and / or a pharmaceutical composition disclosed herein, and optionally instructions for administering the above-mentioned substances. In some embodiments, the kit further comprises an administration device.

[0337] In some embodiments, the present invention provides a kit that may include an antibody or its antigen-binding fragment, nucleic acid molecule, vector, cell, chimeric antigen receptor, genetically modified cell, antibody conjugate or antibody drug conjugate and / or pharmaceutical composition disclosed herein. It may include an antibody or its antigen-binding fragment, nucleic acid molecule, vector, cell, chimeric antigen receptor, genetically modified cell, antibody conjugate or antibody drug conjugate and / or pharmaceutical composition of the present invention in a single common container. In some cases, the kit may include instructions for use, which include information about the antibody or its antigen-binding fragment, nucleic acid molecule, vector, cell, chimeric antigen receptor, genetically modified cell, antibody conjugate or antibody drug conjugate and / or pharmaceutical composition and dosage form in the kit. Typically, such information helps patients and physicians use encapsulated antibodies or their antigen-binding fragments, nucleic acid molecules, vectors, cells, chimeric antigen receptor, genetically modified cells, antibody conjugate or antibody drug conjugate and / or pharmaceutical composition effectively and safely. The container used in such a kit may typically include at least one vial, test tube, flask, bottle, syringe or other suitable container.

[0338] In some embodiments, the present invention provides a kit, which may include an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a cell, a chimeric antigen receptor, a genetically modified cell, an antibody conjugate or an antibody drug conjugate and / or a pharmaceutical composition disclosed herein, instructions for use of the above-mentioned substance, and an administration device for administering the above-mentioned substance. The administration device can introduce a substance into the patient's body through a parenteral route (e.g., intramuscularly, subcutaneously, or intravenously). For example, the administration device can be a syringe (e.g., a syringe pre-filled with an antibody or pharmaceutical composition of the present invention, such as an automatic syringe), which may include a syringe and a needle (which may be used to pierce the skin and / or blood vessels) for accommodating a fluid to be injected (e.g., an antibody or pharmaceutical composition of the present invention). The mode of administration can be changed. The route of administration may include oral administration, intramuscular injection, subcutaneous injection, rectal administration, etc.

[0339] Methods and uses

[0340] In another aspect, the present invention provides a method for treating, alleviating and / or preventing a FOLR1-mediated disease in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, a cell disclosed herein, a chimeric antigen receptor disclosed herein, a genetically modified cell disclosed herein, an antibody conjugate or antibody drug conjugate disclosed herein and / or a pharmaceutical composition disclosed herein.

[0341] In some embodiments, FOLR1-mediated diseases include cancers with high FOLR1 expression. "FOLR1-high-expressing cancer" refers to cancer cells or tumor cells that express significantly higher levels of FOLR1 relative to normal tissues or cells. In some preferred embodiments, the cancer is selected from lung cancer, such as non-small cell lung cancer, ovarian cancer, mesothelioma, breast cancer, such as triple-negative breast cancer, endometrial cancer, fallopian tube cancer, or primary peritoneal cancer.

[0342] As used herein, the term "effective amount" means an amount of a compound that, when administered to a subject for the treatment, alleviation or prevention of a disease, is sufficient to achieve the therapeutic, alleviating or preventive effects described herein. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated. A "therapeutically effective amount" refers to an effective amount for therapeutic treatment. A "prophylactically effective amount" refers to an effective amount for prophylactic treatment.

[0343] In some embodiments, the dosage or effective amount administered to a subject may vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine the effective amount to administer to a human or other subject to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as the activity of the antibody and the route of administration.

[0344] The dosage of the antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, cells, chimeric antigen receptors, genetically modified cells, antibody conjugates or antibody drug conjugates and / or pharmaceutical compositions described herein can be administered to a mammal at once or in a series of sub-doses over an appropriate time period, for example, daily, semi-weekly, weekly, biweekly, semi-monthly, bimonthly, semi-annually or annually as needed. A dosage unit comprising an effective amount of an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, cell, chimeric antigen receptor, genetically modified cell, antibody conjugate or antibody drug conjugate and / or pharmaceutical composition can be administered as a single daily dose, or the total daily dose can be administered as two, three, four or more divided doses administered daily as needed.

[0345] The appropriate mode of administration can be selected by the physician. The route of administration can be parenteral administration, for example, by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. The dosage and method of administration can vary according to the weight, age, condition, etc. of the subject and can be appropriately selected.

[0346] In another aspect, the present invention provides use of an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, a cell disclosed herein, a chimeric antigen receptor disclosed herein, a genetically modified cell disclosed herein, an antibody conjugate or antibody drug conjugate disclosed herein, and / or a pharmaceutical composition disclosed herein in the preparation of a medicament for treating, alleviating, and / or preventing a FOLR1-mediated disease in a subject.

[0347] In some embodiments, FOLR1-mediated diseases include cancers with high FOLR1 expression. "FOLR1-high-expressing cancer" refers to cancer cells or tumor cells that express significantly higher levels of FOLR1 relative to normal tissues or cells. In some preferred embodiments, the cancer is selected from lung cancer, such as non-small cell lung cancer, ovarian cancer, mesothelioma, breast cancer, such as triple-negative breast cancer, endometrial cancer, fallopian tube cancer, or primary peritoneal cancer.

[0348] In some embodiments, the antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, cells, chimeric antigen receptors, genetically modified cells, antibody conjugates or antibody drug conjugates and / or pharmaceutical compositions of the present application can inhibit or delay the development or progression of a disease, can reduce tumor size (or even substantially eliminate the tumor), and / or can alleviate and / or stabilize the disease state.

[0349] In some embodiments of the uses disclosed herein, the antibodies or antigen-binding fragments thereof disclosed herein, the nucleic acid molecules disclosed herein, the vectors disclosed herein, the cells disclosed herein, the chimeric antigen receptors disclosed herein, the genetically modified cells disclosed herein, the antibody conjugates or antibody drug conjugates disclosed herein and / or the pharmaceutical compositions disclosed herein are used in combination with a second therapeutic agent. In some embodiments, the drug comprises a second therapeutic agent. In some embodiments, the drug is used in combination with a second therapeutic agent.

[0350] In some embodiments of the methods and uses disclosed herein, the second therapeutic agent can be selected from antibodies, chemotherapeutic agents, antisense oligonucleotides (such as siRNA), polypeptides and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, LAG3 inhibitors and glucocorticoids.

[0351] In some embodiments, the second therapeutic dose may be an immune checkpoint inhibitor, for example, a PD-1 antibody and / or a PD-L1 antibody. PD-1 antibodies may include, for example, and / or RMP1-14. PD-L1 antibodies may include, for example

[0352] In some embodiments, the second therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthraquinones, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cephalotaxine, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, aziridines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc.), alkaloids, terpenoids, and kinase inhibitors.

[0353] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described below in conjunction with specific examples and accompanying drawings, and the advantages and features of the present invention will become clearer as the description progresses. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are carried out according to conventional conditions in the art, for example, the conditions described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press, or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0354] Example

[0355] Example 1. Acquisition of anti-FOLR1 antibodies

[0356] 1.1 Mouse immunization and serum titer detection

[0357] Prepare 4 6-8 week old BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), and the immunization dose of each mouse is 30-60 μg antigen W3922-hPro1.Fc (human FOLR1 protein, Fc tag). The adjuvant mixture includes alum adjuvant (Pierce, 77161), CpG (mouse) (Shanghai Shenggong) and Titer-Max immune adjuvant (Sigma, T2684). According to the immunization plan in Table 1, mice were immunized by plantar, subcutaneous and intraperitoneal routes, and serum titers were determined by FACS / ELISA. The G1#3 and G1#4 animals with higher titers were given a final booster immunization without adjuvant protein antigen. After 3 days (72 hours), the animals were euthanized and cell fusion was performed using lymph node cells or spleen cells.

[0358] Table 1. Mouse immunization schedule Note: FP, plantar; SC, subcutaneous; IP, intraperitoneal.

[0359] ELISA assays are used to measure serum antibody titers against target antigens. A 96-well high-absorption microplate containing 100 μL / well of W3922-hPro1 / cPro1.his (human FOLR1 protein / monkey FOLR1 protein, His-tag) ELISA (0.5 μg / mL) was incubated at 4°C overnight. After washing, each well was blocked for 1 hour with 200 μL of 2% BSA / 1× PBS. The plate was then washed three times with 1× PBST. Animal serum was diluted 100-fold in blocking buffer and then diluted in a 3-fold series. The diluted serum samples were added to the plate and incubated at 25°C for 2 hours. The plate was then washed and a horseradish peroxidase-conjugated goat anti-mouse IgG-Fc detection antibody (Bethyl, A90-231P) was added and incubated for 1 hour. After six washes, TMB substrate (Invitrogen, 002023) was added for color development for 5-10 minutes, and the reaction was terminated with 1 M H2SO4. The absorbance at 450 nm was read using a microplate reader (Molecular Devices), and the experimental data were analyzed using GraphPad Prism 7 software.

[0360] FACS was used to measure the serum antibody titer against the target antigen. NCI-H2110 cells (ATCC, CRL-5924) endogenously expressing the full-length human FOLR1 protein were cultured at 1×10 5 Cells / well were added to a 96-well standard round-bottom plate and centrifuged at 1500 rpm for 4 minutes at 4°C and the supernatant was removed. Animal serum was diluted 100-fold with blocking buffer and then serially diluted 3-fold. The diluted serum sample (100 μL / well) was added to the plate and mixed with the cells. The cells were incubated at 4°C in the dark for 1 hour. The cells were washed with 180 μL of 1× PBS solution containing 1% BSA. Alexa647 fluorescein-labeled goat anti-mouse IgG detection antibody (Jackson, 109-605-098) was diluted 500-fold with 1% BSA / 1× PBS solution and then added to the cells and mixed by pipetting. The cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice and then resuspended with 100 μL of 1% BSA / 1× PBS solution. Fluorescence values ​​were measured by flow cytometry (BD Canto II) and analyzed by FlowJo software.

[0361] The titers of human / monkey FOLR1 protein (hPro1 or cPro1) and NCI-H2110 cells expressing full-length human FOLR1 protein in G1#3 and G1#4 mice are summarized in Tables 2 and 3. As shown in Tables 2 and 3, both G1#3 and G1#4 mice produced strong immune responses to the target antigens.

[0362] Table 2. Titers of G1 mouse serum against human / monkey FOLR1 protein

[0363] Table 3. Titers of G1 mouse serum against NCI-H2110 cells

[0364] 1.2 Cell fusion

[0365] Lymph nodes and spleens from G1#3 and G1#4 mice were homogenized and filtered to remove blood clots and tissue debris. Sp2 / 0 myeloma cells (ATCC, CRL-1581) in the logarithmic growth phase were collected. TM ), centrifuged and removed the supernatant, then digested with pronase solution (Millipore, 53702). The digestion reaction was terminated by adding bovine serum albumin (FBS) (BovoStar, BSAS1.0), washed, and counted. B cells from lymph nodes and spleen were mixed with Sp2 / 0 myeloma cells at a ratio of 1:1.2 in electrofusion solution, and cell fusion was performed using an electrofusion instrument (BTX, ECM2001). The fused cells were resuspended in DMEM medium containing 20% ​​FBS, 1× HAT (Sigma, H0262), and 1× OPI (Sigma, O5003) and cultured in a 37°C, 5% CO2 incubator for 12 days. The medium was changed twice before the initial screening.

[0366] 1.3 Antibody Screening and Subcloning

[0367] Human FOLR1 protein ELISA binding assays were used for initial screening of 7,680 samples. Based on the initial screening data, 120 positive hybridomas were selected and transferred to 24-well plates for expansion. Supernatants from the 24-well plates were replicated using ELISA binding of monkey FOLR1 protein (W3922-cPro1.His), FACS binding using human FOLR1-expressing cells (NCI-H2110), and FACS internalization assays.

[0368] The W3922-hPro1.his ELISA binding experiment was used for the screening of hybridoma cells. First, 384-well high-adsorption ELISA plates were coated with 30 μL / well of W3922-hPro1.his (0.5 μg / mL) and allowed to stand overnight at 4°C. After blocking with 80 μL / well of 2% BSA / 1×PBS for 1 hour, the plates were washed 3 times with 1×PBST. Hybridoma supernatant was added to the plates and incubated at 25°C for 2 hours. The plates were washed and then horseradish peroxidase-labeled goat anti-mouse IgG-Fc detection antibody was added and incubated for 1 hour. After washing 6 times, TMB substrate was added for color development for 5-10 minutes and the reaction was terminated with 1M H2SO4. The absorbance at 450 nm was read using an enzyme-linked microplate reader.

[0369] The positive wells were retested in the W3922-cPro1.his ELISA binding experiment. 100 μL / well of W3922-cPro1.his (0.5 μg / mL) was used to coat a 96-well high-adsorption ELISA plate and incubated at 4°C overnight. After blocking with 200 μL / well 2% BSA / 1×PBS for 1 hour, the plate was washed 3 times with 1×PBST. Hybridoma supernatant was added to the plate and incubated at 25°C for 2 hours. The plate was then washed, followed by the addition of horseradish peroxidase-labeled goat anti-mouse IgG-Fc detection antibody and incubated for 1 hour. After washing 6 times, TMB substrate was added for color development for 5-10 minutes, and the reaction was terminated with 1M H2SO4. The absorbance at 450 nm was read using a microplate reader.

[0370] The FACS binding assay of NCI-H2110 cells, which stably express the full-length human FOLR1 protein, was used to replicate positive hybridomas. NCI-H2110 cells were plated into a 96-well standard round-bottom plate (1 x 10 5 / well), set the centrifuge to 4 ° C and centrifuge at 1500 rpm for 4 minutes. After removing the supernatant, resuspend the cells with hybridoma supernatant and incubate at 4 ° C in the dark for 1 hour. Wash the well plate twice with 180 μL 1% BSA / 1× PBS solution. Dilute Alexa647 fluorescein-labeled goat anti-mouse IgG detection antibody at 1:500 with 1% BSA / 1× PBS, then add it to the well plate to resuspend the cells and incubate at 4 ° C in the dark for 30 minutes. After centrifugation at 1500 rpm for 4 minutes, wash twice. Finally, resuspend the cells with 100 μL 1% BSA / 1× PBS, measure the fluorescence value by flow cytometry, and analyze the data by FlowJo software.

[0371] The internalization assay of NCI-H2110 cells was also used for hybridoma retesting. NCI-H2110 cells were added to 96-well standard round bottom plates (1 x 10 5 / well), set the centrifuge to 4℃ and centrifuge at 1500rpm for 4 minutes. After removing the supernatant, resuspend the cells with hybridoma supernatant and incubate at 4℃ in the dark for 1 hour. Wash the well plate twice with 180μL 1% BSA / 1×PBS solution. Dilute Alexa647 fluorescein-labeled goat anti-mouse IgG detection antibody at 1:500 with 1% BSA / 1×PBS, then add it to the well plate to resuspend the cells and incubate at 4℃ in the dark for 30 minutes. Wash the cells twice with 180μL of 1% BSA / 1×PBS. Resuspend the cells with 100μL 1% BSA / 1×PBS and incubate at 37℃ or 4℃ in the dark for 3.5 hours. After the incubation, add 100μL / well of quenching buffer (0.1M glycine, 0.15M NaCl, add HCl to pH 2.5) and incubate at 4℃ for 5 minutes. The cells were washed once with 180 μL of 1% BSA / 1× PBS and resuspended in 100 μL of 1% BSA / 1× PBS. Fluorescence was measured by flow cytometry and the data were analyzed using FlowJo software.

[0372] The results of hybridoma cell initial screening and retesting are shown in Table 4.

[0373] Table 4. Initial screening and retest data NOTE: The positive control W3922-BMK1 represents the anti-FOLR1 monoclonal antibody MORAB-003.

[0374] Based on the retest results, hybridoma cell lines were selected for subcloning and cryopreservation. The results of subclone screening and subclone retest are shown in Table 5.

[0375] Table 5. Subclone screening and subclone retest data NOTE: The positive control W3922-BMK1 represents the anti-FOLR1 monoclonal antibody MORAB-003.

[0376] Based on the screening results of the above-mentioned positive hybridoma cells, monoclonal clones were obtained by subcloning. Count the positive hybridoma cells in the logarithmic growth phase, take 200-300 cells and add them to 1.5 mL of semi-solid HAT medium (Sigma, H0262). Gently mix the cells for 5-10 seconds and then inoculate them into 6-well flat-bottom plates. After incubating the plates in a 37°C, 5% CO2 incubator for 7-8 days, pick a single colony and transfer it to a 96-well flat-bottom plate containing DMEM medium containing 10% FBS. After 2-3 days, collect the cell supernatant and screen again to obtain a positive monoclonal cell line.

[0377] 1.4 Antibody subtype detection and hybridoma sequencing

[0378] Antibody isotypes were identified by sandwich ELISA. Isotyping was performed in a 96-well high-adsorption microtiter plate (Thermo Scientific, 442404). 4 μg / mL capture antibody was coated onto a 96-well high-adsorption microtiter plate and incubated overnight at 4°C. After blocking with 200 μL / well of 2% BSA / 1xPBS for 1 hour, the plate was washed three times. Hybridoma supernatant was added to the plate and incubated at 25°C for 2 hours. After washing the plate three times, horseradish peroxidase-conjugated goat anti-mouse Ig kappa / lambda light chain detection antibody (Bethyl, A90-119P; Bethyl, A90-121P) was added and incubated for 1 hour. After washing six times, TMB substrate was added for color development for 5-10 minutes, and the reaction was terminated with 1 M H2SO4. The absorbance at 450 nm was read using a microplate reader.

[0379] Repeat the test using a subtype rapid detection kit (Antagen Pharmaceuticals, ISO-M8a). Add 100 μL of hybridoma supernatant to the sample well of the test strip. Let it sit for approximately 5 minutes and record the test line (T) on the strip. A successful test is indicated when the control line (C) at the far end of the strip develops color.

[0380] The detection results of antibody subtypes are shown in Table 6.

[0381] Table 6. Monoclonal antibody subtype detection results

[0382] Total RNA was extracted from hybridoma cells, and complementary deoxyribonucleic acid (cDNA) was synthesized using a 5'-RACE kit. The antibody VH / VL was then amplified by PCR using a 5' universal primer and a 3' gene-specific primer (GSP). The PCR product was sequenced by Sanger sequencing to obtain the VH / VL coding sequence of the monoclonal antibody. The constant region of the resulting monoclonal antibody was replaced with a human IgG1 constant region to generate a chimeric antibody. The amino acid sequences of its heavy chain (HC) and light chain (LC) are shown in Table 7.

[0383] Table 7. Amino acid sequences of heavy and light chains of anti-FOLR1 chimeric antibodies

[0384] The heavy chain variable region (VH) and light chain variable region sequences of the chimeric antibody are shown in Table 8. The heavy chain complementary determining region (HCDR1, HCDR2 and HCDR3) sequences, light chain complementary determining region (LCDR1, LCDR2 and LCDR3) and framework region (HFR1-HFR4, LFR1-LFR4) sequences are defined according to the Kabat numbering method (see Table 9).

[0385] Table 8. Amino acid sequences of heavy and light chain variable regions of anti-FOLR1 chimeric antibodies

[0386] Table 9. CDR and FR sequences of anti-FOLR1 chimeric antibodies (Kabat numbering)

[0387] Example 2. Humanization of anti-FOLR1 chimeric antibodies and removal of high-risk post-translational modification sites

[0388] 2.1 Design of W3922-1.34.19 Humanization and Removal of High-Risk Post-Translational Modification Sites

[0389] Based on sequence alignment with human germline genes, IGHV1-69*02IGHJ6*01 was selected as the humanization template for the heavy chain variable region, and IGKV2-30*01IGKJ4 was selected as the humanization template for the light chain variable region. Simultaneously, several backmutations were designed based on structural analysis. Combinations of light and heavy chains containing backmutations were used to generate several humanized variants, maintaining affinity for the antigen.

[0390] 2.2 Design of W3922-1.40.2 Humanization and Removal of High-Risk Post-Translational Modification Sites

[0391] Based on sequence alignment with human germline genes, IGHV1-69*02IGHJ6*01 was selected as the humanization template for the heavy chain variable region, and IGKV2-30*01IGKJ4 was selected as the humanization template for the light chain variable region. Simultaneously, several backmutations were designed based on structural analysis. Combinations of light and heavy chains containing backmutations were used to generate several humanized variants, maintaining affinity for the antigen.

[0392] 2.3 Design of W3922-1.45.2 Humanization and Removal of High-Risk Post-Translational Modification Sites

[0393] Based on sequence alignment with human germline genes, IGHV1-69*02IGHJ6*01 was selected as the humanized template for the heavy chain variable region, and IGKV1-33*01IGKJ2 was selected as the humanized template for the light chain variable region. Simultaneously, several backmutations were designed based on structural analysis. Combinations of light and heavy chains containing backmutations were used to generate several humanized variants, maintaining antigen binding affinity.

[0394] After expression and purification of each parental antibody (i.e., chimeric antibody) and humanized antibody, the dissociation rate constant koff of the antibody to the antigen W3922-hPro1.His was measured by SPR binding assay and ranked. The optimal humanized sequence was selected and combined with a mutant with the post-translational modification site removed. The binding ability of each parental antibody and humanized antibody to the antigen was measured by SPR kinetic constant assay. The results are summarized in Tables 10-12.

[0395] Table 10. SPR affinity detection (W3922-1.34.19)

[0396] Table 11. SPR affinity detection (W3922-1.40.2)

[0397] Table 12. SPR affinity detection (W3922-1.45.2)

[0398] Based on affinity testing results, candidate molecule W3922-1.34.19-z7-p3-uIgG1K (z7-p3) was selected as the lead molecule for W3922-1.34.19, candidate molecule W3922-1.40.2-z4-p3-uIgG1K (z4-p3) was selected as the lead molecule for W3922-1.40.2, and candidate molecule W3922-1.45.2-z4-uIgG1K (z4) was selected as the lead molecule for W3922-1.45.2. The amino acid sequences of the above humanized lead molecules are shown in Tables 13-15.

[0399] Table 13. Heavy and light chain amino acid sequences of anti-FOLR1 humanized antibodies

[0400] Table 14. Amino acid sequences of heavy and light chain variable regions of anti-FOLR1 humanized antibodies

[0401] Table 15. CDR and FR sequences of anti-FOLR1 humanized antibodies (Kabat numbering)

[0402] Example 3. Preparation of Antibody Drug Conjugate (ADC)

[0403] The ADC information tested in this application is shown in Table 16.

[0404] Table 16. ADC information tested in this application Note: HFRAO34-HG004-W2 is the maleimide ring-opening product of HFRA34-HG004-W2.

[0405] 3.1 Linker-payload synthesis

[0406] This application utilizes two linker-payloads, HG009 and HG004, to prepare ADCs. These are HG-PL2 and HG-PL4, respectively, derived from International Application No. PCT / CN2022 / 074523 (incorporated herein by reference in its entirety). The drug group of HG009 is MMAE, and the drug group of HG004 is eribulin. The structures and synthesis procedures of the HG009 and HG004 linker-payloads are as follows.

[0407] 3.1.1 Synthesis of HG009 (HG-PL2)

[0408] HG009 has the following structure:

[0409] Step 1: Synthesis of PH-HG-002-1

[0410] To a nitrogen-protected solution of PH-HG-001-3 (3.8 g, 5.1 mmol, 1.0 equiv) in DMF (60 mL) were added N,N-diisopropylethylamine (3.0 g, 23.2 mmol, 4.5 equiv), SM11 (5.7 g, 17.0 mmol, 3.3 equiv), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (12.1 g, 23.3 mmol, 4.5 equiv) in sequence. The reaction mixture was stirred at 25°C for 5 hours, diluted with 300 mL of water, and extracted with ethyl acetate (3 × 300 mL). The combined organic phases were washed with saturated brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was purified by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase water and acetonitrile (10% to 100% acetonitrile, 20 minutes), detector UV 210 nm) to obtain 6.1 g (70%) of colorless oily PH-HG-002-1.

[0411] MS m / z[M+H] + (ESI):1702.93. 1H NMR(400MHz,DMSO-d6)δ:1.30-1.42(s,27H),2.25-2.40(m,4H),3.03-3.08(m ,6H),3.12-3.20(m,4H),3.25-3.29(m,6H),3.35-3.44(m,18H),3.49-3.50(m ,38H),3.71(s,6H),3.88(s,6H),4.21-4.30(m,3H),6.71-6.73(m,3H),7.33( t,J=7.6Hz,3H),7.42(t,J=7.6Hz,2H),7.68-7.75(m,6H),7.88-7.90(m,3H).

[0412] Step 2: Synthesis of PH-HG-002-2

[0413] To a nitrogen-protected solution of PH-HG-002-1 (3.1 g, 1.8 mmol, 1.0 equiv) in dichloromethane (15 mL) was added trifluoroacetic acid (15 mL). The reaction mixture was stirred at 25°C for 3 hours and then concentrated by rotary evaporation. The crude product was purified by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 80% acetonitrile, 20 minutes), UV detector: 210 nm) to afford 1.6 g (64%) of PH-HG-002-2 as a colorless oil.

[0414] MS m / z[M / 2+H]+(ESI):1402.77.

[0415] Step 3: Synthesis of PH-HG-002-3

[0416] To a nitrogen-protected solution of PH-HG-002-2 (3.0 g, 2.1 mmol, 1.0 equiv) in dichloromethane (45 mL) at 0°C were added succinic anhydride (1.9 g, 19.3 mmol, 9.0 equiv), triethylamine (2.6 g, 25.7 mmol, 12.0 equiv), and 4-dimethylaminopyridine (0.8 g, 6.4 mmol, 3.0 equiv). The reaction was stirred at 25°C for 12 hours and then concentrated by rotary evaporation. The crude product was purified by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 80% acetonitrile, 30 min); UV detector: 210 nm) to afford 1.6 g (44%) of PH-HG-002-3 as a colorless oil.

[0417] MS m / z[M / 2+H] + (ESI):1402.77. 1H NMR(300MHz,DMSO-d6)δ:2.34-2.39(m,10H),2.41-2.44(m,6H),3.13-3.28(m,16H),3.37-3.51(m,56H),3.72(s,6H),3.89(s,6 H),4.19-4.31(m,3H),7.32(t,J=7.5Hz,3H),7.43(t,J=7.5Hz,2H),7.68-7.77(m,6H),7.88-7.91(m,6H),12.00-12.06(m,2H).

[0418] Step 4: Synthesis of PH-HG-002-4

[0419] To a nitrogen-protected solution of PH-HG-002-3 (1.6 g, 0.9 mmol, 1.0 equiv) in DMF (30 mL) at 0°C was added Ms-1 (1.7 g, 3.1 mmol, 3.3 equiv), N,N-diisopropylethylamine (546.5 mg, 4.2 mmol, 4.5 equiv), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (1.6 g, 4.2 mmol, 4.5 equiv). The reaction mixture was stirred at 25°C for 5 hours, then diluted with 300 mL of water and extracted with ethyl acetate (3 x 300 mL). The combined organic phases were washed with saturated brine (3 x 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase water (containing 0.1% formic acid) and acetonitrile (10% to 100% acetonitrile, 20 minutes), detector UV 210nm) to obtain 1.7g (55%) of yellow oil PH-HG-002-4.

[0420] MS m / z[M / 2+H] + (ESI):1657.15.

[0421] Step 5: Synthesis of PH-HG-002-5

[0422] To a nitrogen-protected solution of PH-HG-002-4 (1.0 g, 0.3 mmol, 1.0 equiv) in dichloromethane (5 mL) was added formic acid (5.0 mL). The reaction was stirred at 25°C for 48 hours and then concentrated by rotary evaporation. The crude product was purified by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water (containing 0.1% formic acid) and acetonitrile (10% to 80% acetonitrile, 30 minutes); UV detector: 210 nm) to yield 700 mg (74%) of PH-HG-002-5 as a colorless oil.

[0423] MS m / z[M / 2+H] + (ESI):1573.20. 1 H NMR(300MHz, Methanol-d4)δ:2.48-2.58(m,16H),2.65-2.84(m,6H),3.20-3.30(m,3H),3.33-3.47(m,30H),3.53-3.65(m,1 44H),3.88(s,6H),4.03(s,6H),4.20-4.40(m,3H),4.73-4.85(m,3H),7.34-7.42(m,4H),7.67(m,2H),7.83(d,J=7.5Hz,2H).

[0424] Step 6: Synthesis of PH-HG-002-6

[0425] To a nitrogen-protected DMF (4 mL) solution of PH-HG-002-5 (300 mg, 0.1 mmol, 1.0 equivalent) was added diethylamine (0.1 mL) at 0°C. The reaction mixture was stirred at 0°C for 2 hours and then purified by preparative HPLC (conditions: X Select CSH Prep C18 OBD column; dimensions 19 mm × 250 mm, 5 μm; mobile phase, water (0.05% formic acid) and acetonitrile (8.0% to 23.0% acetonitrile, 11 min); detector, UV 200 nm) to afford 140 mg (50%) of the product PH-HG-002-6 as a colorless oil.

[0426] MS m / z[M / 2+H] + (ESI):1461.95.

[0427] Step 7: Synthesis of PH-HG-002-7

[0428] To a nitrogen-protected solution of PH-HG-002-6 (140 mg, 0.1 mmol, 1.0 equiv) in DMF (5 mL) were added hydroxysuccinimide 3-maleimidopropionate (19.1 mg, 0.07 mmol, 1.5 equiv) and N,N-diisopropylethylamine (10.5 mg, 0.14 mmol, 3.0 equiv). The reaction mixture was stirred at 0°C for 4 hours and then purified by preparative HPLC (conditions: XBridge Prep C18 OBD column; dimensions 30 mm × 150 mm, 5 μm; mobile phase: water (0.05% formic acid) and acetonitrile (8.0% to 38.0% acetonitrile, 7 min); detector, UV 200 nm) to afford 75 mg (51%) of the product PH-HG-002-7 as a colorless oil.

[0429] MS m / z[M / 2+H] + (ESI):1537.55.

[0430] Step 8: Synthesis of HG-PL2

[0431] To a nitrogen-protected DMF (4 mL) solution of PH-HG-002-7 (120 mg, 0.039 mmol, 1.0 eq) was added PH-HG-001-8 (144.8 mg, 0.13 mmol, 3.3 eq), N,N-diisopropylethylamine (22.7 mg, 0.18 mmol, 4.5 eq), and 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (PyBOP) (91.4 mg, 0.18 mmol, 4.5 eq) at 0°C. The reaction solution was stirred at 0°C for 2 hours and purified by preparative HPLC (conditions: XBridge Prep C18 OBD column; size 30 mm × 150 mm, 5 μm; mobile phase, water (0.01% formic acid) and acetonitrile (35.0% to 65.0% acetonitrile, 7 min); detector, UV 254 nm) to give 23.4 mg (9%) of white solid product HG-PL2.

[0432] MS m / z[M / 4+H] + (ESI):1598.50. 1 H NMR(400MHz, DMSO-d6)δ:0.75-1.05(m,101H),1.24-1.39(m,7H),1.42-1.54(m,11H),1.64-1.82(m,16H),1.90-2.15(m,13H), 2.21-2.47(m,31H),2.67-2.78(m,5H),2.85-3.09(m,23H),3.12-3.30(m,76H),3.38-3.60(m,96H),3.62-3.76(m,11H),3.88-4 .05(m,14H),4.14-4.37(m,11H),4.39-4.55(m,11H),4.62-4.77(m,4H),4.93-5.12(m,7H),5.37-5.45(m,10H),5.97-6.02(m,3 H),7.00(s,2H),7.18-7.32(m,23H),7.59-7.63(m,8H),7.74-7.77(m,4H),7.88-8.08(m,16H),8.19-8.34(m,8H),9.72(s,3H).

[0433] 3.1.2 Synthesis of HG004 (HG-PL4)

[0434] HG004 has the following structure:

[0435] Step 1: Synthesis of HG-PL4

[0436] To a nitrogen-protected solution of PH-HG-002-7 (65 mg, 0.021 mmol, 1.0 equiv) in DMF (2.0 mL) at 0°C were added PH-HG-003-4 (79.2 mg, 0.069 mmol, 3.3 equiv), N,N-diisopropylethylamine (12.3 mg, 0.095 mmol, 4.5 equiv), and 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (PyBOP) (49.5 mg, 0.095 mmol, 4.5 equiv). The reaction solution was stirred at 0°C for 2 hours and then purified by preparative HPLC (conditions: XBridge Shield RP18 OBD column; size 19 mm × 150 mm, 5 μm; mobile phase, water and acetonitrile (35.0% to 65.0% acetonitrile, 10 min); detector, UV 254 nm) to give 20.9 mg (15%) of white solid product HG-PL4.

[0437] MS m / z[M / 4+H] + (ESI):1606.80. 1 H NMR(400MHz,DMSO-d6)δ:0.75-0.80(m,34H),0.90-0.97(m,22H),1.12-1.43(m,51H),1.59-1.70(m,38H),1.8 4-1.97(m,35H),2.19-2.25(m,41H),2.67-2.77(m,21H),2.90-2.95(m,24H),3.10-3.18(m,53H),3.64-3.68( m,23H),3.76-3.82(m,16H),3.98-4.11(m,30H),4.19-4.26(m,10H),4.49-4.59(m,17H),4.66-4.78(m,6H),4 .85-4.98(m,14H),5.31-5.38(m,5H),6.97-7.23(m,2H),7.58-7.95(m,32H),8.10-8.23(m,10H),9.61(m,6H).

[0438] 3.2 Preparation of ADC

[0439] 3.2.1 Preparation of reference ADC W3922-BMK1-HG012

[0440] W3922-BMK1-HG012 is used as the ADC reference compound of this application, namely MORAB-202. The preparation process of its linker-payload is shown in Cheng et al. (Cheng et al., MORAb-202, an Antibody-Drug Conjugate Utilizing Humanized Anti-human FRα Farletuzumab and the Microtubule-targeting Agent Eribulin, has Potent Antitumor Activity. Mol Cancer Ther. 2018 Dec; 17(12): 2665-2675).

[0441] Antibody BMK1 was reduced with 2.2 equivalents of tris(2-carboxyethyl)phosphine (TCEP) in TEAA buffer (50 mM TEAA, 1 mM EDTA, pH 7.0) and incubated on a shaker at 22°C for 3 hours (60 rpm). The reaction mixture was used directly in the next coupling reaction without removing excess TCEP.

[0442] The reduced antibody was slowly added to TEAA buffer, 10% dimethylacetamide (DMA), and linker-payload (15 equivalents, 10 mM HG012 in DMA) at room temperature. After thorough mixing, the coupling reaction solution was shaken on a shaker at 22°C for 2 hours (shaker speed 60 rpm).

[0443] After the reaction was completed, the ADC solution was washed with 53 mL of HiPrap TM The 26 / 10 desalting column was exchanged with storage buffer (20 mM histidine, pH 5.5), and then filtered through a 0.2 μm PVDF pinhole filter to obtain the ADC product, which was sampled and sent for testing.

[0444] 3.2.2 Preparation of FRA34-HG004-D2 and FRA45-HG004-D2

[0445] The chimeric antibody was reduced with 1.0 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4) and incubated on a shaker at 4°C for 5.5 hours (60 rpm). The reaction solution was used directly in the next coupling reaction without removing excess TCEP.

[0446] After reducing the antibody, slowly add 40% propylene glycol (PG) and a linker-payload solution (3 equivalents, 3 mM HG004 in a 3:1 mixture of dimethylacetamide and water) on ice. After thorough mixing, the coupling reaction mixture was shaken at 4°C for 18 hours (shaker speed 60 rpm).

[0447] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified using a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) for purification.

[0448] Purification steps: Prior to purification, the SP HP column was equilibrated with 25 mL of buffer A (20 mM MES, pH 6.0). The ADC solution was diluted four-fold with buffer (20 mM MES, pH 6.0) to allow ADC binding to the column packing. After loading, the column was washed with buffer A until the absorbance at 280 nm reached baseline equilibrium. Elution was then performed by continuously replacing the buffer system from 100% buffer A (20 mM MES, pH 6.0) to 33.3% buffer B (20 mM MES, 300 mM sodium chloride, pH 6.0) over 30 minutes (buffer flow rate of 5 mL / min). The purified D2 was then concentrated into 20 mM MES, pH 6.0 using an Amicon ultrafiltration tube (10 kDa). The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0449] 3.2.3 Preparation of FRA40-HG004-D2

[0450] The chimeric antibody was reduced with 1.0 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4) and incubated on a shaker at 4°C for 5.5 hours (60 rpm). The reaction solution was used directly in the next coupling reaction without removing excess TCEP.

[0451] After reducing the antibody, slowly add 40% propylene glycol (PG) and a linker-payload solution (3 equivalents, 3 mM HG004 in a 3:1 mixture of dimethylacetamide and water) on ice. After thorough mixing, the coupling reaction mixture was shaken at 4°C for 18 hours (shaker speed 60 rpm).

[0452] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified by a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) and hydrophobic interaction chromatography column (HiTrap TM Octyl FF, 1 mL) was used for purification.

[0453] Purification Step 1: Prior to purification, equilibrate the SP HP column with 25 mL of Buffer A (20 mM MES, pH 6.0). Dilute the ADC solution fourfold with Buffer A (20 mM MES, pH 6.0) to allow ADC binding to the column medium. After loading, wash the column with Buffer A until the absorbance at 280 nm reaches baseline equilibrium. Elution is then performed by continuously replacing the buffer system from 100% Buffer A (20 mM MES, pH 6.0) to 33.3% Buffer B (20 mM MES, 300 mM NaCl, pH 6.0) over 30 minutes (buffer flow rate 5 mL / min).

[0454] Purification Step 2: Prior to purification, an Octyl FF column was equilibrated with 10 mL of Buffer A (20 mM MES, 1.2 M NaCl, pH 6.0). The NaCl concentration in the ADC solution was adjusted to 1.5 M with Buffer A (20 mM MES, 4 M NaCl, pH 6.0) to facilitate ADC binding to the column packing. After loading, the column was washed with Buffer A until the absorbance at 280 nm reached baseline equilibrium. Elution was then performed by continuously replacing the buffer system with 100% Buffer A (20 mM MES, 1.2 M NaCl, pH 6.0) and 100% Buffer B (20 mM MES, pH 6.0) over 2 minutes (buffer flow rate of 1 mL / min).

[0455] The purified D2 was then concentrated into 20 mM MES, pH 6.0 buffer using an Amicon ultrafiltration tube (10 kDa). The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0456] Preparation of FRA40-HG004-B1

[0457] In PBS 7.4 buffer (PBS 7.4, 2mM EDTA, pH 7.4), slowly add 20% propylene glycol (PG) and a linker-payload solution (3 equivalents, 3mM HG004 in a 3:1 mixture of dimethylacetamide and water). After thorough mixing, the coupling reaction solution was shaken at 4°C for 18 hours (shaker speed 60 rpm).

[0458] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and purified using a hydrophobic interaction chromatography column (HiTrap TM Butyl HP, 1 mL) for purification.

[0459] Purification Steps: Prior to purification, equilibrate a Butyl HP column with 10 mL of Buffer A (20 mM PB, pH 7.0). Dilute the ADC solution fivefold with Buffer A (20 mM PB, pH 7.0). After loading, wash the column with Buffer A until the absorbance at 280 nm reaches baseline equilibrium. The resulting flow-through fraction is FRA40-HG004-B1.

[0460] The eluted and purified ADC was then concentrated into 20 mM His, pH 5.5 buffer using an Amicon ultrafiltration tube (10 kDa). The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0461] Preparation of FRA40-HG004-B2

[0462] 5% volume of 1 M Tris-HCl, pH 8 buffer was slowly added to PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4) to adjust the pH of the antibody solution to 8. 150.0 equivalents of dithiothreitol (DTT) were added to reduce the antibody, and the reaction was shaken at 22° C. for 20.5 hours (shaker speed 60 rpm).

[0463] After the reaction, the ADC solution was desalted using a desalting column to remove excess DTT and then replaced with PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4).

[0464] After the reduction, 60.0 equivalents of dehydroascorbic acid (DHAA) were added to the antibody, mixed thoroughly, and then shaken on a shaker at 22° C. for 3 hours (shaker speed 60 rpm).

[0465] The re-oxidized antibody was slowly added to 20% propylene glycol (PG) and a linker-payload solution (3 equivalents, 3 mM HG004 in a 3:1 mixture of dimethylacetamide and water). After thorough mixing, the coupling reaction was shaken at 22°C for 18 hours (shaker speed 60 rpm).

[0466] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified using a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) for purification.

[0467] Purification Procedure: Prior to purification, equilibrate the SP HP column with 25 mL of buffer A (20 mM MES, pH 6.0). Dilute the ADC solution four-fold with purified water to allow ADC binding to the column packing. After loading, wash the column with buffer A until the absorbance at 280 nm reaches baseline equilibrium. Elution is then performed by continuously replacing the buffer system from 100% buffer A (20 mM MES, pH 6.0) to 33.3% buffer B (20 mM MES, 300 mM NaCl, pH 6.0) over 30 minutes (buffer flow rate of 5 mL / min).

[0468] The eluted and purified ADC was then concentrated into 20 mM MES, 10 mM sodium chloride, pH 6.0 buffer using an Amicon ultrafiltration tube (10 kDa). The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0469] Preparation of FRA40-HG004-B3

[0470] 5% volume of 1 M Tris-HCl, pH 8 buffer was slowly added to PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4) to adjust the pH of the antibody solution to 8. 150.0 equivalents of dithiothreitol (DTT) were added to reduce the antibody, and the reaction was shaken at 22° C. for 20.5 hours (shaker speed 60 rpm).

[0471] After the reaction, the ADC solution was desalted using a desalting column to remove excess DTT and then replaced with PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4).

[0472] After the reduction, 60.0 equivalents of dehydroascorbic acid (DHAA) were added to the antibody, mixed thoroughly, and then shaken on a shaker at 22° C. for 3 hours (shaker speed 60 rpm).

[0473] The re-oxidized antibody was slowly added to 20% propylene glycol (PG) and a linker-payload solution (3 equivalents, 3 mM HG004 in a 3:1 mixture of dimethylacetamide and water). After thorough mixing, the coupling reaction was shaken at 22°C for 18 hours (shaker speed 60 rpm).

[0474] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified using a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) for purification.

[0475] Purification Procedure: Prior to purification, equilibrate the SP HP column with 25 mL of buffer A (20 mM MES, pH 6.0). Dilute the ADC solution four-fold with purified water to allow ADC binding to the column packing. After loading, wash the column with buffer A until the absorbance at 280 nm reaches baseline equilibrium. Elution is then performed by continuously replacing the buffer system from 100% buffer A (20 mM MES, pH 6.0) to 33.3% buffer B (20 mM MES, 300 mM NaCl, pH 6.0) over 30 minutes (buffer flow rate of 5 mL / min).

[0476] The eluted and purified ADC (a mixture of D0, B1, and B2) was then concentrated to 3.7 mg / mL using an Amicon ultrafiltration tube (10 kDa).

[0477] ADC (a mixture of D0, B1, and B2) was reduced with 1.0, 1.5, and 2.0 equivalents of tris(2-carboxyethyl)phosphine (TCEP), respectively, and the mixture was shaken at 22°C for 3 hours (60 rpm). The reaction mixture was used directly in the next coupling reaction without removing excess TCEP.

[0478] After reducing the antibody, 20% propylene glycol (PG) and a linker-payload solution (3 / 5 / 6 equivalents, 3 mM HG004 in dimethylacetamide and water, 3:1) were slowly added to the reduced antibody in an ice bath. After thorough mixing, the coupling reaction was incubated on a shaker at 4°C for 16.5 hours (shaker speed: 60 rpm).

[0479] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified by a strong cation exchange chromatography column (HiTrap TM SP HP, 1 mL) for purification.

[0480] Purification Steps: Prior to purification, equilibrate the SP HP column with 5 mL of buffer A (20 mM MES, pH 6.0). Dilute the ADC solution 5-fold with purified water to allow ADC binding to the column packing. After loading the sample, wash the column with buffer A until the absorbance at 280 nm reaches baseline equilibrium. Elution is then performed with 29% buffer B (20 mM MES, 300 mM NaCl, pH 6.0).

[0481] The three batches of ADC obtained by elution and purification were combined and concentrated into 20 mM MES, 10 mM sodium chloride, 6% sucrose, pH 6.0 buffer using an Amicon ultrafiltration tube (10 kDa). Finally, the ADC product was filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0482] 3.2.7. Preparation of HFRA40-HG004-B2 and HFRA40-HG009-B2

[0483] 5% volume of 1 M Tris-HCl, pH 8 buffer was slowly added to PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4) to adjust the pH of the antibody solution to 8. 150.0 equivalents of dithiothreitol (DTT) were added to reduce the antibody, and the reaction was shaken at 22° C. for 20.5 hours (shaker speed 60 rpm).

[0484] After the reaction, the ADC solution was desalted using a desalting column to remove excess DTT and then replaced with PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4).

[0485] 15.0 equivalents of dehydroascorbic acid (DHAA) were added to the reduced antibody, mixed thoroughly, and then shaken on a shaker at 22° C. for 3 hours (shaker speed: 60 rpm).

[0486] The re-oxidized antibody was slowly added to 20% propylene glycol (PG) and 8 equivalents of a linker-payload solution (3 mM HG004 or HG009 in a 3:1 mixture of dimethylacetamide and water). After thorough mixing, the coupling reaction was shaken at 22°C for 18 hours (shaker speed 60 rpm).

[0487] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified using a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) for purification.

[0488] Purification Procedure: Prior to purification, equilibrate the SP HP column with 25 mL of buffer A (20 mM MES, pH 6.0). Dilute the ADC solution four-fold with purified water to allow ADC binding to the column packing. After loading, wash the column with buffer A until the absorbance at 280 nm reaches baseline equilibrium. Elution is then performed by continuously replacing the buffer system from 100% buffer A (20 mM MES, pH 6.0) to 33.3% buffer B (20 mM MES, 300 mM NaCl, pH 6.0) over 30 minutes (buffer flow rate of 5 mL / min).

[0489] The eluted and purified ADC was then concentrated into 10 mM MES, 70 mM sodium chloride, pH 6.0 buffer using an Amicon ultrafiltration tube (10 kDa). The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0490] 3.2.8. Preparation of HFRA34-HG004-D2 and HFRA34-HG009-D2

[0491] Reduce the antibody with 1.0 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4) and incubate on a shaker at 4°C for 5.5 hours (60 rpm). The reaction solution was used directly in the next coupling reaction without removing excess TCEP.

[0492] After reducing the antibody, slowly add 20% propylene glycol (PG) and 3 equivalents of a linker-payload solution (3 mM HG004 or HG009 in a 3:1 mixture of dimethylacetamide and water) to the reduced antibody in an ice bath. After thorough mixing, the coupling reaction mixture was shaken at 4°C for 16.5 hours (shaker speed: 60 rpm).

[0493] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified using a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) for purification.

[0494] Purification Procedure: Prior to purification, equilibrate the SP HP column with 25 mL of buffer A (20 mM MES, pH 6.0). Dilute the ADC solution four-fold with purified water to allow ADC binding to the column packing. After loading, wash the column with buffer A until the absorbance at 280 nm reaches baseline equilibrium. Elution is then performed by continuously replacing the buffer system from 100% buffer A (20 mM MES, pH 6.0) to 33.3% buffer B (20 mM MES, 300 mM NaCl, pH 6.0) over 30 minutes (buffer flow rate of 5 mL / min). The purified D2 was then concentrated into storage buffer (10 mM MES, 55 mM sodium chloride, 6% sucrose, pH 6.0 for HFRA34-HG004-D2 and 10 mM MES, 70 mM sodium chloride, pH 6 for HFRA34-HG009-D2) by exchange using Amicon ultrafiltration tubes (10 kDa). The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0495] 3.2.9 Preparation of HFRA34-HG004-W2

[0496] Referring to WuXiDAR2 technology, an ADC solution of antibody and linker-payload HG004 conjugated was prepared.

[0497] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified using a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) for purification.

[0498] Purification Procedure: Prior to purification, the SP HP column was equilibrated with 25 mL of buffer A (20 mM MES, pH 6.0). The ADC solution was diluted four-fold with purified water to allow ADC binding to the column packing. After loading, the column was washed with buffer A until the absorbance at 280 nm reached baseline equilibrium. Elution was then performed by continuously replacing the buffer system from 100% buffer A (20 mM MES, pH 6.0) to 33.3% buffer B (20 mM MES, 300 mM sodium chloride, pH 6.0) over 30 minutes (buffer flow rate of 5 mL / min). The purified D2 was then concentrated using an Amicon 10 kDa ultrafiltration tube into storage buffer (10 mM MES, 54 mM sodium chloride, 6% sucrose, pH 6.0). The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0499] 3.2.10 Preparation of HFRA45-HG004-D2

[0500] Reduce the antibody with 1.0 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4) and incubate on a shaker at 4°C for 5.5 hours (60 rpm). The reaction solution was used directly in the next coupling reaction without removing excess TCEP.

[0501] After reducing the antibody, slowly add 20% propylene glycol (PG) and a linker-payload solution (3 equivalents, 3 mM HG004 in a 3:1 mixture of dimethylacetamide and water) on ice. After thorough mixing, the coupling reaction mixture is shaken at 4°C for 16.5 hours (shaker speed 60 rpm).

[0502] After the reaction, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified by a protein purifier (AKTA) using a hydrophobic interaction chromatography column (PPG-600M, 5 mL) and a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) for purification.

[0503] Purification Step 1: Prior to purification, equilibrate a PPG-600M column with 25 mL of Buffer A (20 mM PB, 500 mM NaCl, pH 7.0). Adjust the sodium chloride concentration in the ADC solution to 500 mM with Buffer A (20 mM PB, 4 M NaCl, pH 7.0) to facilitate ADC binding to the column packing. After loading, wash the column with Buffer A until the absorbance at 280 nm reaches baseline equilibrium. Subsequently, elute with 100% Buffer B (20 mM PB, 20% propylene glycol, pH 7.0).

[0504] The eluted ADC was filtered through a 0.2 μm PVDF pinhole filter to remove excess linker-payload.

[0505] Purification Step 2: Prior to purification, the SP HP column was equilibrated with 25 mL of buffer A (20 mM MES, pH 6.0). The ADC solution was diluted fourfold with buffer (20 mM MES, pH 6.0) to allow ADC binding to the column packing. After loading, the column was washed with buffer A until the absorbance at 280 nm reached baseline equilibrium. Elution was then performed by continuously replacing the buffer system from 100% buffer A (20 mM MES, pH 6.0) to 33.3% buffer B (20 mM MES, 300 mM sodium chloride, pH 6.0) over 30 minutes (buffer flow rate of 5 mL / min). The purified D2 was then concentrated using an Amicon 10 kDa ultrafiltration tube into 20 mM MES, 6% sucrose, pH 6.0 buffer. The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0506] 3.2.11 Preparation of HFRA45-HG009-D2

[0507] Reduce the antibody with 1.0 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4) and incubate on a shaker at 4°C for 5.5 hours (60 rpm). The reaction solution was used directly in the next coupling reaction without removing excess TCEP.

[0508] After reducing the antibody, slowly add 20% propylene glycol (PG) and a linker-payload solution (3 equivalents, 3 mM HG009 in a 3:1 mixture of dimethylacetamide and water) on ice. After thorough mixing, the coupling reaction mixture is shaken at 4°C for 16.5 hours (shaker speed 60 rpm).

[0509] After the reaction, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified by a protein purifier (AKTA) using a hydrophobic interaction chromatography column (PPG-600M, 5 mL) and a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) for purification.

[0510] Purification Step 1: Prior to purification, equilibrate a PPG-600M column with 25 mL of Buffer A (20 mM PB, 500 mM NaCl, pH 7.0). Adjust the sodium chloride concentration in the ADC solution to 500 mM with Buffer A (20 mM PB, 4 M NaCl, pH 7.0) to facilitate ADC binding to the column packing. After loading, wash the column with Buffer A until the absorbance at 280 nm reaches baseline equilibrium. Subsequently, elute with 100% Buffer B (20 mM PB, 20% propylene glycol, pH 7.0).

[0511] Purification Step 2: Prior to purification, equilibrate the SP HP column with 25 mL of buffer A (20 mM MES, pH 6.0). Dilute the ADC solution fourfold with buffer (20 mM MES, pH 6.0) to allow ADC binding to the column packing. After loading, wash the column with buffer A until the absorbance at 280 nm reaches baseline equilibrium. Elution is then performed by continuously replacing the buffer system from 100% buffer A (20 mM MES, pH 6.0) to 33.3% buffer B (20 mM MES, 300 mM NaCl, pH 6.0) over 30 minutes (buffer flow rate 5 mL / min).

[0512] The eluted ADC was then filtered using activated charcoal to remove excess linker-payload. The eluted ADC was then concentrated using an Amicon 10 kDa ultrafiltration tube into 20 mM MES, 8% sucrose, pH 6.0 buffer. The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0513] 3.2.12 Preparation of W322-1-80-12-xAb-hIgG1-HG009-D2

[0514] The antibody was reduced with 1.0 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PB 7.0 buffer (40 mM PB, pH 7.0) and shaken on a shaker at 22°C for 3 hours (shaker speed 60 rpm). The reaction solution was used directly in the next coupling reaction without removing excess TCEP.

[0515] After reducing the antibody, slowly add 20% propylene glycol (PG) and a linker-payload solution (3 equivalents, a 3:1 mixture of 3 mM HG009 in dimethylacetamide and water) in an ice bath. After thorough mixing, the coupling reaction solution is allowed to react at 4°C for 2 hours.

[0516] After the reaction, the ADC solution was filtered through a 0.22 μm PVDF pinhole filter and then purified using a strong cation exchange chromatography column (HiTrap™ SP HP, 5 mL) on a protein purifier (AKTA).

[0517] Purification Procedure: Prior to purification, equilibrate the SP HP column with 25 mL of buffer A (20 mM histidine buffer, pH 5.5). Dilute the ADC solution fourfold with buffer (20 mM histidine buffer, pH 5.5) to allow ADC binding to the column packing. After loading, wash the column with buffer A until the absorbance at 280 nm reaches baseline equilibrium. Elution is then performed by continuously replacing the buffer system from 100% buffer A (20 mM histidine buffer, pH 5.5) to 60% buffer B (20 mM histidine, 300 mM sodium chloride, pH 5.5) over 60 minutes (buffer flow rate of 5 mL / min).

[0518] The eluted ADC was concentrated into 20 mM histidine, pH 5.5 buffer using an Amicon ultrafiltration tube (10 kDa). The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0519] 3.2.13 Preparation of W322-1-80-12-xAb-hIgG1-HG004-D2

[0520] Reduce the antibody with 1.0 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in histidine 6.0 buffer (20 mM histidine, pH 6.0) and shake on a shaker at 22°C for 2 hours (60 rpm). Use the reaction solution directly in the next coupling reaction without removing excess TCEP.

[0521] After reducing the antibody, slowly add 20% propylene glycol (PG) and a linker-payload solution (3 equivalents, 3 mM HG004 in a 3:1 mixture of dimethylacetamide and water) on ice. After thorough mixing, the coupling reaction mixture was shaken at 4°C for 16.4 hours (shaker speed 60 rpm).

[0522] After the reaction, the ADC solution was filtered through a 0.22 μm PVDF pinhole filter and then purified using a strong cation exchange chromatography column (HiTrap™ SP HP, 5 mL) on a protein purifier (AKTA).

[0523] Purification Procedure: Prior to purification, equilibrate the SP HP column with 25 mL of buffer A (10 mM MES, pH 6.0). Dilute the ADC solution fivefold with buffer (10 mM MES, pH 6.0) to allow ADC binding to the column packing. After loading, wash the column with buffer A until the absorbance at 280 nm reaches baseline equilibrium. Elution is then performed by continuously replacing the buffer system from 100% buffer A (10 mM MES, pH 6.0) to 33% buffer B (10 mM MES, 300 mM NaCl, pH 6.0) over 30 minutes (buffer flow rate 5 mL / min).

[0524] The eluted ADC was concentrated into 10 mM MES, 70 mM sodium chloride, 6% sucrose, pH 6.0 buffer using an Amicon ultrafiltration tube (10 kDa). The ADC product was finally filtered through a 0.2 μm PVDF pinhole filter and sampled for testing.

[0525] 3.2.14 Preparation of HFRAO34-HG004-W2

[0526] The purified HFRA34-HG004-W2 was adjusted to a PB 9.0 buffer system (40 mM PB, pH 9.0) using pure water and PB 10.4 buffer (200 mM Na2HPO4, pH 10.4). After thorough mixing, the maleimide ring-opening reaction solution was shaken on a shaker at 22°C for 24 hours (shaker speed 60 rpm).

[0527] After the reaction was completed, the ADC solution was filtered with a 0.22 μm PVDF pinhole filter and then purified using a strong cation exchange chromatography column (HiTrap TM SP HP, 5mL) for purification.

[0528] Purification Procedure: Prior to purification, the SP HP column was equilibrated with 25 mL of buffer A (10 mM MES, pH 6.0). The ADC solution was diluted fourfold with buffer (a mixture of 800 mM MES, pH 6.0 and purified water at a ratio of 1:240) to allow ADC binding to the column medium. After loading, the column was washed with buffer A until the absorbance at 280 nm reached baseline equilibrium. Elution was then performed by continuously replacing the buffer system from 100% buffer A (10 mM MES, pH 6.0) to 33.3% buffer B (10 mM MES, 300 mM sodium chloride, pH 6.0) over 30 minutes (buffer flow rate of 5 mL / min). The purified ADC was then concentrated using Amicon ultrafiltration tubes (10 kDa) by exchange into storage buffer (10 mM MES, 92 mM sodium chloride, 6% sucrose, pH 6.0). Finally, the ADC product was filtered through a 0.2 μm PVDF pinhole filter and sampled for inspection.

[0529] Table 17. Summary of ADC detection parameters prepared in this application Note: “HIC-DAR” refers to the number of linker-payloads conjugated to a single antibody as determined by hydrophobic interaction chromatography (HIC).

[0530] Example 4. FACS binding assay of anti-FOLR1 chimeric antibodies and ADCs thereof

[0531] NCI-H2110 cells stably expressing full-length human FOLR1 protein were cultured at a rate of 1×10 5Cells were plated into standard 96-well round-bottom plates. Antibodies and ADCs (W3922-1.34.19-uIgG1K, W3922-1.40.2-uIgG1K, W3922-1.45.2-uIgG1K, FRA34-HG004-D2, FRA40-HG004-D2, and FRA45-HG004-D2) at varying concentrations starting at 400 nM were serially diluted fourfold and added to the cells, mixed (total volume 100 μL / well), and incubated at 4°C in the dark for 1 hour. W3922-BMK1 (anti-FOLR1 monoclonal antibody MORAB-003, heavy and light chain sequences are shown in Table 18) was used as a positive control, and a human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×PBS / 1% BSA, Alexa647 fluorescein-labeled goat anti-human IgG detection antibody (1×PBS / 1% BSA, 1:500 dilution) was added and incubated at 4°C in the dark for 0.5 hours. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry, and the data were analyzed using FlowJo. The experimental data were calculated using a four-parameter nonlinear fit in GraphPad Prism 7 software. 50 value.

[0532] Table 18. Reference antibody W3922-BMK1 heavy chain and light chain amino acid sequences

[0533] The results showed that three chimeric antibodies (WBP3922-1.34.19-xIgG1K, WBP3922-1.40.2-xIgG1K, and WBP3922-1.45.2-xIgG1K) and three chimeric antibody ADCs (FRA34-HG004-D2, FRA40-HG004-D2, and FRA45-HG004-D2) exhibited dose-dependent binding to NCI-H2110 cells (Figure 1 and Table 19). WBP3922-1.45.2-uIgG1K exhibited comparable binding activity to the reference antibody W3922-BMK1. The isotype control antibody hIgG1, used as a negative control, showed no significant binding to cells expressing human FOLR1 on the membrane surface.

[0534] Table 19. Binding of chimeric antibodies and ADCs to expressed human FOLR1 protein

[0535] Example 5. Antibody internalization detection of anti-FOLR1 chimeric antibody (acid wash method)

[0536] NCI-H2110 cells stably expressing full-length human FOLR1 protein were cultured at a rate of 1×10 5Cells were plated into 96-well standard round-bottom plates. Different concentrations of antibodies W3922-1.34.19-xIgG1K, W3922-1.34.19-xIgG1K, and W3922-1.45.2-xIgG1K (starting from 400nM, 4-fold serial dilution) were added to the cells and mixed (total volume 100μL / well), and incubated at 4°C in the dark for 1 hour. W3922-BMK1 was used as a positive control, and human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×PBS / 1% BSA, Alexa647 fluorescein-labeled goat anti-human IgG detection antibody (1×PBS / 1% BSA, 1:500 dilution) was added and incubated at 4°C in the dark for 0.5 hour. After washing the cells with 1× PBS / 1% BSA, quenching buffer (0.1 M glycine, 0.15 M NaCl, HCl added to pH 2.5) was added and incubated at 4°C in the dark for 5 minutes. The mean fluorescence intensity of the cells was measured using a flow cytometer (iQue3), and the data were analyzed using GraphPad Prism 7 software. The experimental data were calculated using a four-parameter nonlinear fit with EC. 50 value.

[0537] The results showed that the three chimeric antibodies (WBP3922-1.34.19-xIgG1K, WBP3922-1.40.2-xIgG1K and WBP3922-1.45.2-xIgG1K) showed dose-dependent internalization on NCI-H2110 cells (see Figure 2). 50 The concentrations of WBP3922-1.45.2-xIgG1K were 0.73 to 1.7 nM (Table 20), which was comparable to the reference antibody W3922-BMK1. Cells expressing human FOLR1 on their membrane surface did not significantly internalize the isotype control antibody hIgG1 used as a negative control.

[0538] Table 20. Internalization of chimeric antibodies on NCI-H2110

[0539] Example 6. In vitro killing assay of anti-FOLR1 chimeric antibody ADC

[0540] NCI-H2110 cells were plated at 3 × 10 3The cells were plated in a 96-well plate with 50 μL per well. The cells were then placed in an incubator (temperature set at 37°C, CO2 concentration of 5%) and cultured overnight. The next day, ADC samples of different concentrations (starting from 100 nM, 5-fold gradient dilution to 0.001024 nM) were added to the plate at 50 μL per well, and the plate was placed in an incubator (temperature set at 37°C, CO2 concentration of 5%) and cultured for 6 days. After the culture was completed, 50 μL of CellTiter-Glo was added to each well, and the plate was immediately shaken for 5 minutes to mix thoroughly, and the luminescence intensity (RLU) of each well was read using the Envision multi-function plate reader. Antibody-dependent cytotoxicity was calculated using the following formula: Cytotoxicity % = 100*(RLU 仅细胞 -RLU 样本 ) / RLU 仅细胞 The data were calculated using GraphPad Prism 7 software with a four-parameter nonlinear fit to calculate EC. 50 value.

[0541] The results showed that the three chimeric antibody ADCs (FRA34-HG004-D2, FRA40-HG004-D2 and FRA45-HG004-D2) showed dose-dependent killing on NCI-H2110 cells (see Figure 3). 50 The maximum killing rate was 96% to 97% at concentrations of 0.074 to 0.61 nM (Table 21). The killing rate of the human IgG1 isotype antibody ADC used as a control was relatively weak.

[0542] Table 21. Killing of chimeric antibody ADC on NCI-H2110 Note: W322-1-80-12-xAb-hIgG1-HG009-D2 is an ADC constructed with hIgG1 isotype control antibody and serves as a negative control. WBP3922-BMK1-HG012 (i.e., MORAB-202) serves as a positive control ADC.

[0543] Example 7. FACS binding detection of humanized antibodies and ADCs

[0544] NCI-H2110 cells stably expressing full-length human FOLR1 protein were cultured at a rate of 1×10 5Cells were plated into 96-well standard round-bottom plates. Antibodies and ADCs (W3922-1.34.19-z7-p3-uIgG1K, W3922-1.40.2-z4-p3-uIgG1K, W3922-1.45.2-z4-uIgG1K, HFRA34-HG004-D2, HFRA34-HG009-D2, HFRA34-HG004-W2, HFRA40-HG004-B2, HFRA40-HG009-B2, HFRA45-HG004-D2, and HFRA45-HG009-D2) at different concentrations starting from 400 nM were serially diluted 4-fold and added to the cells (total volume 100 μL / well). The cells were then incubated at 4°C in the dark for 1 hour. W3922-BMK1 was used as a positive control, and human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×PBS / 1% BSA, Alexa647 fluorescein-labeled goat anti-human IgG detection antibody (1×PBS / 1% BSA, 1:500 dilution) was added and incubated at 4°C in the dark for 0.5 hours. The mean fluorescence intensity of the cells was detected by flow cytometry, and the data were analyzed using FlowJo. The experimental data were calculated using a four-parameter nonlinear fit with GraphPad Prism 7 software. 50 value.

[0545] The results showed that three humanized antibodies (W3922-1.45.2-z4-uIgG1K, W3922-1.34.19-z7-p3-uIgG1K and W3922-1.40.2-z4-p3-uIgG1K) and seven humanized antibody ADCs (HFRA34-HG004-D2, HFRA34-HG009-D2, HFRA34-HG004-W2, HFRA40-HG004-B2, HFRA40-HG009-B2, HFRA45-HG004-D2 and HFRA45-HG009-D2) showed dose-dependent binding to NCI-H2110 cells (see Figure 4), EC 50 The concentrations of the isotype control antibody hIgG1 used as a negative control did not significantly bind to cells expressing human FOLR1 on the membrane surface.

[0546] Table 22. Binding of humanized antibodies and their ADCs to cells expressing human FOLR1

[0547] Example 8. Internalization detection of humanized antibodies (acid washing method)

[0548] NCI-H2110 cells stably expressing full-length human FOLR1 protein were cultured at a rate of 1×10 5Cells were plated into 96-well standard round-bottom plates. Antibodies and ADCs (W3922-1.34.19-z7-p3-uIgG1K, W3922-1.40.2-z4-p3-uIgG1K, W3922-1.45.2-z4-uIgG1K, HFRA34-HG004-D2, HFRA34-HG009-D2, HFRA34-HG004-W2, HFRA40-HG004-B2, HFRA40-HG009-B2, HFRA45-HG004-D2, and HFRA45-HG009-D2) at different concentrations starting from 400 nM were serially diluted 4-fold and added to the cells (total volume 100 μL / well). The cells were then incubated at 4°C in the dark for 1 hour. W3922-BMK1 was used as a positive control, and human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×PBS / 1% BSA, Alexa647 fluorescein-labeled goat anti-human IgG detection antibody (1×PBS / 1% BSA, 1:500 dilution) was added and incubated at 4°C in the dark for 0.5 hours. After washing the cells with 1×PBS / 1% BSA, quenching buffer (0.1M glycine, 0.15M NaCl, HCl was added to pH 2.5) was added and incubated at 4°C in the dark for 5 minutes. The mean fluorescence intensity of the cells was detected by flow cytometry, and the data were analyzed using FlowJo. The experimental data were calculated using a four-parameter nonlinear fit of GraphPad Prism 7 software for EC. 50 value.

[0549] The results showed that three humanized antibodies (W3922-1.45.2-z4-uIgG1K, W3922-1.34.19-z7-p3-uIgG1K and W3922-1.40.2-z4-p3-uIgG1K) and their ADCs (HFRA34-HG004-D2, HFRA34-HG009-D2, HFRA34-HG004-W2, HFRA40-HG004-B2, HFRA40-HG009-B2, HFRA45-HG004-D2 and HFRA45-HG009-D2) showed dose-dependent internalization on NCI-H2110 cells (see Figure 5), EC 50 The concentrations of human FOLR1 in cells expressing human FOLR1 on their membrane surface were 0.30 to 2.8 nM (Table 23). The isotype control antibody hIgG1 used as a negative control was not significantly internalized.

[0550] Table 23. Internalization of humanized antibodies and ADCs on NCI-H2110

[0551] Example 9. In vitro killing assay of humanized antibody ADC

[0552] NCI-H2110 cells were plated at 3 × 10 3 The cells were plated in a 96-well plate with 50 μL per well. The cells were then placed in an incubator (temperature set at 37°C, CO2 concentration of 5%) and cultured overnight. The next day, ADC samples of different concentrations (starting from 400nM, 5-fold gradient dilution to 0.00512nM) were added to the plate at 50 μL per well, and the plate was placed in an incubator (temperature set at 37°C, CO2 concentration of 5%) and cultured for 6 days. After the culture was completed, 50 μL of CellTiter-Glo was added to each well, and the plate was immediately shaken for 5 minutes to mix thoroughly, and the luminescence intensity of each well was read using the Envision multi-function plate reader. Antibody-dependent cytotoxicity was calculated using the following formula: Cytotoxicity% = 100*(RLU 仅细胞 -RLU 样本 ) / RLU 仅细胞 The data were calculated using GraphPad Prism 7 software with a four-parameter nonlinear fit to calculate EC. 50 value.

[0553] The results showed that seven humanized antibody ADCs (HFRA34-HG004-D2, HFRA34-HG009-D2, HFRA34-HG004-W2, HFRA40-HG004-B2, HFRA40-HG009-B2, HFRA45-HG004-D2 and HFRA45-HG009-D2) showed dose-dependent killing on NCI-H2110 cells (see Figure 6), EC 50 The maximum killing was 98% to 99% at concentrations ranging from 0.13 to 16 nM (Table 24). The killing effect of the isotype control ADC used as a control was relatively weak.

[0554] Table 24. Killing of humanized antibody ADC on NCI-H2110

[0555] Example 10. Detection of bystander killing effect of humanized antibody ADC

[0556] Raji.Luc (Vitone) and NCI-H2110 cells were cultured in RPMI1640 medium supplemented with 10% FBS and 1% P / S (penicillin-streptomycin double-antibody mixture) and passaged 2-3 times a week. Raji.Luc is a Raji cell line that stably expresses luciferase. 40 μL of each Raji.Luc and NCI-H2110 cell suspension was added to a 96-well cell culture plate at a density of 6.25×10 4 cells / mL and 5×10 4At 10 cells / mL, 20 μL of serially diluted test antibody was added to each well, with 200 μL of sterile DPBS (CORNING, 21-031-CVC) added only to the periphery of the 96-well plate. The plates were incubated in an incubator (37°C, 5% CO2) for 6 days. 50 μL of One-GLO (Promega-E6120) solution was added to each well, and the plates were gently shaken on a plate shaker for approximately half an hour. The luminescence intensity of each well was read using an Envision multi-function plate reader.

[0557] The results showed that 7 humanized antibody ADCs (HFRA34-HG004-D2, HFRA34-HG009-D2, HFRA34-HG004-W2, HFRA40-HG004-B2, HFRA40-HG009-B2, HFRA45-HG004-D2 and HFRA45-HG009-D2) showed dose-dependent bystander killing on Raji.Luc cells (see Figure 7), EC 50 The killing activity of the ADCs was relatively weak compared to the control ADCs used as controls.

[0558] Table 25. Bystander Killing Effect of Humanized Antibody ADC

[0559] Example 11. Detection of ADCC killing effect mediated by humanized antibodies and ADCs

[0560] NCI-H2110 cells were cultured in RPMI1640 medium containing 10% FBS and 1% P / S and passaged 2-3 times a week. 4.0×10 6 Cells were resuspended in 5 μL of BATDA solution and placed in a 37°C, 5% CO2 incubator for about 30 minutes. The cells were then centrifuged and resuspended in RPMI1640 complete medium and washed five times to remove as much BATDA dye as possible. Human PBMC cells (Sai ​​Li-XW0812006W) provided by the supplier were revived in a 37°C water bath and resuspended in RPMI1640 medium containing 10% FBS and 1% P / S. Recombinant human IL-2 was added to a final concentration of 200 UI / mL and placed in a 37°C, 5% CO2 incubator for about 120 minutes. 100 μL of 5×10 4 cells / mL of NCI-H2110 cells, add 50 μL of 7.5×10 6Cells were plated at a concentration of 100 μL / mL of PBMCs, followed by the addition of 50 μL of serially diluted test antibodies to each well. The culture plate was placed in a 37°C, 5% CO2 incubator for approximately 120 minutes. The plate was then centrifuged at 500g for 5 minutes. 20 μL of the supernatant was transferred to the assay plate, and 180 μL of europium solution was added. After shaking, the luminescence intensity of each well was measured using an Envision multi-function plate reader.

[0561] The results showed that three humanized antibodies (W3922-1.34.19-z7-p3-uIgG1K, W3922-1.40.2-z4-p3-uIgG1K and W3922-1.45.2-z4-uIgG1K) and seven humanized antibody ADCs (HFRA34-HG004-D2, HFRA34-HG009-D2, HFRA34-HG004-W2, HFRA40-HG004-B2, HFRA40-HG009-B2, HFRA45-HG004-D2 and HFRA45-HG009-D2) showed dose-dependent mediated killing on NCI-H2110 cells (see Figure 8), EC 50 The killing of the isotype control used as a negative control was almost undetectable.

[0562] Table 26. ADCC killing mediated by humanized antibodies and ADCs on NCI-H2110

[0563] Example 12. Plasma stability test of humanized antibodies

[0564] Human plasma was mixed with the test antibodies (W3922-1.45.2-z4-uIgG1K, W3922-1.34.19-z7-p3-uIgG1K, and W3922-1.40.2-z4-p3-uIgG1K) to a final concentration of 0.5 mg / mL. The plasma-antibody mixture was evenly divided into five 1.5 mL centrifuge tubes and labeled Day 0, Day 1, Day 4, Day 7, and Day 14. The Day 0 sample was immediately snap-frozen in liquid nitrogen and then stored in a -80°C freezer. The remaining samples were incubated in an incubator set at 37°C and 5% CO2. On Days 1, 4, 7, and 14 of the experiment, samples were removed, snap-frozen in liquid nitrogen, and stored in a -80°C freezer.

[0565] A 96-well high-adsorption ELISA plate containing 100 μL / well of W3922-hPro1 / cPro1.his ELISA (0.5 μg / mL) was placed at 4°C overnight. The next day, the frozen sample was removed, diluted (starting from 6.25 nM, diluted 4-fold to a concentration of 0.0000060 nM) and added to the plate at 100 μL per well. The plate was incubated at room temperature for 2 hours, washed, and goat anti-human Fc-HRP (Bethyl, A80-304P) was added and incubated at room temperature for 1 hour. The plate was then washed, TMB was added for color development, and the reaction was terminated with 2 M sulfuric acid. The OD450 nm and OD540 nm of each well were read by a plate reader, and the data were calculated using a four-parameter nonlinear fit using GraphPad Prism 7 software. 50 value.

[0566] The results showed that the binding ability of three humanized antibodies (W3922-1.45.2-z4-uIgG1K, W3922-1.34.19-z7-p3-uIgG1K and W3922-1.40.2-z4-p3-uIgG1K) to FOLR1 after incubation with human plasma was similar to that of the unincubated samples (see Figure 9). 50 The concentrations of the humanized antibodies were 0.035 to 0.065 nM (Table 27), demonstrating that the three humanized antibodies of the present application have long-term stability in plasma.

[0567] Table 27. Plasma stability ELISA test of humanized antibodies

[0568] Example 13. In vivo efficacy determination of humanized antibody ADC in lung cancer animal model

[0569] Mouse lung cancer cells (NCI-H2110) were obtained from Shanghai WuXi Biologics Co., Ltd. and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. NCI-H2110 cells were passaged according to standard procedures. When the cell population reached the desired level, they were harvested, counted, and plated.

[0570] NCI-H2110 cells resuspended in DPBS (cytiva, SH30028.02) were cultured at a concentration of 5×10 7After mixing with Matrigel Matrix (Corning, 354230) at a 1:1 volume ratio, the cells were inoculated subcutaneously on the right flank of CB-17 SCID mice at a volume of 0.2 mL per mouse. On day 8 after cell inoculation, mice with large tumor volume and body weight deviations were removed, and appropriate mice were randomly divided into 13 experimental groups, each with 8 mice. Dosing began on the day the animals were grouped, designated Day 0. The specific dosing schedule is shown in Table 28.

[0571] Table 28. Dosage regimen for NCI-H2110 lung cancer animal model Note: a. The dosing volume was calculated based on the experimental animal's body weight at 10 μL / g. b. Elahere (ImmunoGen, 72903-853) was used as a positive control ADC.

[0572] After administration, the body weight and tumor growth of the experimental animals were continuously observed. During the observation period, the tumor volume and animal weight were measured twice a week and the measured values ​​were recorded. The experiment was terminated after the observation was completed on the 28th day after administration. The in vivo efficacy of the ADC was evaluated by the average body weight change rate and tumor growth inhibition rate of the animals. The calculation formula is as follows:

[0573] 1) Average animal weight change rate: Weight loss rate = 100% × (BW t -BW0) / BW0

[0574] Among them, BW0 represents the mean body weight of animals in the group when the drug was administered; BW t It represents the mean body weight of animals in each group at each measurement.

[0575] 2) Tumor growth inhibition rate TGI (%): TGI (%) = [1-(TV t -TV0) / (CV t -CV0)]×100%

[0576] Among them, TV t represents the mean tumor volume of the treatment group at each measurement; TV0 represents the mean tumor volume of the treatment group at the time of group administration; CV t CV0 represents the mean tumor volume of the negative control group at each measurement; CV0 represents the mean tumor volume of the negative control group when grouped and dosed.

[0577] Results are expressed as mean ± SEM. GraphPad Prism software was used to plot line graphs of time after administration (X-axis) and animal body weight or tumor volume (Y-axis). T-test or two-way ANOVA analysis was performed on the data from each group. *P < 0.05 was considered significant. Both statistical and biological significance were considered in the analysis of results.

[0578] The weight of animals in the negative control group (DPBS) gradually decreased within the permitted animal welfare range, with no weight loss exceeding 20%, indicating that tumor-bearing animals had some impact on weight stability. During the 28-day observation period following group dosing, the average weight loss of tumor-bearing animals in the other dosing groups did not exceed 20%, demonstrating that the ADC was generally well tolerated by the experimental animals. The weight changes and weight change rates of the animals in each group after dosing are shown in Figure 10 and Table 29.

[0579] Table 29. Average body weight change rate of tumor-bearing mice in each group after administration

[0580] The tumor volumes and tumor growth inhibition rates of the animals after drug administration are shown in Figure 11 and Table 30. The higher the tumor growth inhibition rate (TGI%), the better the inhibitory effect of the corresponding drug on tumors. On day 28 after group administration, the TGI% of the positive control groups G2 W3922-BMK1-HG012 3 mg / kg and G3 W3922-BMK2 (Elahere) 3 mg / kg were 69.22% and 43.64%, respectively. The high-dose administration groups G4 HFRA34-HG004-D2 3 mg / kg, G5 HFRA40-HG004-B2 3 mg / kg, G6 HFRA34-HG004-W2 3 mg / kg, G7 HFRA45-HG004-D2 3 mg / kg, and G8 HFRA45-HG009-D2 3 mg / kg showed no significant difference in TGI. The TGI% at 3 mg / kg were 73.12%, 72.10%, 91.03%, 93.67% and 57.95%, respectively, which were statistically different from those in the negative control group, indicating that the high-dose groups all showed different degrees of tumor inhibition effects, among which the tumor inhibition effects of the G4 HFRA34-HG004-D2 3 mg / kg, G5 HFRA40-HG004-B2 3 mg / kg, G6 HFRA34-HG004-W2 3 mg / kg, and G7 HFRA45-HG004-D2 3 mg / kg groups at the same dosage were significantly better than those of the positive control group.

[0581] The TGI% of the low-dose administration groups G9 HFRA34-HG004-D2 1 mg / kg, G10 HFRA40-HG004-B2 1 mg / kg, G11 HFRA34-HG004-W2 1 mg / kg, G12 HFRA45-HG004-D2 1 mg / kg and G13 HFRA45-HG009-D2 1 mg / kg were 41.56%, 45.82%, 52.09%, 53.43% and 45.41%, respectively, all showing different degrees of tumor inhibition effect. Among them, the tumor inhibition effect of the G11 HFRA34-HG004-W2 1 mg / kg and G12 HFRA45-HG004-D2 1 mg / kg administration groups at the same dose was the best.

[0582] Table 30. Tumor growth inhibition rate of each group of tumor-bearing mice after administration

[0583] In summary, the ADC was well tolerated by animals in all dosing groups. At a dose of 3 mg / kg, all dosing groups demonstrated effective tumor inhibition, with the G4 HFRA34-HG004-D2, G5 HFRA40-HG004-B2, G6 HFRA34-HG004-W2, and G7 HFRA45-HG004-D2 groups exhibiting significantly superior tumor inhibition compared to the positive control group. Furthermore, HFRA34-HG004-W2 and HFRA45-HG004-D2 demonstrated the best tumor inhibition at both high and low doses.

[0584] Example 14 Binding Detection of Humanized Antibody ADC to Ovarian Cancer Cell Lines

[0585] Cell culture

[0586] The ovarian cancer cell line OV-90 was cultured in RPMI-1640+10% FBS+1% Antibiotic-Antimycotic (Gibco, #15240-062) in a 37°C, 5% CO2 incubator. After the cell density reached 80%-90%, the cells were collected for subsequent experimental plating.

[0587] Flow cytometry detection of the binding ability of the test substance on the surface of OV-90 cell line

[0588] 1) After treating the cells with trypsin, count the cells and ensure that the cell viability is above 90.0%.

[0589] 2) In a 96-well V-bottom plate, press 2×10 5 OV-90 cells were added to each well (duplicate wells).

[0590] 3) Wash cells twice with FACS staining buffer, centrifuge at 450 g for 5 minutes, and discard the supernatant. In the first round, add 100 μL of the test substance MORAb-202, IMGN-853, HFRA34-HG004-W2, HFRAO34-HG004-W2, HFRA34-HG004-D2, or isotype control (starting at 300 nM, 3-fold dilution, 6 concentration gradients) to the corresponding wells. In the second round, add 100 μL of the test substance IMGN-853, HFRA34-HG004-W2, HFRAO34-HG004-W2, HFRA34-HG004-D2, or isotype control (starting at 300 nM, 2-fold dilution, 6 concentration gradients) to the corresponding wells and incubate at 4°C for 60 minutes.

[0591] 4) Wash cells twice with FACS staining buffer, centrifuge at 450g for 5 minutes, and discard the supernatant. Add PE anti-human IgG Fc antibody (1:200 dilution) to each well and incubate at 4°C for 40 minutes in the dark.

[0592] 5) After incubation, wash the cells twice with FACS staining buffer, centrifuge at 450g for 5 minutes, and discard the supernatant. Resuspend the cells in 200 μL FACS staining buffer and prepare for FACS analysis.

[0593] Data Analysis

[0594] Data were analyzed using FlowJo and Graphpad Prism 10 to calculate binding curves and EC values. 50 .

[0595] result

[0596] In the first round of experiments, 6 concentration points were set in the range of 1.23nM to 300nM, and the binding ability of the 6 test substances on OV-90 cells was evaluated. The results are shown in Figure 12 and Tables 31-32. Figure 12 shows the binding curves of the test substances on OV-90 cells. Table 31 lists the MFI (first round) of the test substances on OV-90, and Table 32 lists the EC values ​​of the test substances on OV-90 cells. 50 (First round).

[0597] Table 31 MFI of the test substances on OV-90 (first round)

[0598] Table 32 EC values ​​of the test substances on OV-90 50 (First Round)

[0599] The first round of results showed that HFRA34-HG004-W2, HFRAO34-HG004-W2 and HFRA34-HG004-D2 had strong binding ability on OV-90 cells, with the plateau MFI values ​​greater than 1100, and the corresponding EC 50 The values ​​were 3.865nM, 3.579nM and 3.532nM respectively; the binding affinity of IMGN-853 on OV-90 was relatively weak, with the plateau MFI value around 700, and the corresponding EC 50 The binding of MORAb-202 to OV-90 cells within this concentration range did not reach the upper and lower plateaus, so further analysis of this test substance was cancelled in the second round of experiments.

[0600] The second round of experiments set 6 concentration points in the range of 0.938nM to 30nM to further analyze the binding ability of the five test substances on OV-90 cells. The experimental results were basically consistent with the first round. The binding EC values ​​of HFRA34-HG004-W2, HFRAO34-HG004-W2, HFRA34-HG004-D2 and IMGN-853 on OV-90 cells 50 The values ​​were 3.581nM, 3.560nM, 3.515nM and 2.247nM, respectively. Figure 13 shows the binding analysis of the test substances on OV-90 cells. Table 33 lists the MFI (Round I) of the test substances on OV-90 cells, and Table 34 lists the EC values ​​of the test substances on OV-90 cells. 50 (Round II).

[0601] Table 33. MFI of test substances on OV-90 (second round)

[0602] Table 34 EC50 of the test substances on OV-90 cells (second round)

[0603] Example 15 Detection of the cell proliferation inhibitory effect of humanized antibody ADC on human ovarian cancer cell line OV-90

[0604] Cell culture

[0605] Ovarian cancer cell line OV-90 was cultured in RPMI-1640 + 10% FBS + 1% Antibiotic-Antimycotic (Gibco, #15240-062) in a 37°C, 5% CO2 incubator. Cells were passaged regularly and cells in the logarithmic growth phase were used for plating.

[0606] Cell plating

[0607] Cells were stained with trypan blue and viable cells were counted. Adjust the cell concentration to the appropriate level and add 135 μL of cell suspension to each well of the culture plates (density: 4000 cells / well, incubate for 7 days). Incubate blank wells with culture medium without cells. Incubate the plates overnight in an incubator at 37°C, 5% CO2, and 100% relative humidity.

[0608] Compound storage plate preparation

[0609] Prepare a 10x compound storage plate: Dilute compounds IMGN853, MORAb-202, Eribulin, HFRA34-HG004-W2, HFRAO34-HG004-W2, and HFRA34-HG004-D2 in cell culture medium from the highest concentration (100 nM) to the lowest concentration (0.015 nM), using a 3-fold dilution gradient for a total of 9 concentrations (100 nM, 33.33 nM, 11.11 nM, 3.703 nM, 1.234 nM, 0.411 nM, 0.137 nM, 0.045 nM, and 0.015 nM). Prepare the plate freshly each time.

[0610] Compound-treated cells

[0611] Dosing: Add 15 μL of 10x compound working solution to a cell culture plate containing culture medium. Dilute the compound concentration 10-fold. In duplicate, plate each well at each concentration for each compound. Add 15 μL of cell culture medium to the vehicle and blank controls. Return the 96-well plate to the incubator and culture for 7 days.

[0612] CellTiter-Glo luminescent cell viability assay

[0613] Cell viability was detected according to the instructions of the Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega-G7573).

[0614] Data Analysis

[0615] The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1 – (RLU compound – RLU blank control) / (RLU vehicle control – RLU blank control)) * 100%. The inhibition rate of the compound at different concentrations was calculated in Excel, and then the inhibition curve was plotted and relevant parameters, including minimum inhibition rate, maximum inhibition rate, and IC, were calculated using GraphPad Prism software. 50 .

[0616] result

[0617] The parameters of cell proliferation inhibition by compounds in cell activity experiments are summarized in Table 35

[0618] Table 35. Parameters of cell proliferation inhibition by compounds

[0619] Figure 14 shows the inhibitory growth curves of Eribulin, MORAb-202, HFRAO34-HG004-W2, HFRA34-HG004-W2, HFRA34-HG004-D2, and IMGN853 against the human ovarian cancer cell line OV-90. The curves were fitted using the log(inhibitor) vs. response variable slope equation in Prism software. Data points represent the inhibition rate (n=2). Compounds were diluted threefold with a starting concentration of 10 nM. Curves were plotted with the log value of compound concentration as the X value.

[0620] As can be seen from Figure 14 and Table 35, the highest inhibition rates of the test compounds HFRAO34-HG004-W2, HFRA34-HG004-W2, and HFRA34-HG004-D2 on OV-90 cell proliferation were all above 70%.

[0621] Example 16 In vivo efficacy determination of humanized antibody ADC in an ovarian cancer animal model

[0622] Human ovarian cancer OV-90 cells were cultured in vitro in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibacterial / antifungal agents at 37°C and 5% CO2. Cells were passaged every 2-3 days at a 1:2 ratio. When the cell saturation reached 80%-90%, the cells were harvested, counted, and inoculated. 0.2 mL (5 × 10 6 OV-90 cells (solvent PBS) were mixed with Matrigel at a volume ratio of 1:1 and inoculated subcutaneously into the right back of each nude mouse (Balb / c nude mice, 6-8 weeks old). The average tumor volume reached 147 mm 3 The drug was administered in groups according to Table 36.

[0623] Table 36 Animal groups and dosing regimens for in vivo efficacy experiments Note: N is the number of mice

[0624] The health status and mortality of the animals were monitored daily. Routine examinations included observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as behavioral activity, food and water intake (visual observation only), weight changes (weight was measured twice a week), physical signs or other abnormal conditions. The number of deaths and side effects of animals in each group was recorded based on the number of animals in each group. The tumor diameter was measured with a vernier caliper twice a week. The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0625] The tumor inhibition effect of the compound is evaluated by TGI (%) or relative tumor proliferation rate T / C (%). TGI (%) reflects the tumor growth inhibition rate. Calculation of TGI (%): TGI (%) = [1-(average tumor volume of a certain treatment group at the end of drug administration - average tumor volume of the treatment group at the beginning of drug administration) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the beginning of treatment)] × 100%. Relative tumor proliferation rate T / C (%): Calculation formula is as follows: T / C% = T / C × 100%. T and C are the average tumor volumes of the drug administration group and the vehicle control group on the same day, respectively. After the end of the experiment, the tumor weight will be measured and T / C will be calculated. weight Percentage, T weight and C weight Represent the tumor weights of the drug-treated group and the vehicle control group, respectively.

[0626] Mortality, morbidity, and weight change

[0627] Animal body weight was used as an indirect indicator for assessing drug toxicity. In this model, no significant weight loss was observed in any of the dosing groups (Figure 15 and Table 37). No morbidity or mortality was observed. The effects of the tested ADC on body weight in female Balb / c nude mice bearing subcutaneous OV-90 cell xenografts are shown in Figures 15 and 16.

[0628] Tumor volume

[0629] Table 37 Tumor volume of each group at different time points a. Mean ± SEM; b. Days after administration

[0630] Tumor growth curve

[0631] The tumor growth curve of the female Balb / c nude mouse model bearing subcutaneous OV-90 cell xenograft tumors after treatment with the tested ADC is shown in FIG17 .

[0632] Anti-tumor efficacy evaluation indicators

[0633] The anti-tumor efficacy was calculated and evaluated based on tumor volume on day 35 after dosing. The results are shown in Table 38. As shown in Table 38, each test substance and the control ADC IMGN853 had a significant anti-tumor effect on OV-90 ovarian cancer subcutaneous xenografts, with no significant differences in efficacy between the dosing groups.

[0634] Table 38 Evaluation of the anti-tumor efficacy of the tested ADCs on the OV-90 xenograft tumor model Note: a. Mean ± SEM. b. Tumor growth inhibition was determined by T / C and TGI (TGI (%) = [1-(T 35 -T0) / (V 35 The cp value was calculated based on the tumor volume.

[0635] The results showed that all test substances and the control ADC IMGN853 had significant tumor inhibitory effects on OV-90 ovarian cancer subcutaneous xenografts.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to FOLR1 protein, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein (1) the VH comprises the heavy chain complementary determining region (HCDR) 1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 49; and the VL comprises the light chain complementary determining region (LCDR) 1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 50; (2) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 51; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 52; (3) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 53; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 54; (4) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 7; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 8; (5) the VH comprises the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 9; and the VL comprises the LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 10; or (6) The VH comprises the heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 of VH as shown in SEQ ID NO: 11; and the VL comprises the LCDR1, LCDR2 and LCDR3 of VL as shown in SEQ ID NO:

12.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein (1) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 62, 21, and 22, respectively; (2) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 16, and 15, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 62, 21, and 22, respectively; (3) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 17, 18, and 19, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively; (4) the VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22, respectively; or (5) The VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 16 and 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein (1) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50; (2) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52; (3) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 53, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 54; (4) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 7, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 8; (5) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 10; or (6) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

12.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is of an isotype selected from IgG, IgA, IgM, IgE and IgD; preferably, the antibody is of IgG isotype.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is of a subtype selected from IgG1, IgG2, IgG3 and IgG4; preferably, the antibody is of IgG1 subtype.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein (1) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:43, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:44; (2) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 45, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 46; (3) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 47, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 48; (4) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2; (5) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:3, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:4; or (6) the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:5, and the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

6.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv and ds-scFv.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a monovalent, bivalent or multivalent antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the antibody is a bispecific antibody, and the bispecific antibody further comprises a second antigen-binding region that binds to a second antigen; preferably, the second antigen is selected from cancer-associated antigens, immune cell antigens, and immune checkpoint molecules.

12. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

13. A vector comprising the nucleic acid molecule of claim 12.

14. A cell comprising the nucleic acid molecule of claim 12 or the vector of claim 13.

15. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the method comprising: (1) culturing the cell of claim 14 under conditions suitable for expression of the antibody or antigen-binding fragment thereof; (2) isolating the antibody or antigen-binding fragment thereof from the culture medium and / or culture supernatant of the cell.

16. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

17. A genetically modified cell comprising the chimeric antigen receptor of claim 16; preferably, the genetically modified cell is an immune cell, such as a T cell or a NK cell.

18. An antibody conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, and a chemical moiety coupled to the antibody or antigen-binding fragment thereof; preferably, the chemical moiety is selected from the group consisting of a cytotoxic drug, an immunostimulatory molecule, and a detectable label.

19. An antibody drug conjugate (ADC), wherein the antibody drug conjugate has the structure of the following formula (I): Ab-(L-(D) v ) w (I) in, Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, L is a linker, D is a drug group, v is 1, 2 or 3, preferably 3; w ranges from about 1 to about 20, preferably 1 to 8, and more preferably w is 2 or 4.

20. The antibody drug conjugate of claim 19, wherein said -L-(D) v Selected from the compound of formula (II), or its tautomer, stereoisomer or pharmaceutically acceptable salt: in, Indicates -L-(D) v The site of attachment to Ab; a, b, c, d, e, and f are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably 0, 1, 2, 3, 4, or 5; preferably 0, 1, 2, or 3; preferably 1 or 2; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably 1, 2, 3, 4 or 5; preferably 1, 2 or 3; preferably 1; m is 2 or 3; X is C, N or Si; A is selected from R is Where D is the drug group; n2 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; preferably 7, 8, 9, 10 or 11; Y is a bond or wherein n1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably 1, 2, 3, 4 or 5; preferably 1, 2 or 3; b1, c1, d1 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably 0, 1, 2, 3, 4 or 5; preferably 0, 1, 2 or 3; preferably 1 or 2; When X is C or Si, m is 3; When X is N, m is 2.

21. The antibody drug conjugate according to claim 19 or 20, wherein The-L-(D) v A compound selected from formula (III), (III-1) or (III-2), or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof: wherein R is as defined in claim 20.

22. The antibody drug conjugate according to any one of claims 19 to 21, wherein R is Preferably, n2 is 7 or 11.

23. The antibody drug conjugate according to any one of claims 19 to 22, wherein The drug group is derived from eribulin, methyl auristatin E or SN-38, and the eribulin, methyl auristatin E and SN-38 have the following structures: Preferably, the drug group is selected from: More preferably 24. The antibody drug conjugate according to any one of claims 19 to 23, wherein the -L-(D) v Selected from the following specific compounds or their tautomers, stereoisomers or pharmaceutically acceptable salts, which are represented by the structure shown in formula (IV) in, Rx is And n2, * and Payload are defined as follows: The following compounds are particularly preferred: Where Rx is: More preferably, The Rx is selected from: The Rx is more preferably:

25. The antibody drug conjugate according to any one of claims 19 to 24, wherein The antibody drug conjugate is selected from: (1) in, a. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO:43, the light chain comprises the amino acid sequence set forth in SEQ ID NO:44, and w is 2; b. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO:45, the light chain comprises the amino acid sequence set forth in SEQ ID NO:46, and w is 2; c. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO:47, the light chain comprises the amino acid sequence set forth in SEQ ID NO:48, and w is 2; d. The heavy chain of Ab comprises the amino acid sequence shown in SEQ ID NO: 1, the light chain comprises the amino acid sequence shown in SEQ ID NO: 2, and w is 2; e. The heavy chain of Ab comprises the amino acid sequence shown in SEQ ID NO: 3, the light chain comprises the amino acid sequence shown in SEQ ID NO: 4, and w is 1, 2 or 3; or f. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO: 5, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 6, and w is 2; or (2) in, a. The heavy chain of Ab comprises the amino acid sequence set forth in SEQ ID NO:43, the light chain comprises the amino acid sequence set forth in SEQ ID NO:44, and w is 2; b. The heavy chain of Ab comprises the amino acid sequence shown in SEQ ID NO:45, the light chain comprises the amino acid sequence shown in SEQ ID NO:46, and w is 2; or c. The heavy chain of Ab comprises the amino acid sequence shown in SEQ ID NO: 47, the light chain comprises the amino acid sequence shown in SEQ ID NO: 48, and w is 2.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the nucleic acid molecule of claim 12, the vector of claim 13, the chimeric antigen receptor of claim 16, the genetically modified cell of claim 17, the antibody conjugate of claim 18, or the antibody drug conjugate of any one of claims 19 to 25, and optionally a pharmaceutically acceptable carrier or excipient.

27. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the nucleic acid molecule of claim 12, the vector of claim 13, the chimeric antigen receptor of claim 16, the genetically modified cell of claim 17, the antibody conjugate of claim 18, the antibody drug conjugate of any one of claims 19 to 25, or the pharmaceutical composition of claim 26; optionally, the kit further comprises instructions for use and / or an administration device.

28. A method for treating, alleviating and / or preventing a FOLR1-mediated disease in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the vector according to claim 13, the chimeric antigen receptor according to claim 16, the genetically modified cell according to claim 17, the antibody conjugate according to claim 18, the antibody drug conjugate according to any one of claims 19 to 25, or the pharmaceutical composition according to claim 26.

29. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the nucleic acid molecule of claim 12, the vector of claim 13, the chimeric antigen receptor of claim 16, the genetically modified cell of claim 17, the antibody conjugate of claim 18, the antibody drug conjugate of any one of claims 19 to 25, or the pharmaceutical composition of claim 26 in the preparation of a medicament for treating, alleviating and / or preventing a FOLR1-mediated disease in a subject.

30. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the nucleic acid molecule of claim 12, the vector of claim 13, the chimeric antigen receptor of claim 16, the genetically modified cell of claim 17, the antibody conjugate of claim 18, the antibody drug conjugate of any one of claims 19 to 25, the pharmaceutical composition of claim 26, or the kit of claim 27, for use in treating, alleviating and / or preventing a FOLR1-mediated disease in a subject.

31. The method of claim 28, the use of claim 29, or the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, chimeric antigen receptor, genetically modified cell, antibody conjugate, antibody drug conjugate, pharmaceutical composition or kit of claim 30, wherein: The FOLR1-mediated disease includes cancers with high FOLR1 expression; preferably, the cancer is selected from lung cancer such as non-small cell lung cancer, ovarian cancer, mesothelioma, breast cancer such as triple-negative breast cancer, endometrial cancer, fallopian tube cancer or primary peritoneal cancer.

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