Biparatopic fr-α antibodies and immunoconjugates

Abstract

To provide biparatopic antibodies comprising polypeptides that bind to folate receptor α (FRα) and compositions comprising such biparatopic antibodies.SOLUTION: In a specific aspect, biparatopic antibodies bind to FRα and modulate FRα activity. The present disclosure also provides methods for treating disorders, such as cancer, by administering a biparatopic antibody that specifically binds to FRα and modulates FRα activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The field of the disclosure relates generally to biparatopic antibodies and immunoconjugates that bind to the human folate receptor 1 (FRα). [Background technology]

[0002] Cancer is one of the leading causes of death in the developed world, with over one million people diagnosed with cancer and half a million deaths annually in the United States alone. Overall, it is estimated that more than one in three people will develop some form of cancer in their lifetime.

[0003] Antibody-drug conjugates (ADCs), which consist of highly cytotoxic drugs conjugated to antibodies that bind to tumor-associated antigens, are a promising therapeutic strategy to enhance the efficacy of tumor-targeting antibodies. ADCs offer the possibility to combine the favorable pharmacokinetics, biodistribution, and tumor-targeting properties of antibodies with the potent cell-killing mechanisms provided by the attached small molecule or payload.

[0004] Folate receptor-α (FRα or FOLR1) is a glycosylphosphatidylinositol-linked cell surface glycoprotein with high affinity for folate. Its physiological role in normal and cancerous tissues has not yet been fully elucidated. Most normal tissues do not express FRα, and physiological folate transport into most cells is thought to be mediated by several other proteins, particularly the reduced folate carrier. High levels of FRα have been found in serous and endometrioid epithelial ovarian cancers, endometrial adenocarcinomas, and non-small cell lung cancers of the adenocarcinoma subtype. Importantly, expression of FRα is maintained in metastatic lesions and recurrent cancers in ovarian cancer patients, as well as after chemotherapy in epithelial ovarian and endometrial cancers. These properties, together with the highly restricted expression of FRα in normal tissues, make FRα a very promising target for targeted therapies such as ADCs.

[0005] Mirvetuximab soravtansine (IMGN853), a folate-targeted ADC comprising an FRα-targeting antibody conjugated to DM4, a potent tubulin-active maytansinoid, was recently evaluated in the clinic in patients with platinum-resistant ovarian cancer with intermediate and high FRα levels. The FORWARD I phase 3 trial randomized 366 patients 2:1 to receive either mirvetuximab soravtansine or physician's choice of single-agent chemotherapy (pegylated liposomal doxorubicin, topotecan, or weekly paclitaxel). Although the trial did not meet its primary endpoint of improved progression-free survival (PFS) (overall population hazard ratio (HR) = 0.98, p = 0.897), the prespecified high FRα subpopulation (218 / 366) demonstrated an overall response rate of 24% with IMGN853 treatment compared with 10% with standard of care chemotherapy. Additionally, in the pre-specified high FRα subpopulation, patients who received IMGN853 had longer PFS compared to chemotherapy (HR=0.69, p-value=0.049), and patients who received IMGN853 had longer overall survival compared to chemotherapy (HR=0.62, p-value=0.033). While these results are promising for patients expressing high levels of FRα, the results also demonstrated the limitations of IMGN853 in improving progression-free survival across a broader patient population.

[0006] Thus, there remains a need to identify additional folate-targeting ADCs that may result in more effective treatments and higher ADC delivery. Summary of the Invention [Means for solving the problem]

[0007] Provided herein is a biparatopic antibody or antigen-binding fragment thereof that specifically binds to human folate receptor 1 (FRα), the antibody or antigen-binding fragment thereof comprising: (a) a first FRα-binding domain comprising a first variable heavy chain (VH) and a first variable light chain (VL) that binds to a first epitope on FRα; and (b) a second FRα-binding domain comprising a second VH and a second VL that binds to a second epitope on FRα.

[0008] In some embodiments, the first FRα-binding domain specifically binds to the same epitope of FRα as an antibody comprising a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 26, and a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21. In some embodiments, the first FRα-binding domain specifically binds to the same epitope of FRα as an antibody comprising a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 57, and 26, and a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21. In some embodiments, the first FRα-binding domain competitively inhibits binding to the same epitope of FRα as an antibody comprising a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 26, and a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21. In some embodiments, the first FRα-binding domain competitively inhibits binding to the same epitope of FRα as an antibody comprising a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 57, and 26, and a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21. In some embodiments, the second FRα-binding domain specifically binds to the same epitope of FRα as an antibody comprising a VH amino acid sequence of SEQ ID NO: 22 or 23, and a VL amino acid sequence of SEQ ID NO: 17 or 18. In some embodiments, the second FRα-binding domain competitively inhibits binding to the same epitope of FRα as an antibody comprising a VH amino acid sequence of SEQ ID NO: 22 or 23, and a VL amino acid sequence of SEQ ID NO: 17 or 18.

[0009] In some embodiments, the first VH comprises VH CDR1-3 comprising the amino acid sequences of (a) SEQ ID NOs: 10-12 or (b) SEQ ID NOs: 15, 16, and 12, respectively, and the first VL comprises VL CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 4-6, respectively. In some embodiments, the first VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 26, and / or the first VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21. In some embodiments, the first FRα-binding domain competitively inhibits binding to the same epitope of FRα as an antibody comprising a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 57, and 26, and a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21. In some embodiments, the second VH comprises VH CDR1-3 comprising the amino acid sequences of (a) SEQ ID NOs: 7-9 or (b) SEQ ID NOs: 13, 14, and 9, respectively, and the second VL comprises VL CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 1-3, respectively. In some embodiments, the second VH comprises the amino acid sequence of SEQ ID NO: 22 or 23, and / or the second VL comprises the amino acid sequence of SEQ ID NO: 17 or 18.

[0010] In some embodiments, the first VH and VL pair and / or the second VH and VL pair are murine, non-human, humanized, chimeric, resurfaced, or human. In some embodiments, the antibody or antigen-binding fragment thereof binds to human FRα but does not bind to FOLR2 or FOLR3. In some embodiments, the first FRα antigen-binding domain is a single chain variable fragment (scFv). In some embodiments, the first FRα binding domain scFv has a peptide orientation of VH-linker-VL. In some embodiments, the first FRα binding domain scFv has a peptide orientation of VL-linker-VH. In some embodiments, the second FRα binding domain is a single chain variable fragment (scFv). In some embodiments, the second FRα binding domain In some embodiments, the scFv of the second FRα-binding domain has a peptide orientation of VH-linker-VL. In some embodiments, the scFv of the second FRα-binding domain has a peptide orientation of VL-linker-VH. In some embodiments, the linker is a glycine-serine linker.

[0011] In some embodiments, the second FRα-binding domain comprises an amino acid sequence selected from SEQ ID NOs: 27-29. In some embodiments, the first FRα-binding domain comprises an amino acid sequence selected from SEQ ID NOs: 30-32. In some embodiments, a biparatopic antibody or antigen-binding fragment thereof disclosed herein comprises the amino acid sequence of (i) SEQ ID NOs: 33 and 34, (ii) SEQ ID NOs: 35 and 36, (iii) SEQ ID NOs: 37 and 38, or (iv) SEQ ID NOs: 39 and 40.

[0012] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:41-43.

[0013] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:44-46.

[0014] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof is a tetravalent biparatopic antibody or antigen-binding fragment thereof. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof is a bivalent biparatopic antibody or antigen-binding fragment thereof. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises an FRα-binding domain selected from the group consisting of a tandem scFv, a diabody, a triabody, a tetrabody, and a knob-in-hole structure. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof has a knob-in-hole (KIH) structure.

[0015] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises an FRα-binding domain, the FRα-binding domain comprising SEQ ID NOs: 1-3 and 7-9, and is located on the knob side of the KIH structure. In some embodiments, the FRα-binding domain comprising SEQ ID NOs: 1-3 and 7-9, and is located on the hole side of the KIH structure. In some embodiments, the FRα-binding domain comprising SEQ ID NOs: 4-6 and 10-12, and is located on the knob side of the KIH structure. In some embodiments, the FRα-binding domain comprising SEQ ID NOs: 4-6 and 10-12, and is located on the hole side of the KIH structure.

[0016] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises a full-length antibody. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises a first FRα-binding domain, and the first FRα-binding domain is a full-length antibody. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises a second FRα-binding domain, and the second FRα-binding domain is a full-length antibody. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises an antigen-binding fragment. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises a first FRα-binding domain, and the first FRα-binding domain is an antigen-binding fragment. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises a second FRα-binding domain, and the second FRα-binding domain is an antigen-binding fragment. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 41-43.

[0017] In some embodiments, provided herein is a combination of isolated nucleic acid molecules encoding the biparatopic antibodies or antigen-binding fragments thereof disclosed herein.

[0018] In some embodiments, provided herein is an isolated vector comprising one of the nucleic acid molecules disclosed herein.

[0019] In some embodiments, provided herein is a host cell comprising an isolated nucleic acid molecule disclosed herein, or a combination of isolated vectors disclosed herein. In some embodiments, the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

[0020] In some embodiments, provided herein is a pharmaceutical composition comprising a combination of a biparatopic antibody or antigen disclosed herein, a nucleic acid molecule(s) disclosed herein, a vector disclosed herein, or a host cell disclosed herein, and a pharma- ceutically acceptable carrier or excipient. In some embodiments, provided herein is a pharmaceutical composition comprising a biparatopic antibody disclosed herein, and a pharmaceutical carrier or excipient. In some embodiments, the pharmaceutical composition comprises an average of 1-10 drugs per antibody or antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition comprises an average of 2-5 drugs per antibody or antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition comprises an average of 3-4 drugs per antibody or antigen-binding fragment thereof.

[0021] In some embodiments, provided herein is a method of making a biparatopic antibody disclosed herein, comprising: (a) culturing cells that express the antibody; and (b) isolating the antibody from the cultured cells. In some embodiments, the cultured cells are eukaryotic.

[0022] In some embodiments, provided herein is an immunoconjugate represented by the formula: [ka] or a pharma- ceutically acceptable salt thereof, CB is any biparatopic antibody or antigen-binding fragment thereof provided herein; L2 is represented by one of the following formulas: [ka] During the ceremony, R x , R y , R x’ , and R y’ is, for each occurrence, independently H, -OH, halogen, -O-(C 1-4 alkyl), -SO3H, -NR 40 R 41 R 42 + or optionally -OH, halogen, SO3H, or NR 40 R 41 R 42 + C replaced by 1-4 alkyl, where R 40 , R 41 , and R 42 are each independently H or C 1-4 is alkyl; l and k each independently represent an integer of 1 to 10; l1 is an integer from 2 to 5; k1 is an integer from 1 to 5; s1 indicates the site connected to the cell-binding agent CB, and s3 indicates the site connected to the A group; A is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1-3 is alkyl; L1 is expressed by the following formula: -CR 3 R 4 -(CH2) 1-8 -C(=O)- In the formula, R 3 and R 4are each independently H or Me, and the -C(=O)- moiety of L1 is connected to D; D is expressed as follows: [ka] q is an integer from 1 to 20. In some embodiments, q is an integer from 1 to 10. In some embodiments, q is an integer from 2 to 5. In some embodiments, q is an integer from 3 to 4.

[0023] In some embodiments, the R of the immunoconjugate x , R y , R x’ , and R y’ are all H; and l and k are each independently an integer from 2 to 6. In some embodiments, A of the immunoconjugate is a peptide comprising 2 to 5 amino acid residues.

[0024] In some embodiments, A of the immunoconjugate is Gly-Gly-Gly, Ala-Val, Val-Ala, D-Val-Ala, Val-Cit, D-Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Al a-Ala-D-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 54), β-Ala-Leu-Ala-Leu (SEQ ID NO: 55), Gly-Phe-Leu-Gly (SEQ ID NO: 56), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val- D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, D-Ala-Pro, and D-Ala-tBu-Gly, wherein the first amino acid of each peptide is connected to the L2 group and the last amino acid of each peptide is -NH-CR 1 R 2 In some embodiments, the R 1 and R 2 are both H. In some embodiments, L1 of the immunoconjugate is -(CH2) 4-6 -C(=O)-.

[0025] In some embodiments, D of the immunoconjugate is represented by the formula: [ka]

[0026] In some embodiments, the immunoconjugate has the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein: [ka] is any biparatopic antibody or antigen-binding fragment thereof provided herein, wherein L is connected to the L2 group via the amine group of Lys; [ka] is any of the biosynthetic compounds provided herein, wherein the L2 group is connected to the thiol group of Cys. a paratopic antibody or an antigen-binding fragment thereof; R 3 and R 4 are each independently H or Me; m1, m3, n1, r1, s1, and t1 each independently represent an integer of 1 to 6; m2, n2, r2, s2, and t2 each independently represents an integer of 1 to 7; t3 is an integer from 1 to 12; D1 is expressed by the following formula: [ka]

[0027] In some embodiments, the immunoconjugate is represented by the formula: [ka] During the ceremony: m1 and m3 each independently represent an integer of 2 to 4; m2 is an integer from 2 to 5; r1 is an integer from 2 to 6; r2 is an integer from 2 to 5.

[0028] In some embodiments, A of the immunoconjugate is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D -Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0029] In some embodiments, the immunoconjugate has the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein: A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly; D1 is expressed by the following formula: [ka]

[0030] In some embodiments, the immunoconjugate is represented by the formula: [ka] In the formula, D1 is represented by the following formula: [ka]

[0031] In some embodiments, the immunoconjugate is represented by the formula: [ka] During the ceremony, CBA is any biparatopic antibody or antigen-binding fragment thereof provided herein; q is an integer from 1 to 10, for example, 1 or 10; D1 is expressed by the following formula: [ka]

[0032] In some embodiments, q is an integer from 2 to 5. In some embodiments, q is an integer from 3 to 4.

[0033] In some embodiments, the immunoconjugate has the following formula: [ka] or a pharma- ceutically acceptable salt thereof, CBA is a biparatopic antibody or antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 41-43; D1 is expressed by the following formula: [ka] q is an integer from 1 to 10. In some embodiments, q is an integer from 2 to 5. In some embodiments, q is an integer from 3 to 4.

[0034] In some embodiments, disclosed herein is an immunoconjugate having the formula (A)-(L)-(C), wherein: (A) is any biparatopic antibody or antigen-binding fragment provided herein, (L) is a linker; (C) is a cytotoxic agent and a linker (L) connects (A) to (C).

[0035] In some embodiments, the linker of the immunoconjugates disclosed herein is selected from the group consisting of cleavable linkers, non-cleavable linkers, hydrophilic linkers, and dicarboxylic acid-based linkers. In some embodiments, the linker is selected from the group consisting of N-(γ-maleimidobutryloxy)sulfosuccinimide ester (sulfo-GMBS or sGMBS), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) or N-succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate (SPP). N-Succinimidyl 4-(2-pyridyldithio)butanoate (Sulfo-SPP); N-Succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC); N-Sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (Sulfo-SMCC); N-Succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); and N-Succinimidyl-[(N-maleimidopropionate) PEG-4-maleimide)-tetraethylene glycol] ester (NHS-PEG4-maleimide).

[0036] In some embodiments, the linker is sulfo-GMBS.

[0037] In some embodiments, the linker is GMBS.

[0038] In some embodiments, the linker is sulfo-SPDB.

[0039] In some embodiments, the cytotoxic agent of the immunoconjugates disclosed herein is selected from the group consisting of maytansinoids, maytansinoid analogs, benzodiazepines, taxoids, CC-1065, CC-1065 analogs, duocarmycins, duocarmycin analogs, calicheamicins, dolastatins, dolastatin analogs, auristatins, tomaymycin derivatives, and leptomycin derivatives or prodrugs of the agents. In some embodiments, the cytotoxic agent is a maytansinoid.

[0040] In some embodiments, the immunoconjugate further comprises a second (C). In some embodiments, the immunoconjugate further comprises a third (C). In some embodiments, the immunoconjugate further comprises a fourth (C).

[0041] In some embodiments, provided herein is a composition comprising at least one immunoconjugate disclosed herein, wherein the immunoconjugate comprises an average of 3-4 Cs per A.

[0042] In some embodiments, a pharmaceutical composition is provided comprising an immunoconjugate provided herein and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an average of 1-10 drugs per antibody or antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition comprises an average of 2-5 drugs per antibody or antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition comprises an average of 3-4 drugs per antibody or antigen-binding fragment thereof.

[0043] In some embodiments, provided herein is a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein, an immunoconjugate disclosed herein, or a pharmaceutical composition disclosed herein.

[0044] In some embodiments, provided herein are methods of treating cancer. In some embodiments, the cancer is ovarian cancer, uterine cancer, peritoneal cancer, fallopian tube cancer, endometrial cancer, lung cancer, or brain cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is platinum-resistant epithelial ovarian cancer. In some embodiments, the ovarian cancer is recurrent epithelial ovarian cancer. In certain embodiments, the ovarian cancer is platinum-refractory epithelial ovarian cancer. In some embodiments, the cancer is uterine cancer. In some embodiments, the cancer is peritoneal cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is IMGN853-resistant.

[0045] In some embodiments, the method further comprises administration of a steroid. [Brief description of the drawings]

[0046] [Figure 1] Binding competition of huMov19-biotin with folate receptor antibody FR57; FRα antibody A ("FRα-A"); FRα antibody B ("FRα-B"); FRα antibody C ("FRα-C"); and non-biotinylated huMov19 ("huMov19") is shown by FACS. (See Example 1.) [Diagram 2] Exemplary molecules, characteristics, and schematic diagrams of conventional monospecific antibodies (such as huMov19 and FR57), bivalent biparatopic knob-in-hole (KIH) antibodies, and tetravalent biparatopic (Morrison) antibodies are shown. (See Example 1.) [Diagram 3]A gel of transfection ratios of several heavy and light chain plasmids used to generate asymmetric Fc-based molecules: L1: transfection with FR57scFv-knob only; L2: transfection with Mov19LC:Mov19HC-hole:FR57scFv-knob at 4:4:1; L3: transfection with Mov19LC:Mov19HC-hole:FR57scFv-knob at 6:2:1; L4: transfection with Mov19LC:Mov19HC-hole:FR57scFv-knob at 6:6:1; L5: transfection with Mov19LC:Mov19HC-hole:FR57scFv-knob at 9:3:1. C: Mov19HC-hole:FR57scFv-knob transfection; L6: Mov19LC: Mov19HC-hole:FR57scFv-knob transfection at 2:3:1; L7: Mov19LC: Mov19HC-hole:FR57scFv-knob transfection at 1:1:1; L8: Mov19LC: Mov19-hole transfection at 3:1; L9: isotype human IgG1 transfection. (See Example 1.) [Figure 4A] Figure 1 shows the binding activity of Morrison's antibody or fragment thereof by competitive FACS (see Example 2). In particular, the binding activity of Mov19-G1-FR57scFv1 (M9346A-FR57scFv) is shown. [Figure 4B] 1 shows the binding activity of Morrison's antibody or fragment thereof by competitive FACS (see Example 2). In particular, the binding activity of FR57-G1-Mov19scFv1 (FR57-M9346AscFv) is shown. [Figure 4C] 1 shows the binding activity of Morrison's antibody or fragment thereof by competitive FACS (see Example 2). In particular, the binding activity of Mov19-G1-FRα antibody-A-scFv1 (M9346A-FR-α-A:scFv) is shown. [Figure 4D] 1 shows the binding activity of Morrison's antibody or fragment thereof by competitive FACS (see Example 2). In particular, the binding activity of FRα antibody-A-G1-Mov19scFv1 (FR-α-A:M9346AscFv) is shown. [Figure 4E] 1 shows the binding activity of Morrison's antibody or fragment thereof by competitive FACS (see Example 2). In particular, the binding activity of FRα antibody-A-scFv2-G1-Mov19 (FR-α-A:scFv-M9346A) is shown. [Figure 4F] 1 shows the binding activity of Morrison's antibody or fragment thereof by competitive FACS (see Example 2). In particular, the binding activity of FRα antibody-B-scFv2-G1-Mov19 (FR-α-B:scFV-M9346A) is shown. [Figure 4G] 1 shows the binding activity of Morrison's antibody or fragment thereof by competitive FACS (see Example 2). In particular, the binding activity of FRα antibody-C-scFv2-G1-Mov19 (FR-α-C:scFv-M9346A) is shown. [Figure 4H] Figure 1 shows the binding activity of Morrison's antibody or fragment thereof by competitive FACS (see Example 2). In particular, the binding activity of FR57scFv2-G1-Mov19 (FR57scFv-M9346A) is shown. [Diagram 5] Shown is an SDS PAGE gel of three purified preparations (P1, P2, and P3) of FR57scFv2-knob-Mov19-hole antibody under non-reducing and reducing conditions. The FR57scFv2-knob-Mov19-hole antibody is a biparatopic antibody in knob-in-hole (KIH) format, with the FR57 scFv on the knob side of the antibody and the huMov19 antibody sequence on the hole side of the antibody. (See Example 2.) [Figure 6] Shows an overlay of size exclusion chromatography results from day 0 and day 14 samples of FR57scFv2-Knob-Mov19-Hole antibody. mAU: milliabsorbance units. (See Example 2.) [Figure 7] Shown are the binding (A and B), internalization and processing (C and D), and degradation (E and F) of knob-in-hole (KIH) biparatopic antibodies (A, C, and E), or tetravalent biparatopic antibodies (B, D, and F) compared to the huMov19 ("parent") antibody. (See Example 2.) [Figure 8] 1 shows the median tumor volume in an OV-90 xenograft model after administration of vehicle, an immunoconjugate containing a tetravalent biparatopic antibody ("tetravalent-s-SPDB-DM4"), or an immunoconjugate containing a huMov19 antibody ("Ms-SPDB-DM4") (see Example 4). [Figure 9] 1 shows the median tumor volume in an Igrov-1 xenograft model after administration of vehicle, tetravalent-s-SPDB-DM, or Ms-SPDB-DM4. (See Example 4.) [Figure 10] 1 shows the median tumor volume in an OV-90 xenograft model following administration of vehicle, an immunoconjugate containing a knob-in-hole biparatopic antibody ("KIH-s-SPDB-DM4"), or Ms-SPDB-DM4. (See Example 4.) [Figure 11] The cytotoxic activity of immunoconjugates containing FR57 antibody (FR57-L-DM21) or huMov19 antibody (ML-DM21) against KB cells is shown. (See Example 5.) [Figure 12] Cytotoxic activity of biparatopic KIH-DM21 immunoconjugate, huMov19 immunoconjugate M-DM21, and huMov19 immunoconjugate Ms-SPDB-DM4 against a panel of FRα-positive cell lines including KB cells (A), Igrov-1 cells (B), JEG-3 cells (C), T47D cells (D), and JHOS-4 cells (E) (see Example 5). [Figure 13] 1 shows the in vitro bystander killing activity of biparatopic KIH-L-DM21 immunoconjugate, huMov19 immunoconjugate ML-DM21, and huMov19 immunoconjugate Ms-SPDB-DM4 in target-negative cells Namalwa / luc mixed with KB cells (A), Igrov-1 cells (B), JEG-3 cells (C), and T47D cells (D). (See Example 5.) [Figure 14A]1 shows the median tumor volumes following administration of biparatopic immunoconjugate KIH-L-DM21 and huMov19 immunoconjugate ML-DM21 to an OV-90 xenograft model (see Example 6). [Figure 14B] 1 shows the median tumor volumes following administration of biparatopic immunoconjugate KIH-s-SPDB-DM4 and huMov19 immunoconjugate Ms-SPDB-DM4 to an OV-90 xenograft model (see Example 6). [Figure 15] 1 shows the median tumor volume following administration of biparatopic immunoconjugate KIH-L-DM21 immunoconjugate compared to vehicle, huMov19 immunoconjugate ML-DM21, or huMov19 immunoconjugate Ms-SPDB-DM4 ("IMGN853") to the Ishikawa xenograft model. (See Example 6.) [Figure 16] 1 shows the median tumor volume following administration of biparatopic KIH-L-DM21 immunoconjugates compared to vehicle, huMov19 immunoconjugate ML-DM21, or huMov19 immunoconjugate IMGN853 in an Igrov-1 xenograft model (see Example 6). [Figure 17] 1 shows the median tumor volume following administration of biparatopic KIH-L-DM21 immunoconjugates compared to vehicle, huMov19 immunoconjugate ML-DM21, or huMov19 immunoconjugate Ms-SPDB-DM4 in a KB xenograft model (see Example 7). [Figure 18] A and B show the toxicity of the biparatopic KIH-sSPDB-DM21 immunoconjugate (A) and the huMov19 immunoconjugate Ms-SPDB-DM4 ("IMGN853") (B) compared to whole antibodies (TAb: whole antibodies, conjugated and unconjugated). (See Example 7.) [Figure 19]1 shows the median tumor volume following administration of biparatopic KIH-L-DM21 immunoconjugate compared to vehicle, huMov19 immunoconjugate M9346A-DM21-L, or huMov19 immunoconjugate IMGN853 on an IMGN853-resistant KB human cervical cancer xenograft model. (See Example 6.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] I. Definition To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0048] Unless otherwise indicated, the term "human folate receptor 1", "FRα", "folate receptor alpha (FR-α)", or "FOLR1" as used herein refers to any naturally occurring human FRα polypeptide. The term "FRα" encompasses "full-length" unprocessed FRα polypeptides, as well as any form of FRα polypeptide resulting from processing within a cell. The term also encompasses naturally occurring variants of FRα, such as those encoded by splice variants and allelic variants. The FRα polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or can be prepared by recombinant or synthetic methods. When specifically indicated, "FRα" can be used to refer to a nucleic acid encoding a FRα polypeptide. Human FRα sequences are known and include, for example, the sequence publicly available at UniProtKB Accession No. P15328 (including isoforms). As used herein, the term "human FRα" refers to an FRα comprising the sequence of SEQ ID NO:53. [ka]

[0049] The term "anti-FRα antibody" or "antibody that binds to FRα" refers to an antibody that can bind to FRα with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting FRα. As used herein, such antibodies include, for example, bispecific (e.g., biparatopic) antibodies. Unless otherwise specified, the extent of binding of an anti-FRα antibody to unrelated non-FRα proteins is less than about 10% of the binding of the antibody to FRα, as measured, for example, by radioimmunoassay (RIA). Examples of FRα antibodies are known in the art and are disclosed in U.S. Published Application Nos. 2012 / 0009181 and 2012 / 0282175, as well as U.S. Patent No. 9,200,073 B2, and PCT Publication WO 2011 / 106528 A1, each of which is incorporated herein by reference in its entirety. Sequences of exemplary anti-FRα antibodies and antigen-binding fragments thereof are provided in Tables 1-8.

[0050] The term "IMGN853" (also known as "mirvetuximab soravtansine") refers to an immunoconjugate described herein that includes the huMov19 (or M9346A) antibody, a sulfoSPDB linker, and the DM4 maytansinoid. The "v19" (or "M9346A") antibody is an anti-FRα antibody that comprises a full-length heavy chain of SEQ ID NO: 47 (including the variable heavy chain sequence SEQ ID NO: 24, which is underlined in relation to SEQ ID NO: 47 below) and a full-length light chain of SEQ ID NO: 48 (including the variable light chain sequence SEQ ID NO: 19, which is underlined in relation to SEQ ID NO: 48 below). [ka]

[0051] The huMov19(M9346A) antibody is encoded by a plasmid deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110, on April 7, 2010, and has ATCC deposit numbers PTA-10772 and PTA-10773 or 10774. DM4 refers to N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl) maytansinoid. "SulfoSPDB" refers to N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate) linker.

[0052] The terms "elevated" FRα, "increased expression" of FRα, or "overexpression" of FRα in a particular tumor, tissue, or cell sample refer to FRα (an FRα polypeptide or a nucleic acid encoding such a polypeptide) that is present at a level higher than that present in a healthy or non-diseased (native, wild-type) tissue or cell of the same type or origin. Such increased expression or overexpression can be caused, for example, by mutation, gene amplification, increased transcription, increased translation, or increased protein stability.

[0053] FRα expression can be measured immunohistochemically and given a "staining intensity score" or "staining uniformity score" in comparison to a calibration control showing a defined score (e.g., an intensity score of 3 is given to the test sample if the intensity is comparable to the calibration control at level 3, and an intensity score of 2 is given to the test sample if the intensity is comparable to the calibration control at level 2). For example, a score of 1, 2, or 3, preferably a score of 2 or 3 by immunohistochemistry, indicates increased expression of FRα. Staining uniformity, whether heterogeneous or uniform, also indicates FRα expression. Staining intensity and staining uniformity scores can be used alone or in combination (e.g., 2 homo, 2 hetero, 3 homo, 3 hetero, etc.). The uniformity of staining can also be expressed as the percentage (%) of cells stained at a particular intensity (e.g., 25% of cells stained at an intensity of 1, 2, or 3; 50% of cells stained at an intensity of 1, 2, or 3; 70% of cells stained at an intensity of 1, 2, or 3). In another example, an increase in FRα expression can be determined by detection of at least a 2-fold, at least a 3-fold, or at least a 5-fold increase compared to a control value (e.g., expression levels in tissues or cells from subjects without cancer or with cancer that do not have elevated FRα values). FRα expression can be measured by immunohistochemistry and a visual score is given, with FRα positivity referring to 50% or more of the tumor cells having membrane staining for FRα visualized under a 10x microscope objective or less.

[0054] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, e.g., a protein, a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, or a combination thereof, through at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antibody, and any other modified immunoglobulin molecule so long as the antibody exhibits the desired biological activity. As used herein, such antibodies include, for example, bispecific (e.g., biparatopic) antibodies. Antibodies can be of any of the five major immunoglobulin classes, IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the distinctiveness of the heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, such as toxins, radioisotopes, etc.

[0055] The term "antibody fragment" or "antibody fragment thereof" refers to a portion of an intact antibody. An "antigen-binding fragment" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment may include an antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antibody fragments can be naked or conjugated to other molecules, such as toxins, radioisotopes, etc.

[0056] A "monoclonal" antibody or antigen-binding fragment thereof refers to a population of homogeneous antibodies or antigen-binding fragments involved in highly antigen-specific recognition and binding of a single antigenic determinant, i.e., epitope. This is in contrast to polyclonal antibodies, which usually contain different antibodies against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecule that contains an antigen recognition site. Furthermore, a "monoclonal" antibody or antigen-binding fragment thereof refers to antibodies and antigen-binding fragments thereof produced by any method, including, but not limited to, hybridomas, phage selection, recombinant expression, and transgenic animals.

[0057] The term "humanized" antibody or antigen-binding fragment thereof refers to forms of non-human (e.g., murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from a complementarity-determining region (CDR) are replaced by residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and function ("CDR granules"). Humanization is a process known as "humanization" (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding residues in an antibody or fragment from a non-human species having the desired specificity, affinity, and function. The humanized antibody or antigen-binding fragment thereof can be further modified by substitution of additional residues either within the Fv framework regions and / or the replaced non-human residues to improve and optimize the specificity, affinity, and / or function of the antibody or antigen-binding fragment thereof. Generally, a humanized antibody or antigen-binding fragment thereof comprises substantially all of at least one, typically two or three, variable domains, including all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of a human immunoglobulin consensus region. A humanized antibody or antigen-binding fragment thereof may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody.

[0058] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on sequence variation between species (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.), "Kabat"); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al, J. Molec. Biol. 273:927-948 (1997)). Additionally, some in the art use a combination of these two methods to determine CDRs.

[0059] The "constant" region is not involved directly in binding the antibody to an antigen, but exhibits various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0060] The Kabat numbering system is commonly used to refer to residues within the variable domain (roughly residues 1-107 in the light chain and residues 1-113 in the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed., 1991, National Institutes of Health, Bethesda, Md.) ("Kabat").

[0061] Kabat amino acid position numbering refers to the numbering system used for the heavy or light chain variable domains of an antibody arrangement in Kabat et al. (Sequences of Immunological Interest. 5th Ed., 1991, National Institutes of Health, Bethesda, Md.), ("Kabat"). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) as well as inserted residues after heavy chain FR residue 82 (such as residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence at regions of homology with the "standard" Kabat numbered sequence, whereas Chothia refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. [Table A]

[0062] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof produced by a human, an antibody or antigen-binding fragment thereof having an amino acid sequence that corresponds to an antibody or antigen-binding fragment thereof produced by a human, made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0063] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequences are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and function, and the constant regions are homologous to sequences of an antibody or antigen-binding fragment thereof from another species (usually human) to avoid eliciting an immune response in that species.

[0064] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and from non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope typically comprises at least 3, more commonly at least 5, or 8-10 amino acids in a unique spatial conformation.

[0065] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens faster and remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure.

[0066] "Or better" as used herein to refer to binding affinity refers to stronger binding between a molecule and its binding partner. "Or better" as used herein refers to stronger binding represented by a smaller numerical Kd value. For example, an antibody with an affinity for an antigen of "0.6nM or better" means that the affinity of the antibody for the antigen is <0.6nM, i.e., 0.59nM, 0.58nM, 0.57nM, etc., or any value less than 0.6nM.

[0067] "Specifically binds" generally means that an antibody binds to an epitope through its antigen-binding domain, and that the binding involves a degree of complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope through its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B", or antibody "A" may be said to bind epitope "C" with higher specificity than to the related epitope "D".

[0068] "Preferentially binds" means that an antibody specifically binds to an epitope more readily than it binds to a related, similar, homologous, or analogous epitope. Thus, an antibody that "preferentially binds" to a given epitope is more likely to bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.

[0069] An antibody is said to "competitively inhibit" the binding of a reference antibody to a given epitope if it preferentially binds to that epitope, or to an overlapping epitope, to such an extent that it blocks the reference antibody from binding to the epitope to some extent. Competitive inhibition can be measured by any method known in the art, for example, competitive ELISA assay. An antibody can be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0070] As used herein, the phrases "substantially similar" or "substantially the same" refer to a sufficiently high degree of similarity between two numerical values ​​(generally one associated with an antibody of the present disclosure and the other associated with a reference / comparator antibody) such that one of skill in the art would consider the difference between the two values ​​to have little biological and / or statistical significance in the biological characteristic measured by the values ​​(e.g., Kd values). The difference between the two values ​​can be less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%, depending on the value of the reference / comparator antibody.

[0071] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, including, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within this definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), and other modifications known in the art. Because the polypeptides of this disclosure are based on antibodies, it is understood that in certain embodiments the polypeptides can occur as single chains or linked chains.

[0072] The terms "polynucleotide" or "nucleic acid," as used interchangeably herein, refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those containing intercalators (e.g., acridine, psoralens, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional bonds to additional nucleotides or conjugated to a solid support. The 5' and 3' terminal OH may be phosphorylated or replaced with an amine or an organic capping group moiety of 1-20 carbon atoms. The other hydroxyl may be derivatized to a standard protecting group.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (--O--) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0073] The term "vector" refers to a construct capable of delivering and optionally expressing one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors bound to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0074] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0075] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0076] The term "identical" or "percent identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are identical or have a certain percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. One such non-limiting example of a sequence alignment algorithm is described in Karlin et al, Proc. Natl. Acad. Sci., 87:2264-2268 (1990), and Karlin Gapped BLAST is the algorithm modified in Altschul et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993) and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, gapped BLAST is performed using the algorithm described in Altschul et al., Nucleic Acids Res. Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program of the GCG software (e.g., with a NWSgapdna.CMP matrix, using a gap weight of 40, 50, 60, 70, or 90, and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program of the GCG software package, incorporating the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)), can be used to determine percent identity between two amino acid sequences (e.g., using gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, or 5, with either a Blossum 62 matrix or a PAM250 matrix). Alternatively, in certain embodiments, percent identity between nucleotide or amino acid sequences can be determined using the algorithm of Myers and Miller (CA BIOS, 4:11-17 (1989)) algorithm. For example, percent identity can be determined using the ALIGN program (version 2.0) using PAM120 with a residue table, a gap length penalty of 12, and a gap penalty of 4. Appropriate parameters for maximum alignment with a particular alignment software can be determined by one of skill in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percent identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y / Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or by a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.

[0077] As a non-limiting example, whether a particular polynucleotide has a particular percentage of "sequence identity" to a reference sequence (e.g., at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% identical) can be determined in certain embodiments using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)) to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, parameters are set such that the percentage of identity is calculated over the entire length of the reference nucleotide sequence and allows gaps in homology of up to 5% of the total number of nucleotides in the reference sequence.

[0078] "Conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain.Families of amino acid residues having similar side chains are defined in the art and include amino acid residues having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).For example, replacing tyrosine with phenylalanine is a conservative substitution. In some embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not inhibit the binding of the polypeptide or antibody comprising the amino acid sequence to the antigen(s) to which the polypeptide or antibody binds, i.e., FRα. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1 187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0079] A "bispecific antibody" refers to an antibody that binds to two different epitopes. , may be on the same target antigen or on different target antigens.

[0080] A "biparatopic antibody" is a bispecific antibody that binds to two different, non-overlapping epitopes on the same target antigen (eg, FRα).

[0081] In some embodiments, the FRα antibody or antigen-binding fragment thereof disclosed herein is a multivalent molecule. As used within this application, the term "valent" refers to the presence of a certain number of binding sites in an antibody molecule. For example, a natural or full-length antibody according to the present invention has two binding sites and is "bivalent". The term "tetravalent" refers to the presence of four binding sites in an antigen-binding protein. The term "trivalent" refers to the presence of three binding sites in an antibody molecule. As used herein, the term "bispecific, tetravalent" refers to an antigen-binding protein according to the present invention that has four antigen-binding sites, at least one of which binds to a first antigen and at least one of which binds to a second antigen or another epitope of the antigen.

[0082] As used herein, the term "immunoconjugate" or "conjugate" refers to a compound or derivative thereof linked to a cell-binding agent and defined by the general formula: CLA, where C = cytotoxin, L = linker, and A = antibody or antigen-binding fragment thereof (e.g., an anti-FRα antibody or antibody fragment). Immunoconjugates can also be defined by the general formula in the reverse order: ALC.

[0083] A "linker" is any chemical moiety capable of linking a compound, usually a drug such as a maytansinoid, to a cell-binding agent, such as an anti-FRα antibody or an antigen-binding fragment thereof, in a stable covalent bond. The linker may be sensitive or substantially resistant to cleavage (e.g., acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, or disulfide bond cleavage) under conditions under which the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups and thioether groups.

[0084] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents one or more cellular functions and / or causes cell death. In some embodiments, the cytotoxic agent is a maytansinoid, e.g., DM21. Immunoconjugates comprising DM21 are disclosed in WO2018 / 160539A1, which is incorporated herein by reference in its entirety.

[0085] The immunoconjugate can include a site-specific DM21 conjugate of "DM21C," represented by the following structural formula: [ka] Wherein D1 is: [ka] It is.

[0086] The immunoconjugates may also include lysine-linked DM21, "L-DM21," "DM21-L," or "DM21L," represented by the following structural formula: [ka] wherein D1 is shown above and is attached to the antibody by a linker, such as a γ-maleimidobutyric acid N-succinimidyl ester (GMBS) or N-(γ-maleimidobutryloxy)sulfosuccinimide ester (sulfo-GMBS or sGMBS) linker. GMBS and sulfo-GMBS (or sGMBS) linkers are known in the art and can be represented by the following structural formula: [ka]

[0087] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, and thus the present application includes cases where the circumstance occurs and cases where it does not occur. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus the present application includes structures where the non-hydrogen substituent is present and structures where the non-hydrogen substituent is not present.

[0088] The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal characterized by uncontrolled cell proliferation of a cell population. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancer include fallopian tube cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. The cancer may be a cancer that expresses FRα ("FRα-expressing cancer").

[0089] The terms "cancer cells", "tumor cells", and grammatical equivalents refer to the total population of cells derived from a tumor or precancerous lesion, including both non-tumorigenic cells and tumorigenic stem cells (cancer stem cells), which constitute the majority of the tumor cell population. As used herein, the term "tumor cells" is modified by the term "non-tumorigenic" when it refers only to tumor cells that lack the ability to regenerate and differentiate, thus distinguishing them from cancer stem cells.

[0090] An "advanced" cancer is one that has spread outside the original site or organ, either by local invasion or metastasis. The term "advanced" cancer includes both locally advanced and metastatic disease.

[0091] "Metastatic" cancer refers to cancer that has spread from one part of the body to another part of the body.

[0092] A "refractory" cancer is one that progresses despite anti-tumor agents, such as chemotherapy agents, being administered to the cancer patient.

[0093] A "recurrent" cancer is one that has regrown at the original site or a distant site after responding to initial therapy.

[0094] A "relapsed" patient is one who has signs or symptoms of cancer after remission. Optionally, the patient has relapsed after adjuvant or neoadjuvant therapy.

[0095] The term "maintenance therapy" refers to therapy given to help prevent cancer from coming back after the cancer has gone away following initial treatment.

[0096] The term "subject" refers to any animal (e.g., mammal) that is to receive a particular treatment, including, but not limited to, humans, non-human primates, rodents, etc. The terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0097] The term "pharmaceutical formulation" refers to a formulation that is in a form such that the biological activity of the active ingredient is effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. The formulation can be sterile.

[0098] An "effective amount" of an antibody, immunoconjugate, or other agent disclosed herein is an amount sufficient to carry out a specifically stated purpose.

[0099] The term "therapeutically effective amount" refers to an amount of an antibody, immunoconjugate, or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells, reduce tumor size or tumor burden, inhibit (i.e., slow to some extent and / or inhibit) the invasion of cancer cells into peripheral organs, and / or inhibit the proliferation of cancer cells into peripheral organs. In certain embodiments, the drug may inhibit (i.e., slow to some extent and, in certain embodiments, halt) tumor metastasis, inhibit to some extent tumor growth, alleviate to some extent one or more symptoms associated with cancer, and / or result in a favorable response, such as an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), a complete response (CR), a partial response (PR), in some cases an increase in stable disease (SD), a decrease in progressive disease (PD), a decrease in time to progression (TTP), or any combination thereof. See the definition of "treating" herein. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic.

[0100] The terms "treating" or "treatment" or "treat" or "palliating" or "alleviating" and the like refer to a therapeutic measure that cures, slows, relieves the symptoms of, and halts the progression of, a diagnosed pathological condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having a disorder. In certain embodiments, a subject has been successfully "treated" for cancer according to the methods of the present disclosure if the patient exhibits one or more of the following: a reduction in the number or complete absence of cancer cells; a reduction in tumor size; an inhibition or absence of cancer cell invasion into peripheral organs, including, for example, spread of cancer to soft tissue and bone; an inhibition or absence of tumor metastasis; an inhibition or absence of tumor growth; an alleviation of one or more symptoms associated with a particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in the tumorigenicity, tumorigenic frequency, or tumorigenic potential of a tumor; a reduction in the number or frequency of cancer stem cells within a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), a complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a shortened time to progression (TTP), or any combination thereof.

[0101] As used herein, the terms "administer," "administering," "administration," and the like refer to methods that can be used to enable delivery of the immunoconjugate to a desired site of biological action. Administration techniques that can be used with the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, PA. In one aspect, the immunoconjugate is administered intravenously.

[0102] The term "instructing" means providing written instructions for applicable treatments, medications, therapies, therapeutic regimens, etc., by any means, for example, in the form of a package insert or other written promotional material.

[0103] The terms "pretreat" and "pretreatment" refer to a therapeutic procedure that occurs prior to administration of a therapeutic antibody, antigen-binding fragment thereof, or immunoconjugate. For example, as described in more detail herein, a steroid (e.g., a corticosteroid) can be administered as a prophylactic agent about 1 week, about 5 days, about 3 days, about 2 days, or about 1 day, or 24 hours prior to administration of the immunoconjugate. The steroid can also be administered prior to the immunoconjugate on the same day as the immunoconjugate.

[0104] Unless otherwise stated or clear from the context, the term "about" as used herein is understood to mean within normal tolerances in the art, e.g., within two standard deviations of the mean. About means 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0105] The recitation of a list of chemical groups within any definition of a variable herein includes the definition of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0106] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0107] Whenever an embodiment is described herein with the word "comprising," it may also be described with the words "consisting of" and / or "consisting essentially of." It is understood that similar embodiments described with the term "essentially of" are also provided. In this disclosure, "comprises," "comprising," "containing," "having," and the like may have their meanings ascribed to them in U.S. Patent Law, and may mean "includes," "including," and the like, and "consisting essentially of," or "consists essentially of," and the like may similarly have the meanings ascribed to them in U.S. Patent Law, and the terms are open-ended, allowing for the presence of more than what is recited, but excluding prior art embodiments, so long as the presence of more than what is recited does not change the basic or novel characteristics of what is recited.

[0108] Unless otherwise stated or clear from the context, as used herein, the term "or" should be understood to be inclusive. When used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A," and "B." Similarly, when used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0109] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0110] II. Biparatopic Antibodies Provided herein are biparatopic anti-FRα antibodies and antigen-binding fragments thereof. These biparatopic antibodies and antigen-binding fragments thereof include a first FRα-binding domain that binds to a first epitope of FRα and a second FRα-binding domain that binds to a second epitope of FRα. The first and second epitopes of FRα are non-overlapping epitopes. These biparatopic antibodies and antigen-binding fragments may include additional FRα-binding domains. For example, a tetravalent biparatopic antibody or antigen-binding fragment thereof may have two FRα-binding domains that bind to a first epitope and two FRα-binding domains that bind to a second epitope. Exemplary biparatopic antibodies and antigen-binding fragments thereof are shown in FIG. 1.

[0111] A. FRα-binding domain Disclosed herein are FRα binding domains that can be used to construct biparatopic antibodies or antigen-binding fragments thereof. The FRα binding domains have six complementary The FRα antigen-binding domain can include variable heavy chain (VH) CDR1, VH CDR2, VH CDR3, variable light chain (VL) CDR1, VL CDR2, and VL CDR3. The FRα antigen-binding domain can include a variable heavy chain (VH) and a variable light chain (VL). The VH and VL can be separate polypeptides or can be part of the same polypeptide (e.g., in an scFv).

[0112] FRα antibodies and antigen-binding fragments thereof are known in the art and are disclosed, for example, in PCT Application Publication Nos. WO2011 / 106528A1; WO2012 / 135675A3; WO2012 / 138749A1; WO2014 / 036495A3; and WO2015 / 031815A2, each of which is incorporated herein by reference in its entirety. Additional FRα antibodies are disclosed in U.S. Patent Nos. 8,557,966B2, 8,709,432B2, 9,702,881B2; and 9,637,547B2; and U.S. Patent Application Publication No. US-2012-0282282A1, each of which is incorporated herein by reference in its entirety. Additionally, the FRα antibody huMov19(M9346A) antibody is encoded by a plasmid deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, VA 20110 on April 7, 2010 and has ATCC deposit numbers PTA-10772 and PTA-10774. As provided herein, an FRα binding domain can be the FRα binding domain (e.g., the six CDRs or VH and VL) of any of these antibodies or antigen-binding fragments thereof.

[0113] As an example, the FRα binding domain can include the CDR sequences, VH sequences, and / or VL sequences of the huMov19 and / or FR57 antibodies. The CDR sequences of the huMov19 and FR57 antibodies are shown in Tables 1 and 2 below.

[0114] In some embodiments, the FRα-binding domain disclosed herein comprises one or more polypeptides comprising one or more of the CDR sequences described herein. For example, the FRα-binding domain can comprise one or more of the light chain CDR sequences (i.e., LC CDR1, LC CDR2, and LC CDR3) and / or one or more of the heavy chain CDR sequences (i.e., HC CDR1, HC CDR2, and HC CDR3) shown in Tables 1 and 2 below. [Table 1] [Table 2]

[0115] In some embodiments, the FRα-binding domain comprises (a) a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 1 to 3, respectively, and (b) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 7 to 9, respectively. In some embodiments, the FRα-binding domain comprises (a) a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 1 to 3, respectively, and (b) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 9, respectively. In some embodiments, the FRα-binding domain comprises (a) a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 4 to 6, respectively, and (b) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 10 to 12, respectively. In some embodiments, the FRα binding domain comprises (a) a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NOs: 4 to 6, respectively, and (b) a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NOs: 15, 16, and 12, respectively.

[0116] As an example, the FRα binding domain can include the CDR sequences, VH sequences, and / or VL sequences of the huMov19 and / or FR57 antibodies. The CDR sequences of huMov19 and FR57 are shown in Tables 1 and 2 below.

[0117] In some embodiments, the FRα-binding domain disclosed herein comprises one or more polypeptides comprising one or more of the CDR sequences described herein. For example, the FRα-binding domain can comprise one or more of the light chain CDR sequences (i.e., LC CDR1, LC CDR2, and LC CDR3) and / or one or more of the heavy chain CDR sequences (i.e., HC CDR1, HC CDR2, and HC CDR3) shown in Tables 1 and 2 below.

[0118] In some embodiments, the FRα binding domain comprises the light chain variable sequence and / or the heavy chain variable sequence of the huMov19 and / or FR57 antibodies. The light chain variable sequences and heavy chain variable sequences of huMov19 and FR57 are shown in Tables 3 and 4 below. [Table 3] [Table 4]

[0119] In some embodiments, the FRα binding domain comprises a VL having at least 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%, at least about 99%, or 100% sequence identity to SEQ ID NO: 17, and optionally the VL comprises the sequences of VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NOs: 1 to 3, respectively. In some embodiments, the FRα binding domain comprises a VL having at least 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%, at least about 99%, or 100% sequence identity to SEQ ID NO: 19, and optionally the VL comprises the sequences of VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NOs: 4 to 6, respectively.

[0120] In some embodiments, the FRα-binding domain comprises a VH having at least 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%, at least about 99%, or 100% sequence identity to SEQ ID NO:22, and optionally the VH comprises the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NOs:7-9, respectively, or SEQ ID NOs:13, 14, and 9, respectively. In some embodiments, the FRα-binding domain comprises at least 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%, at least about 99%, or 100% sequence identity to SEQ ID NO:24. and a VH having about 99% or 100% sequence identity, and optionally the VH comprises the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NOs: 10-12, respectively, or SEQ ID NOs: 15, 16, and 12, respectively.

[0121] In some embodiments, the FRα-binding domain comprises a VL and a VH, wherein (i) the VL has at least 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%, at least about 99%, or 100% sequence identity to SEQ ID NO: 17, and optionally the VL has a VL CDR1, a VL CDR2, and a VL H of SEQ ID NOs: 1-3, respectively. and (ii) the VH has at least 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%, at least about 99%, or 100% sequence identity to SEQ ID NO: 22, and optionally the VH comprises the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NOs: 7-9, respectively, or SEQ ID NOs: 13, 14, and 9, respectively.

[0122] In some embodiments, the FRα-binding domain comprises a VL and a VH, wherein (i) the VL has at least 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%, at least about 99%, or 100% sequence identity to SEQ ID NO: 19, and optionally the VL has a VL CDR1, VL CDR2, VL H, or VL H of SEQ ID NOs: 4-6, respectively. and (ii) the VH has at least 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%, at least about 99%, or 100% sequence identity to SEQ ID NO: 24, and optionally the VH comprises the VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NOs: 10-12, respectively, or SEQ ID NOs: 15, 16, and 12, respectively.

[0123] In some embodiments, the FRα-binding domain comprises a VL and a VH. The VL and VH can be separate polypeptides. The VL and VH can be part of the same polypeptide, for example, a polypeptide comprising a VL, a linker, and a VH. The polypeptide comprising a VL, a linker, and a VH can be in the orientation of VL-linker-VH or VH-linker-VL.

[0124] Thus, in some embodiments, an FRα-binding domain (e.g., an scFv) comprises, from N-terminus to C-terminus, a VL comprising the amino acid sequence of SEQ ID NO: 17, a linker (e.g., a glycine-serine linker), and a VH comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, an FRα-binding domain comprises, from N-terminus to C-terminus, a VH comprising the amino acid sequence of SEQ ID NO: 22, a linker (e.g., a glycine-serine linker), and a VL comprising the amino acid sequence of SEQ ID NO: 17.

[0125] In some embodiments, the FRα-binding domain (e.g., an scFv) comprises, from N-terminus to C-terminus, a VL comprising the amino acid sequence of SEQ ID NO: 19, a linker (e.g., a glycine-serine linker), and a VH comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the FRα-binding domain comprises, from N-terminus to C-terminus, a VH comprising the amino acid sequence of SEQ ID NO: 24, a linker (e.g., a glycine-serine linker), and a VL comprising the amino acid sequence of SEQ ID NO: 19.

[0126] Linkers that can be used to connect VH and VL are known in the art. For example, the linker can be a glycine-serine linker. In embodiments, the linker can be of any length and can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, or 60 or more amino acids. In other embodiments, linkers useful in the present disclosure have at least one amino acid and fewer than 100 amino acids, fewer than 90 amino acids, fewer than 80 amino acids, fewer than 70 amino acids, fewer than 60 amino acids, fewer than 50 amino acids, fewer than 40 amino acids, fewer than 30 amino acids, fewer than 20 amino acids, fewer than 19 amino acids, fewer than 18 amino acids, fewer than 17 amino acids, fewer than 16 amino acids, fewer than 15 amino acids, fewer than 14 amino acids, fewer than 13 amino acids, or fewer than 12 amino acids. In some embodiments, the linker sequence includes a glycine amino acid residue. In another example, the linker sequence includes a combination of glycine and serine amino acid residues.

[0127] In some embodiments, the FRα binding domain comprises a linker fused in-frame between the VH and VL. In some embodiments, such a glycine / serine linker comprises the peptide GGGS (SEQ ID NO: 49) or GGGGS (SEQ ID NO: 50), or any combination of amino acid residues thereof, including but not limited to, repeats thereof, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeats of these given peptides. The glycine / serine linkers disclosed herein are (GS) n , (GGS) n , (GGGS) n , (GGGGS) n , or (GGGGS) n wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker sequence is GGGGSGGGSGGGGGS (SEQ ID NO:51) (also referred to as Gly4Ser)3). In another embodiment, the linker sequence is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:52) (also referred to as Gly4Ser)4).

[0128] In some embodiments, the FRα-binding domain is an scFv. Exemplary scFv FRα-binding domains are shown in Table 5 below. [Table 5]

[0129] In some embodiments, the FRα binding domain has a sequence identity of at least 70%, at least about 75%, at least about 80%, at least about 8% to SEQ ID NO: 27, 28, or 29. 5%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity, and optionally the scFv comprises VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NOs: 1-3, respectively, and VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NOs: 7-9, respectively, or SEQ ID NOs: 13, 14, and 9, respectively.

[0130] In some embodiments, the FRα binding domain comprises an scFv comprising an amino acid sequence that is at least 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%, at least about 99%, or 100% sequence identity to SEQ ID NO: 30, 31, or 32, and optionally the scFv comprises VL CDR1, VL CDR2, and VL CDR3 sequences of SEQ ID NOs: 4-6, respectively, and VH CDR1, VH CDR2, and VH CDR3 sequences of SEQ ID NOs: 10-12, respectively, or SEQ ID NOs: 15, 16, and 12, respectively.

[0131] In a specific embodiment, the FRα binding domain binds to the same epitope of FRα as an antibody comprising the amino acid sequences of SEQ ID NO:17 and SEQ ID NO:22.

[0132] In a specific embodiment, the FRα binding domain binds to the same epitope of FRα as an antibody comprising the amino acid sequences of SEQ ID NO:19 and SEQ ID NO:24.

[0133] In certain embodiments, the FRα-binding domain is a murine, chimeric, or humanized FRα-binding domain. As used herein, a humanized FRα-binding domain may be a resurfaced FRα-binding domain.

[0134] In certain embodiments, the FRα-binding domain binds to human FRα but does not bind to FOLR2 or FOLR3.

[0135] B. Biparatopic Antibody Format A biparatopic anti-FRα antibody or antigen-binding fragment thereof may comprise a combination of the above-mentioned FRα-binding domains, where the FRα-binding domains bind to non-overlapping epitopes of FRα.

[0136] Many different types of bispecific constructs are known in the art and can be used with the biparatopic anti-FRα antibodies or antigen-binding fragments thereof provided herein.

[0137] Early attempts at constructing bispecific antibodies utilized either chemical cross-linking or hybrid hybridomas or quadromas to link the two halves of two different antibodies together. Although these techniques are useful for generating bispecific antibodies, they are associated with production issues such as the production of mixed populations containing various combinations of antigen binding sites, difficulties in protein expression, the need for purification of the desired bispecific antibody, low yields, and the cost of production.

[0138] More recent approaches utilize genetically engineered constructs that can produce homogenous products of single bispecific antibodies without the need for extensive purification to remove unwanted by-products. Such constructs include tandem scFvs, diabodies, tandem diabodies, dual variable domain antibodies, and heterodimerization using motifs such as Ch1 / Ck domains or DNL® (Chames & Baty, 2009, Curr Opin Drug Discov Devel 12:276-83; Chames & Baty, mAbs 1:539-47). BITE (registered trademark) The term "bispecific antibody" refers to tandem scFvs linked by a short peptide linker (Chames & Baty, mAbs 1:539-47). Other approaches to bispecific antibody production include tetravalent IgG-scFv fusions (Dong et al., 2011, MAbs 3:273-88); dual acting Fab (DAF) antibodies (Bostrom et al., 2009, Science 323:1610-14); Igg-like dual variable domain antibodies (DVD-Ig) (Wu et al., 2007, Nat Biotechnol 25:1290-97); and the use of dynamic exchange between IgG4 molecules (van der Neut Kolfschoten et al., 2007, Science 317:1554-57).

[0139] DOCK-AND-LOCK® (DNL®) complexes (see, e.g., U.S. Pat. Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,901,680; 7,906,118; 7,981,398; 8,003,111) represent another bispecific antibody format. Standard DNL® complexes include trimers with two DDD-linked molecules bound to one AD-linked molecule, although complex structural variations allow for the formation of dimers, trimers, tetramers, pentamers, hexamers, and other multimers.

[0140] In some embodiments, disclosed herein are biparatopic constructs with asymmetric Fc molecules, including "knobs-in-holes" structures. See Kontermann, MAbs., 4(2):182-97 (2012). In knobs-into-holes (KIH) technology, the CH3 domain is engineered to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization. KIH technology is described, for example, in Ridgway et al., Protein Engineering 9(7):617-721 (1996); US5,731,168; US5,807,706; US5,821,333, each of which is incorporated herein by reference in its entirety. The "CrossMab" technology further involves swapping heavy and light chain domains within the Fab half of a bispecific antibody, making the two arms so different that light-heavy mispairing does not occur (Schaefer et al., 2011, Proc Natl. Acad Sci USA 108:11187-92). The knobs-into-holes approach introduces an amino acid in the CH3 domain of one heavy chain with a bulky side chain that fits into a properly designed cavity in the CH3 domain of the other heavy chain. The combination of approaches prevents mismatches in both heavy-heavy and heavy-light chain interactions, resulting in a predominantly single product.

[0141] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof is bivalent (see, e.g., the "knobs-in-holes" example shown in FIG. 1). A bivalent biparatopic anti-FRα antibody or antigen-binding fragment thereof can comprise, for example, two FRα-binding domains comprising an scFv, two FRα-binding domains comprising a VH and a VL on separate polypeptide chains, or one FRα-binding domain comprising an scFv and one FRα-binding domain comprising a VH and a VL on separate polypeptide chains.

[0142] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof is trivalent.

[0143] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof is trivalent (see, e.g., the example of "Morrison" shown in FIG. 1). Tetravalent antibodies are described, for example, in MJ Coloma, SL Morrison, Nat. Biotechnol., 15(2):159-63 (1997), which is incorporated herein by reference in its entirety. is incorporated herein by reference.

[0144] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an FRα-binding domain that is an scFv. In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an FRα-binding domain that comprises a VH and a VL on separate polypeptides. In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an FRα-binding domain that is an scFv and an FRα-binding domain that comprises a VH and a VL on separate polypeptides.

[0145] In some embodiments, the bivalent biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a single FRα-binding domain that is an scFv and a single FRα-binding domain that comprises a VH and a VL on separate polypeptides. In such embodiments, the scFv can be fused to a heavy chain constant region and the VH can be fused to a heavy chain constant region. In some embodiments, the constant region has a "knob and hole" sequence. The "knob" sequence can be in the heavy chain constant region fused to the scFv and the "hole" sequence can be fused to the constant region fused to the VH. Alternatively, the "hole" mutation can be in the heavy chain constant region fused to the scFv and the "knob" sequence can be fused to the constant region fused to the VH. Exemplary biparatopic anti-FRα antibodies or antigen-binding fragments thereof in such formats are shown in Table 7.

[0146] In some embodiments, the tetravalent biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises two FRα-binding domains that are scFvs and two FRα-binding domains that comprise a VH and a VL on separate polypeptides. In such embodiments, the scFvs can be fused to the N-terminus or C-terminus of a polypeptide that comprises a VH. The scFvs can also be fused to the N-terminus or C-terminus of a polypeptide that comprises a VL.

[0147] A tetravalent biparatopic anti-FRα antibody or antigen-binding fragment thereof can comprise two polypeptides, a first polypeptide comprising a heavy chain constant region, a VH, and an scFv, and a second polypeptide comprising a light chain constant region and a VL. A tetravalent biparatopic anti-FRα antibody or antigen-binding fragment thereof can also comprise two polypeptides, a first polypeptide comprising a heavy chain constant region and a VH, and a second polypeptide comprising a light chain constant region, a VL, and an scFv. Sequences of exemplary biparatopic anti-FRα antibodies or antigen-binding fragments thereof in such formats are found in Table 6.

[0148] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof is a bispecific heterodimeric diabody, e.g., a tetrameric bispecific heterodimeric diabody. As used herein, the term "bispecific heterodimeric diabody" refers to a complex of two or more polypeptide chains or proteins, each of which can contain at least one antibody VL and one antibody VH domain, and the VL and VH domains of each polypeptide chain are derived from different antibodies.

[0149] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof disclosed herein comprises one or more resurfaced FRα-binding domains. In some embodiments, all of the FRα-binding domains of the biparatopic antibody or antigen-binding fragment thereof are resurfaced.

[0150] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues from a complementarity determining region (CDR) have been replaced by residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and function ("CDR grafted") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 321:522-525 (1986); al., Nature 332:323-327(1988); Verhoeyen et al., Science 239:1534-1536(1988)).

[0151] In a further embodiment, the biparatopic antibody or antigen-binding fragment thereof is a CDR-grafted antibody or a resurfaced antibody comprising at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 7 to 9, respectively, and the light chain variable region comprises three complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 1 to 3, respectively.

[0152] In a further embodiment, the biparatopic antibody or antigen-binding fragment thereof is a CDR-grafted or resurfaced antibody comprising at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 13, 14, and 9, respectively, and the light chain variable region comprises three complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 1 to 3, respectively.

[0153] In a further embodiment, the biparatopic antibody or antigen-binding fragment thereof is a CDR-grafted antibody or a resurfaced antibody comprising at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 10 to 12, respectively, and the light chain variable region comprises three complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 4 to 6, respectively.

[0154] In a further embodiment, the biparatopic antibody or antigen-binding fragment thereof is a CDR-grafted antibody or a resurfaced antibody comprising at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 15, 16, and 12, respectively, and the light chain variable region comprises three complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 4 to 6, respectively.

[0155] In a further embodiment, there is provided an antibody or antigen-binding fragment thereof having a humanized (e.g., resurfaced, CDR-grafted) heavy chain variable region sharing at least 90% sequence identity with the amino acid sequence corresponding to SEQ ID NOs: 22-26, more preferably 95% sequence identity with SEQ ID NOs: 22-26, and most preferably 100% sequence identity with SEQ ID NOs: 22-26. In certain embodiments, the antibody contains conservative mutations in the framework regions outside the CDRs.

[0156] Similarly, antibodies are provided that have a humanized (e.g., resurfaced, CDR-grafted) light chain variable region that shares at least 90% sequence identity with the amino acid sequences corresponding to SEQ ID NOs: 17-21, more preferably 95% sequence identity with SEQ ID NOs: 17-21, and most preferably 100% sequence identity with SEQ ID NOs: 17-21. In certain embodiments, the antibodies contain conservative mutations in the framework regions outside the CDRs.

[0157] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In some embodiments, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, in some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof can comprise a light chain constant region that is either a kappa light chain constant region or a lambda light chain constant region. In some embodiments, the light chain constant region is a kappa light chain constant region.

[0158] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 19 and 24; 20 and 25; and 21 and 26, respectively. and a second FRα-binding domain that does not compete with huMov19 for binding to FRα. In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a first FRα-binding domain that comprises the VL and VH sequences of SEQ ID NOs: 20 and 57, respectively, and a second FRα-binding domain that does not compete with huMov19 for binding to FRα.

[0159] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a first FRα-binding domain comprising VL and VH sequences selected from the group consisting of SEQ ID NOs: 17 and 22; and 18 and 23, respectively, and a second FRα-binding domain that does not compete with FR57 for binding to FRα.

[0160] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an FRα-binding domain that competitively inhibits binding to the same FRα epitope as an antibody comprising the VH amino acid sequence of SEQ ID NO: 22 and the VL amino acid sequence of SEQ ID NO: 17.

[0161] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an FRα-binding domain that competitively inhibits binding to the same FRα epitope as an antibody comprising the VH amino acid sequence of SEQ ID NO:24 and the VL amino acid sequence of SEQ ID NO:19.

[0162] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises (i) an antibody comprising the VH amino acid sequence of SEQ ID NO: 22 and the VL amino acid sequence of SEQ ID NO: 17 and a first FRα-binding domain that competitively inhibits binding to the same FRα epitope, and (ii) an antibody comprising the VH amino acid sequence of SEQ ID NO: 24 and the VL amino acid sequence of SEQ ID NO: 19 and a second FRα-binding domain that competitively inhibits binding to the same FRα epitope.

[0163] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an FRα-binding domain that binds to the same FRα epitope as an antibody comprising the VH amino acid sequence of SEQ ID NO:22 and the VL amino acid sequence of SEQ ID NO:17.

[0164] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an FRα-binding domain that binds to the same FRα epitope as an antibody comprising the VH amino acid sequence of SEQ ID NO:24 and the VL amino acid sequence of SEQ ID NO:19.

[0165] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises (i) an antibody comprising a VH amino acid sequence of SEQ ID NO: 22 and a VL amino acid sequence of SEQ ID NO: 17, and a first FRα-binding domain that binds to the same FRα epitope, and (ii) an antibody comprising a VH amino acid sequence of SEQ ID NO: 24 and a VL amino acid sequence of SEQ ID NO: 19, and a second FRα-binding domain that binds to the same FRα epitope.

[0166] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 24, respectively).

[0167] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 25, respectively). The antibody or antigen-binding fragment thereof comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 57, respectively).

[0168] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 26, respectively).

[0169] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 24, respectively).

[0170] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 57, respectively).

[0171] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 26, respectively).

[0172] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 24, respectively).

[0173] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 57, respectively).

[0174] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 26, respectively).

[0175] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 24, respectively).

[0176] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 57, respectively).

[0177] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 26, respectively).

[0178] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 24, respectively).

[0179] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 57, respectively).

[0180] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 26, respectively).

[0181] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 24, respectively).

[0182] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 57, respectively).

[0183] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 26, respectively).

[0184] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises: The variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 24, respectively).

[0185] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 17 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 57, respectively).

[0186] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 26, respectively).

[0187] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 24, respectively).

[0188] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 57, respectively).

[0189] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 26, respectively).

[0190] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 24, respectively).

[0191] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 57, respectively).

[0192] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or a variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or SEQ ID NO: 20, respectively). 22) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NOs: 21 and / or 26, respectively).

[0193] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 24, respectively).

[0194] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 22, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 57, respectively).

[0195] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 19 and / or 26, respectively).

[0196] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 24, respectively).

[0197] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises a variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and a variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 57, respectively).

[0198] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 20 and / or 26, respectively).

[0199] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 24, respectively).

[0200] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 25, respectively). In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 25, respectively). and a variant heavy chain (e.g., SEQ ID NO: 21 and / or 57, respectively).

[0201] In some embodiments, a biparatopic antibody or antigen-binding fragment thereof of the present disclosure comprises the variable light chain and / or variable heavy chain of FR57 (e.g., SEQ ID NO: 18 and / or 23, respectively) and the variable light chain and / or variable heavy chain of huMOV19 (e.g., SEQ ID NO: 21 and / or 26, respectively).

[0202] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises (i) an scFv that binds to the same epitope as FR57 and (ii) an scFv that binds to the same epitope as huMov19. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27 and SEQ ID NO:30. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27 and SEQ ID NO:31. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27 and SEQ ID NO:32.

[0203] In some embodiments, the anti-FRα biparatopic antibody or antigen-binding fragment thereof comprises SEQ ID NO: 28 and SEQ ID NO: 30. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 28 and SEQ ID NO: 31. In some embodiments, the anti-FRα biparatopic antibody or antigen-binding fragment thereof comprises SEQ ID NO: 28 and SEQ ID NO: 32.

[0204] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 29 and SEQ ID NO: 30. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 29 and SEQ ID NO: 31. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 29 and SEQ ID NO: 32.

[0205] It should be understood that the VH and VL sequences of SEQ ID NOs: 27-32 can be arranged in different orders. For example, the N-terminal to C-terminal orientation as set forth in SEQ ID NO: 27 is VH-(G4S)4-VL. However, disclosed herein are scFv polypeptide sequences in which the VH and VL sequences are swapped around the glycine-serine linker (e.g., VL-(G4S)4-VH).

[0206] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:19, and SEQ ID NO:24. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:19, and SEQ ID NO:25. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:19, and SEQ ID NO:57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:19, and SEQ ID NO:26.

[0207] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:20, and SEQ ID NO:24. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:20, and SEQ ID NO:25. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:20, and SEQ ID NO:57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:20, and SEQ ID NO:26.

[0208] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof is (i) an scFv that binds to the same epitope as FR57, and (ii) an FRα-binding domain comprising a VH and a VL on separate polypeptides that bind to the same epitope as huMov19. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:21, and SEQ ID NO:24. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:21, and SEQ ID NO:25. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:21, and SEQ ID NO:57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:27, SEQ ID NO:21, and SEQ ID NO:26.

[0209] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:19, and SEQ ID NO:24. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:19, and SEQ ID NO:25. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:19, and SEQ ID NO:57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:19, and SEQ ID NO:26.

[0210] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:20, and SEQ ID NO:24. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:20, and SEQ ID NO:25. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:20, and SEQ ID NO:57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:20, and SEQ ID NO:26.

[0211] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:21, and SEQ ID NO:24. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:21, and SEQ ID NO:25. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:21, and SEQ ID NO:57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:28, SEQ ID NO:21, and SEQ ID NO:26.

[0212] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:19, and SEQ ID NO:24. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:19, and SEQ ID NO:25. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:19, and SEQ ID NO:57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:19, and SEQ ID NO:26.

[0213] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:20, and SEQ ID NO:24. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:20, and SEQ ID NO:25. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:20, and SEQ ID NO:57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:20, and SEQ ID NO:26.

[0214] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:21, and SEQ ID NO:24. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:21, and SEQ ID NO:25. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:21, and SEQ ID NO:57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:29, SEQ ID NO:21, and SEQ ID NO:26.

[0215] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises (i) an scFv that binds to the same epitope as huMov19 and (ii) an FRα-binding domain comprising a VH and a VL on separate polypeptides that bind to the same epitope as FR57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:30, SEQ ID NO:17, and SEQ ID NO:22. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:30, SEQ ID NO:17, and SEQ ID NO:23.

[0216] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 30, SEQ ID NO: 18, and SEQ ID NO: 22. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 30, SEQ ID NO: 18, and SEQ ID NO: 23.

[0217] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises (i) an scFv that binds to the same epitope as huMov19 and (ii) an FRα-binding domain comprising a VH and a VL on separate polypeptides that bind to the same epitope as FR57. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:31, SEQ ID NO:18, and SEQ ID NO:22. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO:31, SEQ ID NO:18, and SEQ ID NO:23.

[0218] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 31, SEQ ID NO: 17, and SEQ ID NO: 22. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 31, SEQ ID NO: 17, and SEQ ID NO: 23.

[0219] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 32, SEQ ID NO: 17, and SEQ ID NO: 22. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 32, SEQ ID NO: 17, and SEQ ID NO: 23.

[0220] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 32, SEQ ID NO: 18, and SEQ ID NO: 22. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises SEQ ID NO: 32, SEQ ID NO: 18, and SEQ ID NO: 23.

[0221] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises a polypeptide sequence disclosed in Table 6 below. [Table 6-1] [Table 6-2] [Table 6-3]

[0222] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises the polypeptide sequence of SEQ ID NO: 33 and SEQ ID NO: 34. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a polypeptide sequence selected from SEQ ID NO: 35 and SEQ ID NO: 36. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a polypeptide sequence selected from SEQ ID NO: 37 and SEQ ID NO: 38. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a polypeptide sequence selected from SEQ ID NO: 39 and SEQ ID NO: 40.

[0223] In some embodiments, the biparatopic antibody or antigen-binding fragment thereof comprises a polypeptide sequence disclosed in Table 7 below. [Table 7-1] [Table 7-2]

[0224] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises the polypeptide sequence of SEQ ID NO: 41-43. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises the polypeptide sequence of SEQ ID NO: 44-46.

[0225] The biparatopic antibody or antigen-binding fragment thereof of the present disclosure may further comprise a linker. In some embodiments, the linker may link the first antibody or antigen-binding fragment thereof to the second antibody or antigen-binding fragment thereof from the N-terminus to the C-terminus. In other embodiments, the linker may link the second polypeptide to the first polypeptide from the N-terminus to the C-terminus.

[0226] In one embodiment, the biparatopic antibody or antigen-binding fragment thereof comprises a linker sequence located between the first peptide, antibody, or antigen-binding fragment thereof and the second peptide, antibody, or antigen-binding fragment thereof. The linker can be of any length and can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, or 60 or more amino acids. In other embodiments, linkers useful in the present disclosure have at least one amino acid and fewer than 100 amino acids, fewer than 90 amino acids, fewer than 80 amino acids, fewer than 70 amino acids, fewer than 60 amino acids, fewer than 50 amino acids, fewer than 40 amino acids, fewer than 30 amino acids, fewer than 20 amino acids, fewer than 19 amino acids, fewer than 18 amino acids, fewer than 17 amino acids, fewer than 16 amino acids, fewer than 15 amino acids, fewer than 14 amino acids, fewer than 13 amino acids, or fewer than 12 amino acids. In some embodiments, the linker sequence includes a glycine amino acid residue. In other examples, the linker sequence includes a combination of glycine and serine amino acid residues.

[0227] In some embodiments, such glycine / serine linkers can comprise the peptides GGGS (SEQ ID NO:49) or GGGGS (SEQ ID NO:50), or any combination of amino acid residues including, but not limited to, repeats thereof, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeats of those given peptides. The glycine / serine linkers disclosed herein are represented by the formula: (GS) n , (GGS) n , (GGGS) n , (GGGGS) n , or (GGGGS) nwherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker sequence is GGGGSGGGSGGGGGS (SEQ ID NO:51) (also referred to as Gly4Ser)3). In another embodiment, the linker sequence is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:52) (also referred to as Gly4Ser)4).

[0228] In some embodiments, the biparatopic anti-FRα antibody comprises an altered (e.g., mutated or engineered) Fc region. For example, in some aspects, the Fc region is altered to modify the serum half-life or other functional properties of the antibody to reduce or enhance the effector function of the antibody. Reducing or eliminating effector function is desirable in certain cases, for example, for antibodies whose mechanism of action involves blocking or antagonizing rather than killing cells bearing the target antigen. Increasing effector function is generally desirable when targeting unwanted cells such as tumors and foreign cells that express low levels of FcγR, for example, tumor-specific B cells that have low levels of FcγRIIB (e.g., non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma). The immunoconjugates of the present invention with such conferred or altered effector function activity are useful for treating and / or preventing diseases, disorders, or infections in which enhanced potency of effector function activity is desired. In some embodiments, the Fc region is an isotype selected from IgM, IgA, IgG, IgE, or other isotypes.

[0229] Although the Fc region of a biparatopic anti-FRα antibody or antigen-binding fragment thereof may have the ability to bind to one or more Fc receptors (e.g., FcγR(s)), in certain embodiments, the antibody or antibody fragment comprises a variant Fc region with altered binding (compared to the binding exhibited by the wild-type Fc region) to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b), e.g., with enhanced binding to activating receptors and / or with significantly reduced or no ability to bind to inhibitory receptor(s). Thus, the Fc region of a biparatopic anti-FRα antibody or antigen-binding fragment thereof may comprise a portion or all of the CH2 domain and / or the CH3 domain of the complete Fc region. The Fc region may comprise some or all of the three domains, or may comprise a variant CH2 and / or variant CH3 sequence (e.g., one or more insertions and / or one or more deletions relative to the CH2 or CH3 domains of an intact Fc region). Such an Fc region may include a non-Fc polypeptide portion, or may include a portion of a non-naturally occurring intact Fc region, or may include a non-naturally occurring orientation of the CH2 and / or CH3 domains (e.g., two CH2 domains or two CH3 domains, or a CH3 domain linked to a CH2 domain in an N-terminal to C-terminal direction, etc.).

[0230] Fc region modifications identified as altering effector function, including modifications that increase binding to activating receptors (e.g., FcγRIIA (CD16A)) and decrease binding to inhibitory receptors (e.g., FcγRIIB (CD32B)), are known in the art (see, e.g., Stavenhagen, et al., Cancer Res. 57(18):8882-8890 (2007)). Table 8 shows exemplary single, double, triple, quadruple, and quintuple substitutions of exemplary modifications that increase binding to activating receptors and / or decrease binding to inhibitory receptors (numbering is that of the EU index as set forth in Kabat, and substitutions are relative to the amino acid sequence of SEQ ID NO:59). [Table 8]

[0231] Exemplary variants of human IgG1 Fc regions with reduced binding to CD32B and / or increased binding to CD16A include substitutions of F243L, R292P, Y300L, V305I, or P396L, numbering being that of the EU index as set forth in Kabat. These amino acid substitutions may be present in the human IgG1 Fc region in any combination. In one embodiment, the variant human IgG1 Fc region includes substitutions of F243L, R292P, and Y300L. In another embodiment, the variant human IgG1 Fc region includes substitutions of F243L, R292P, Y300L, V305I, and P396L.

[0232] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain constant region that comprises a modification that reduces effector function (e.g., Idusogie et al., J. Immunol. 166:2571-2575 (2001); Sazinsky et al., PNAS USA 105:2016 7-20172(2008);Davis et al.,J.Rheumatol.34:2204-2210(2007);Bolt et al.,Eur.J.Immunol.23:403-411(1993);Alegre et al.,Transplantation 57:1537-1543(1994);Xu et al. al.,Cell Immunol.200:16-26(2000);Cole et al.,Transplantation 68:563-571(1999);Hutchins et al.,PNAS USA 92:11980-11984(1995);Reddy et al. al., J. Immunol. 164:1925-1933 (2000); WO 97 / 11971, and WO 07 / 106585; U.S. Patent Application Publication No. 2007 / 0148167A1; McEarchern et al., Blood 109:1185-1192 (2007); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Kumagai et al., J. Clin. Pharmacol. 47:1489-1497 (2007), the contents of each of which are incorporated herein by reference in their entireties.

[0233] In some embodiments, it is preferred for the Fc region of the biparatopic anti-FRα antibody or antigen-binding fragment thereof to exhibit reduced binding (or substantially no binding) to effector receptors selected from the group consisting of FcγRIA (CD64), FcγRIIA (CD32A) (allotypes R131 and H131), FcγRIIB (CD32B), FcγRIIIA (CD16a) (allotypes V158 and F158), and FcγRIIIB (CD16b) (allotypes FcγIIIb-NA1 and FcγIIIb-NA2) compared to the binding exhibited by the wild-type IgG Fc region (SEQ ID NO: 59). In some embodiments, the effector receptor binding affinity of the Fc region variant of the biparatopic anti-FRα antibody or antigen-binding fragment thereof is reduced by at least 10-fold, at least 50-fold, or at least 100-fold compared to the binding affinity of the corresponding antibody or antibody-binding fragment comprising the wild-type Fc region of the corresponding immunoglobulin.

[0234] In certain embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment comprises an IgG Fc region that exhibits reduced effector function (e.g., decreased ADCC) and comprises a modification at one or more amino acid positions selected from the group consisting of 233, 234, 235, 236, 237, 238, 239, 265, 266, 267, 269, 270, 271, 295, 296, 297, 298, 300, 324, 325, 327, 328, 329, 331, and 332, where the numbering of the amino acid positions is according to the EU index as set forth in Kabat. In one embodiment, the CH2-CH3 domain of the biparatopic anti-FRα antibody or antigen-binding fragment comprises any one, two, three or four of the following substitutions: L234A, L235A, D265A, N297Q, N297A and N297G, numbering being as per the EU index as set forth in Kabat. In another embodiment, the CH2-CH3 domain comprises an N297Q substitution, an N297A substitution, or an L234A and an L235A substitution, these mutations being for abolishing FcR binding. Alternatively, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a CH2-CH3 domain of a naturally occurring Fc region that essentially exhibits reduced (or substantially absent) binding to FcγRIIIA (CD16a) and / or reduced effector function (compared to the binding and effector function exhibited by the wild-type IgG1 Fc region (SEQ ID NO:59)). In certain embodiments, the Fc constant region of the biparatopic anti-FRα antibody comprises an IgG2 Fc region (SEQ ID NO: 60) or an IgG4 Fc region (SEQ ID NO: 61). Since the N297A, N297G, N297Q, L234A, L235A, and D265A substitutions abolish effector function, these substitutions would preferably not be used in situations where effector function is desired.

[0235] Biparatopic anti-FRα antibodies containing Fc regions with reduced or abolished effector functions A preferred IgG1 sequence for the CH2 and CH3 domains of the antibody or antigen-binding fragment thereof contains the substitutions L234A / L235A (underlined) (SEQ ID NO: 62): [ka]

[0236] A preferred IgG1 sequence for the CH2 and CH3 domains of a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprising an Fc region with reduced or abolished effector function contains the substitution N297A (underlined) (SEQ ID NO: 63): [ka]

[0237] A preferred IgG1 sequence for the CH2 and CH3 domains of a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprising an Fc region with reduced or abolished effector function contains the substitution N297Q (underlined) (SEQ ID NO: 64): [ka]

[0238] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an Fc (immunoglobulin) sequence selected from SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an Fc (immunoglobulin) sequence with reduced or abolished effector function (e.g., comprising the substitutions set forth in SEQ ID NO: 62, SEQ ID NO: 63, and / or SEQ ID NO: 64 above) and one or more knob-in-hole mutations as described herein. In some embodiments, the Fc sequence comprises a knob mutation as disclosed herein. In some embodiments, the Fc sequence comprises a hole mutation as disclosed herein.

[0239] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof is selected from the group consisting of IgG1-C220S, C226S, C229S, P238S; IgG1-C226S, C229S; IgG1-C226S, C229S, E233P, L234V, L235A; IgG1-L234A, L235A; IgG1-L234F, L235E, P331S ;IgG1-L234F, L235E, P331S;IgG1-H268Q, A330S, P331S;IgG1-G236R, L328R;IgG1-L235G, G236R , IgG1-N297A; IgG1-N325A, L328R; IgG1-N325L, L328R; IgG1-K326W, E333S; IgG2-V234A, G237A; IgG2-E333S; IgG2 H268Q, V309L, A330S, A331S; IgG4-S228P, L236E; IgG4-F234A, L235A; IgG4-F234A, G237A, E318A; IgG4-L235A, G237A, E318A; IgG4-L236E; IgG2-EU sequence 118 to 260; and IgG4-EU sequence 261 to 447, where the numbering of the positions is according to the EU index as set out in Kabat.

[0240] In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain immunoglobulin constant domain that has reduced CDC activity. In certain embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain constant region comprising a mutation that reduces CDC activity (see, e.g., WO 1997 / 11971 and WO 2007 / 106585; U.S. Patent Application Publication No. 2007 / 0148167A1; McEarchern et al., Blood 109:1185-1192 (2007); Hayden-Ledbetter et al., Clin. Cancer 15:2739-2746 (2009); Lazar et al., PNAS USA 103:4005-4010 (2006); Bruckheimer et al., Neoplasia 11:509-517 (2009); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Sazinsky et al., J. Med. 1999, 14:1111-1112 (2009)). al., PNAS USA 105:20167-20172 (2008), each of which is incorporated herein by reference in its entirety. Examples of heavy chain constant domain sequence modifications that reduce CDC include one or more modifications corresponding to, according to the EU index, IgG1-C226S, C229S, E233P, L234V, L235A; IgG1-C226S, P230S; IgG1-L234F, L235E, P331S; IgG1-S239D, A330L, I332E; IgG2 EU sequence 118-260; IgG4-EU sequence 261-447; and IgG2-H268Q, V309L, A330S, A331S.

[0241] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof provided comprises a heavy chain immunoglobulin constant domain that comprises one or more half-life-enhancing amino acid modifications (e.g., substitutions).Many mutations that can increase the half-life of molecules that comprise Fc regions are known in the art and are included as components of the biparatopic anti-FRα antibody or antigen-binding fragment thereof provided herein.See, for example, U.S. Patent Nos. 6,277,375, 7,083,784, 7,217,797, and 8,088,376, U.S. Patent Application Publication Nos. 2002 / 0147311; and 2007 / 0148164; and PCT Publication Nos. WO1998 / 23289; WO2009 / 058492; and WO2010 / 033279 (the contents of each of which are incorporated herein in their entirety by reference).

[0242] The serum half-life of a protein comprising an Fc region can be increased by increasing the binding affinity of the Fc region to FcRn. As used herein, the term "half-life" refers to a pharmacokinetic property of a molecule that is a measure of the average survival time of the molecule after their administration. Half-life can be expressed as the time required to eliminate 50% of a known amount of a molecule from the body of a subject (e.g., a human patient or other mammal) or a specific compartment thereof, for example, when measured in serum (i.e., circulating half-life) or other tissues. In general, an increase in half-life results in an increase in the mean residence time (MRT) of an administered molecule in the circulation.

[0243] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof is and 436, where the numbering of the amino acid positions is according to the EU index. In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, where the numbering of the amino acid positions is according to the EU index. In some embodiments, a biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises one or more of: a substitution of the amino acid at Kabat position 252 with Tyr, Phe, Trp, or Thr; a substitution of the amino acid at Kabat position 254 with Thr; a substitution of the amino acid at Kabat position 256 with Ser, Arg, Gln, Glu, Asp, or Thr; a substitution of the amino acid at Kabat position 257 with Leu; a substitution of the amino acid at Kabat position 309 with Pro; a substitution of the amino acid at Kabat position 311 with Ser; a substitution of the amino acid at Kabat position 428 with Thr, Leu, Phe, or Ser; a substitution of the amino acid at Kabat position 433 with Arg, Ser, Iso, Pro, or Gln; or a substitution of the amino acid at Kabat position 434 with Trp, Met, Ser, His, Phe, or Tyr. More specifically, the biparatopic anti-FRα antibody or antigen-binding fragment domain thereof can include amino acid substitutions relative to the constant domain of wild-type human IgG, including substitution of the amino acid at Kabat position 252 with Tyr, substitution of the amino acid at Kabat position 254 with Tyr, and substitution of the amino acid at Kabat position 256 with Glu.

[0244] In some embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises at least one substitution selected from T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, N434S, N434H, N434Y, H435K, and Y436I, where the numbering is that of the EU index as set forth in Kabat. In further embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises substitutions selected from: (a) M252Y, S254T, and T256E; (b) M252Y and S254T; (c) M252Y and T256E; (d) T250Q and M428L; (e) T307Q and N434A; (f) A378V and N434A; (g) N434A and Y436I; (h) V308P and N434A; and (i) K288D and H435K.

[0245] In a preferred embodiment, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a variant IgG Fc region comprising any one, two, or three of the substitutions M252Y, S254T, and T256E. The present disclosure further provides a biparatopic anti-FRα antibody or antigen-binding fragment thereof having a variant Fc region comprising: (a) one or more mutations that modify effector function and / or FcγR; and (b) one or more mutations that extend serum half-life. [Table 9]

[0246] III. Biparatopic Antibody Production Biparatopic antibodies or antigen-binding fragments thereof that immunospecifically bind to FRα can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, for example, Sambrook J. t al.,(2001)Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY;Ausubel FM et al.,Current Protocols in Molecular Biology,John Wiley & Sons(1987 and annual updates);Current Protocols in Immunology,John Wiley & Sons(1987 and annual updates)Gait(ed.)(1984)Oligonucleotide Synthesis:A Practical Approach,IRL Press;Eckstein(ed.)(1991)Oligonucleotides and Analogues:A Practical Approach,IRL Press;Birren B et al.,(eds.)(1999)Genome Analysis:A See Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0247] The biparatopic antibodies or antigen-binding fragments thereof provided herein can be prepared by chemically linking two different monoclonal antibodies or by fusing two hybridoma cell lines to produce a hybrid hybridoma.

[0248] In certain embodiments, the described biparatopic antibodies or antigen-binding fragments thereof are prepared, expressed, created, or isolated by any means, including, for example, synthesis of DNA sequences, creation via genetic engineering, etc. In certain embodiments, such biparatopic antibodies or antigen-binding fragments thereof comprise sequences (e.g., DNA sequences, or amino acid sequences) that do not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.

[0249] Methods for making bispecific, bivalent antibodies or antigen-binding fragments thereof are described, for example, in U.S. Patent Nos. 5,731,168, 5,807,706, and 5,821,333, and U.S. Patent Application Publication Nos. 2003 / 020734 and 2002 / 0155537, each of which is incorporated herein by reference in its entirety. Bispecific tetravalent antibodies and methods for making them are described, for example, in International Application Publication Nos. WO02 / 096948 and WO00 / 44788, the disclosures of both of which are incorporated herein by reference in their entireties. See generally, International Application Publication Nos. WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, and WO 92 / 05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992), each of which is incorporated by reference in its entirety.

[0250] One method for generating bispecific antibodies is called the "knobs-into-holes" strategy (see, for example, International Publication WO 2006 / 028936). In this technique, mispairing of Ig heavy chains is reduced by mutating selected amino acids that form the interface of the CH3 domain of IgG. At positions in the CH3 domain where the two heavy chains directly interact, an amino acid with a small side chain (hole) is introduced into the sequence of one heavy chain, and an amino acid with a large side chain (knob) is introduced at the position of the corresponding interacting residue on the other heavy chain. In some embodiments, the compositions of the invention have immunoglobulin chains whose CH3 domains have been modified to preferentially form bispecific antibodies by mutating selected amino acids that interact at the interface between the two polypeptides. Bispecific Antibodies can be composed of immunoglobulin chains of the same subclass (eg, IgG1 or IgG3) or of different subclasses (eg, IgG1 and IgG3, or IgG3 and IgG4).

[0251] In one embodiment, the biparatopic antibody or antigen-binding fragment thereof comprises a T366W mutation in the "knob chain" and a T366S, L368A, Y407V mutation in the "hole chain", and optionally an additional interchain disulfide bridge between the CH3 domains. These include, for example, a Y349C mutation in the "knob chain" and an E356C or S354C mutation in the "hole chain"; a R409D, K370E mutation in the "knob chain" and a D399K, E357K mutation in the "hole chain"; a T366W mutation in the "knob chain" and a T366S, L368A, Y407V mutation in the "hole chain"; a R409D, K370E mutation in the "knob chain", and by introducing a D399K, E357K mutation in the "hole strand"; a Y349C, T366W mutation in one strand and an E356C, T366S, L368A, Y407V mutation in the corresponding strand; and a Y349C, T366W mutation in one strand and an S354C, T366S, L368A, Y407V mutation in the corresponding strand (numbering according to the EU numbering system).

[0252] The bispecific antibodies described herein can also be produced according to the DuoBody technology platform (Genmab A / S), as described, for example, in International Publication Nos. WO2011 / 131746, WO2011 / 147986, WO2008 / 119353, and WO2013 / 060867, and Labrijn AF et al., (2013) PNAS 110(13):5145-5150. The DuoBody technology can be used to combine one half of a first FRα binding domain, which comprises two heavy chains and two light chains, with one half of a second FRα binding domain, which comprises two heavy chains and two light chains. The resulting heterodimer comprises one heavy chain and one light chain from the first FRα binding domain, paired with one heavy chain and one light chain from the second FRα binding domain.

[0253] Biparatopic antibodies or antigen-binding fragments thereof optionally comprise heterodimers of IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG2, IgG4 and IgG3, or IgG1 and IgG3 chains. Such heterodimeric heavy chain antibodies can be routinely engineered, for example, by modifying selected amino acids that form the interface of the CH3 domains of human IgG4 and IgG1 or IgG3 to promote heterodimeric heavy chain formation.

[0254] In certain embodiments, the biparatopic antibody or antigen-binding fragment thereof may comprise a chimeric FRα binding domain or a humanized FRα binding domain. In certain embodiments, the biparatopic antibody or antigen-binding fragment thereof may be a F(ab')2 fragment. The F(ab')2 fragment contains two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds in the hinge region.

[0255] The biparatopic antibodies or antigen-binding fragments thereof described herein can be produced by any technique known to those skilled in the art. For example, the F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using an enzyme such as pepsin.

[0256] In certain embodiments, provided herein are methods of making a biparatopic antibody or antigen-binding fragment thereof, comprising culturing one or more cells described herein. In certain embodiments, provided herein are methods of making a biparatopic antibody or antigen-binding fragment thereof, comprising culturing one or more cells described herein. In certain embodiments, provided herein are methods of making a biparatopic antibody or antigen-binding fragment thereof, comprising culturing one or more cells described herein, using a cell or host cell (e.g., a cell or host cell that contains a polynucleotide encoding an antibody described herein). In certain embodiments, the method further comprises purifying the antibody or antigen-binding fragment obtained from the cell or host cell by using a recombinant humanized antibody or antigen-binding fragment to express (e.g., recombinantly express) the antibody or antigen-binding fragment. In certain embodiments, the cell is an isolated cell. In certain embodiments, an exogenous polynucleotide has been introduced into the cell. In certain embodiments, the method further comprises purifying the antibody or antigen-binding fragment obtained from the cell or host cell.

[0257] The FRα antigen-binding domain can be prepared, for example, from monoclonal antibodies, using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technology, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques known in the art, including those taught in, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced recombinantly from a host cell that exogenously expresses the antibody described herein. The monoclonal antibodies described herein can be made, for example, by the hybridoma method described in Kohler G & Milstein C (1975) Nature 256:495, or can be isolated, for example, from phage libraries, for example, using the techniques described herein. Other methods for preparation of clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 in Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0258] In addition, the FRα binding domains described herein can also be generated using various phage display methods known in the art. In phage display methods, proteins are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of diseased tissue). The DNA encoding the VH and VL domains is recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli is infected with helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are usually recombinantly fused to either phage gene III or gene VIII. Phages expressing antibodies or fragments that bind to a particular antigen can be selected or identified by antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to generate the antibodies described herein include those described in Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT Application No. PCT / GB91 / 001134; International Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403, 484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0259] As described in the above references, after phage selection, antibody coding regions from the phage can be isolated and FRα-binding domains, including human FRα-binding domains, can be produced and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described below. For example, techniques for recombinantly producing FRα-binding domains, such as Fab, Fab', and F(ab')2 fragments, can be employed using methods known in the art, such as those disclosed in PCT Publication WO92 / 22324, Mullinax RL et al., (1992) BioTechniques 12(6):864-9; Sawai H et al., (1995) Am J Reprod Immunol 34:26-34; and Better M et al., (1988) Science 240:1041-1043.

[0260] In one embodiment, to produce an FRα binding domain or antibody, PCR primers containing VH or VL nucleotide sequences, restriction sites, and flanking sequences to protect the restriction sites can be used to amplify VH and VL sequences from a template, e.g., an scFv clone. Using cloning techniques known to those skilled in the art, the PCR amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line to generate a stable or transient cell line expressing an antibody, e.g., an IgG, using techniques known to those skilled in the art.

[0261] IV. Polynucleotides Encoding Biparatopic Antibodies In certain embodiments, the disclosure encompasses a polynucleotide comprising a nucleic acid encoding a biparatopic anti-FRα antibody or an antigen-binding fragment thereof, or a domain of such an antibody or fragment, e.g., a VH, a VL, a VL and a VH (e.g., in an scFv), a heavy chain, a light chain, a heavy chain with an scFv, a light chain with an scFv, a constant region, or a constant region with an scFv.

[0262] Thus, provided herein are polynucleotides encoding SEQ ID NOs: 17-40. Also provided herein are compositions comprising combinations of polynucleotides encoding any biparatopic anti-FRα antibody or antigen-binding fragment thereof (e.g., a composition comprising a polynucleotide encoding SEQ ID NO: 17 and a polynucleotide encoding SEQ ID NO: 22, a composition comprising a polynucleotide encoding SEQ ID NO: 18 and a polynucleotide encoding SEQ ID NO: 23, a composition comprising a polynucleotide encoding SEQ ID NO: 19 and a polynucleotide encoding SEQ ID NO: 24, a composition comprising a polynucleotide encoding SEQ ID NO: 20 and a polynucleotide encoding SEQ ID NO: 25, a composition comprising a polynucleotide encoding SEQ ID NO: 21 and a polynucleotide encoding SEQ ID NO: 26, a composition comprising a polynucleotide encoding SEQ ID NO: 33 and a polynucleotide encoding SEQ ID NO: 34, a composition comprising a polynucleotide encoding SEQ ID NO: 35 and a polynucleotide encoding SEQ ID NO: 36, a composition comprising a polynucleotide encoding SEQ ID NO: 37 and a polynucleotide encoding SEQ ID NO: 38, a composition comprising a polynucleotide encoding SEQ ID NO: 39 and a polynucleotide encoding SEQ ID NO: 40, a composition comprising a polynucleotide encoding SEQ ID NO: 41 and a polynucleotide encoding SEQ ID NO: 42 and a polynucleotide encoding SEQ ID NO: 43, 3, or a composition comprising a polynucleotide encoding SEQ ID NO:44, a polynucleotide encoding SEQ ID NO:45, and a polynucleotide encoding SEQ ID NO:46. Also provided herein are compositions comprising combinations of polynucleotides encoding any biparatopic anti-FRα antibody or antigen-binding fragment thereof (e.g., a composition comprising a polynucleotide encoding SEQ ID NO:20 and a polynucleotide encoding SEQ ID NO:57).

[0263] In certain embodiments, the biparatopic anti-FRα antibody or antigen-binding fragment thereof is encoded by a plasmid deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, VA 20110, having ATCC accession numbers PTA-10774 (deposited on April 7, 2010), PTA-125915 ("Mov19-Fc-hole"; deposited with the ATCC on April 29, 2019 and received by the ATCC on April 30, 2019), and PTA-125916 ("FR57scFv2-Fc-knob"; deposited with the ATCC on April 29, 2019 and received by the ATCC on April 30, 2019).

[0264] The polynucleotide of the present invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and can be double-stranded or single-stranded, where the single strand can be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide is a cDNA or DNA that lacks one or more endogenous introns.

[0265] In some embodiments, the polynucleotide is a non-naturally occurring polynucleotide, hi some embodiments, the polynucleotide is recombinantly produced.

[0266] In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure. In some embodiments, the polynucleotide is purified from natural components.

[0267] In some embodiments, the polynucleotides provided herein are codon-optimized (changing the codons of human mRNA to those preferred by a bacterial host, such as E. coli) for expression in a particular host.

[0268] V. Cells and Vectors Vectors and cells comprising the polynucleotides described herein are also provided.

[0269] In certain aspects, provided herein are cells (e.g., host cells) that express (e.g., recombinantly) the antibodies, antigen-binding fragments thereof described herein that specifically bind to FRα, and that contain related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) that contain polynucleotides that contain nucleotide sequences encoding such anti-FRα antibodies, or fragments thereof, for recombinant expression in a host cell, preferably a mammalian cell. Also provided herein are host cells that contain vectors for recombinantly expressing the anti-FRα antibodies, or antigen-binding fragments thereof, described herein. In certain aspects, provided herein are methods for producing the antibodies, or antigen-binding fragments thereof, described herein, comprising expressing such antibodies, or antigen-binding fragments thereof, in a host cell.

[0270] Recombinant expression of the antibodies or antigen-binding fragments thereof described herein can be achieved by expressing the antibodies or fragments thereof (e.g., heavy or light chains), fusion proteins comprising the heavy or light chains (e.g., one or more This includes the construction of expression vectors comprising a polynucleotide encoding a heavy or light chain fused to a variable domain (e.g., scFv), a polypeptide comprising a variable domain, a VH and a VL (e.g., scFv), a constant domain, and / or a fusion protein comprising a constant domain (e.g., a constant domain fused to one or more variable domains (e.g., ScFv)). Once a polynucleotide encoding an antibody or fragment thereof described herein is obtained, vectors for the production of the antibody or fragment thereof can be generated by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for the preparation of a protein by expressing a polynucleotide that is a nucleotide sequence encoding an antibody or fragment thereof. Methods well known to those skilled in the art can be used to construct expression vectors comprising the coding sequence for an antibody or fragment thereof and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody or fragment thereof operably linked to a promoter. Such vectors can include, for example, nucleotide sequences encoding the constant region of an antibody molecule (e.g., International Publication Nos. WO86 / 05807, and WO89 / 01036; and U.S. Patent No. 5,122,464), and antibody variable domains can be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains. Nucleotide sequences encoding additional variable or FRα-binding domains (e.g., scFvs) can also be cloned into such vectors for expression of fusion proteins comprising the heavy or light chain fused to the FRα-binding domain or a fragment thereof (e.g., VH or VL).

[0271] The expression vector can be introduced into a cell (e.g., a host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody or fragment (e.g., heavy or light chain) described herein, a fusion protein comprising a heavy or light chain (e.g., a heavy or light chain fused to one or more variable domains (e.g., scFv)), a polypeptide comprising a variable domain, a VH and a VL (e.g., scFv), a constant domain, and / or a fusion protein comprising a constant domain (e.g., a constant domain fused to one or more variable domains (e.g., ScFv)). Thus, provided herein is a host cell comprising a polynucleotide encoding an antibody or fragment thereof described herein, operably linked to a promoter for expression of such a sequence in the host cell.

[0272] In certain embodiments, for expression of multi-chain antibodies, vectors encoding all of the chains individually can be co-expressed in a host cell for expression of the entire immunoglobulin molecule.

[0273] In certain embodiments, the host cell comprises a vector comprising a polynucleotide encoding all of the chains of an antibody or antigen-binding fragment thereof described herein, hi certain embodiments, the host cell comprises a plurality of different vectors encoding all of the chains of an antibody or antigen-binding fragment thereof described herein.

[0274] A vector or combination of vectors can include a polynucleotide encoding two polynucleotides that interact to form an antibody or antigen-binding fragment thereof described herein: for example, a first polynucleotide encoding a fusion protein comprising a heavy chain and an scFv and a second polynucleotide encoding a light chain; a first polynucleotide encoding a fusion protein comprising a light chain and an scFv and a second polynucleotide encoding a heavy chain; a first polynucleotide encoding a fusion protein comprising a heavy chain and a VH and a second polynucleotide encoding a fusion protein comprising a light chain and a VL, etc. When two polypeptides are encoded by polynucleotides in two separate vectors, the vector can include a polynucleotide encoding a fusion protein comprising a heavy chain and a second polynucleotide encoding a fusion protein comprising a light chain, etc. The polynucleotides encoding the fusion protein are transfected into the host cells at a ratio of 1:1.

[0275] A vector or combination of vectors can include polynucleotides encoding three polypeptides that interact to form an antibody or antigen-binding fragment thereof described herein: for example, a first polynucleotide encoding a heavy chain, a second polynucleotide encoding a light chain, and a third polynucleotide encoding a fusion protein comprising a heavy chain constant domain, a VH, and a VL (optionally, VH and VL are scFvs). When the three polypeptides are encoded by polynucleotides in three separate vectors, the vectors can be transfected into a host cell in a ratio of 6 polynucleotides encoding the heavy chain:3 polynucleotides encoding the light chain:1 polynucleotide encoding the fusion protein.

[0276] A vector or combination of vectors can include polynucleotides encoding four polypeptides that interact to form an antibody or antigen-binding fragment thereof described herein: for example, a first polynucleotide encoding a first heavy chain, a second polynucleotide encoding a second heavy chain, a third polynucleotide encoding a first light chain, and a fourth polynucleotide encoding a second light chain.

[0277] In some embodiments, the host cells comprise the vector or combination of vectors described above, while in other embodiments, two host cells, three host cells, or four host cells comprise the vector or combination of vectors described above.

[0278] A variety of host expression vector systems can be utilized to express the antibody molecules or fragments thereof (e.g., heavy or light chains) described herein, fusion proteins comprising heavy or light chains (e.g., heavy or light chains fused to one or more variable domains (e.g., scFv)), polypeptides comprising variable domains, VH and VL (e.g., scFv), constant domains, and / or fusion proteins comprising constant domains (e.g., constant domains fused to one or more variable domains (e.g., ScFv)). Such host expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequences, can express the antibodies or fragments thereof described herein in situ. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., Chlamydomonas sp.) infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) containing the antibody coding sequences or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences. reinhardtii); or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) carrying recombinant expression constructs comprising promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).In certain embodiments, cells for expressing an antibody or antigen-binding fragment thereof described herein are CHO cells, such as CHO cells from GS System™ (Lonza). In certain embodiments, expression of a nucleotide sequence encoding an antibody that immunospecifically binds to FRα (e.g., human FRα) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0279] The antibody molecules or fragments thereof (e.g., heavy or light chains, variable domains, and / or polypeptides comprising VH and VL (e.g., scFv)) described herein have been produced by recombinant expression and can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, chromatography (e.g., by ion exchange, affinity, particularly affinity for a particular antigen following Protein A, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for the purification of proteins. Additionally, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0280] VI. Immunoconjugates Containing Biparatopic Antibodies In one aspect, the present disclosure relates to an immunoconjugate comprising a biparatopic FRα-binding agent (e.g., an antibody or antigen-binding fragment thereof) described herein and a cytotoxic agent. The cytotoxic agent can be linked or conjugated to the FRα-binding agent directly or indirectly via a linker using techniques known in the art to generate an "immunoconjugate," "conjugate," or "ADC."

[0281] A. Exemplary Immunoconjugates In a first embodiment, the immunoconjugate provided herein comprises a biparatopic FRα antibody or antigen-binding fragment thereof as described herein covalently attached to a maytansinoid compound as described herein via the epsilon amino groups of one or more lysine residues located on the biparatopic FRα antibody or antigen-binding fragment thereof. In one embodiment, the immunoconjugate has formula (I): [ka] or a pharma- ceutically acceptable salt thereof, CB is a biparatopic anti-FRα antibody or an antigen-binding fragment thereof; L2 is represented by one of the following formulas: [ka] During the ceremony, R x , R y , R x’ , and R y’ is, for each occurrence, independently H, -OH, halogen, -O-(C 1-4 alkyl), -SO3H, -NR 40 R 41 R 42 + or optionally -OH, halogen, SO3H, or NR 40 R 41 R 42 + C replaced by 1-4 alkyl, where R 40 , R 41 , and R 42 are each independently H or C 1-4 is alkyl; l and k each independently represent an integer of 1 to 10; l1 is an integer from 2 to 5; k1 is an integer from 1 to 5; s1 indicates the site connected to the cell-binding agent CB, and s3 indicates the site connected to the A group; A is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1-3 is alkyl; L1 is expressed by the following formula: -CR 3 R 4 -(CH2) 1-8 -C(=O)- In the formula, R 3 and R 4 are each independently H or Me, and the -C(=O)- portion of L1 Min is connected to D; D is expressed as follows: [ka] q is an integer from 1 to 20. In some embodiments, q is In some embodiments, q is an integer from 1 to 10. In some embodiments, q is an integer from 2 to 5. In some embodiments, q is an integer from 3 to 4.

[0282] In a first particular embodiment of the first embodiment, the immunoconjugate provided herein is represented by formula (I) above, wherein R x , R y , R x’ , and R y’ are all H; l and k are each independently an integer from 2 to 6; and the remainder of the variables are as described above for formula (I).

[0283] In a second particular embodiment of the first embodiment, the immunoconjugate provided herein is represented by formula (I) above, where A is a peptide comprising 2-5 amino acid residues; the remaining variables are as described above for formula (I) in the first embodiment or the first particular embodiment. In some embodiments, A is a peptide cleavable by a protease. In some embodiments, A is a peptide cleavable by a protease expressed in tumor tissue. In some embodiments, A is -NH-CR, -NH-CR-C ... 1 R 2 In some embodiments, the peptide has an amino acid covalently attached to -S-L1-D. 1 R 2 The amino acids connected to -S-L1-D are L-amino acids.

[0284] In a third specific embodiment of the first embodiment, the immunoconjugate provided herein is represented by formula (I) above, wherein A is Gly-Gly-Gly, Ala-Val, Val-Ala, D-Val-Ala, Val-Cit, D-Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N9-Tosyl-Arg, Phe-N9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys , Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Ala-Leu-Ala-Leu Column number 54), β-Ala-Leu-Ala-Leu (SEQ ID NO: 55), Gly-Phe-Leu-Gly (SEQ ID NO: 56), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-L and D-Ala-tBu-Gly, wherein the first amino acid of each peptide is connected to the L2 group and the last amino acid of each peptide is connected to -NH-CR1R2-S-L1-D, and the remaining variables are as described above for formula (I) in the first embodiment or the first specific embodiment.

[0285] In a fourth specific embodiment of the first embodiment, the immunoconjugate provided herein is represented by formula (I) above, wherein R 1 and R 2 are both H; the remainder of the variables are as described above for formula (I) in the first embodiment or the first, second, or third specific embodiments.

[0286] In a fifth specific embodiment of the first embodiment, the immunoconjugate provided herein is represented by formula (I) above, wherein L1 is -(CH2) 4-6 the remainder of the variables are as described above for Formula (I) in the first embodiment, or the first, second, third, or fourth specific embodiment.

[01] In a sixth specific embodiment of the first embodiment, the immunoconjugate provided herein is represented by formula (I) above, wherein D is represented by the following formula: [ka] The remainder of the variables are as described above for Formula (I) in the first embodiment or the first, second, third, fourth, or fifth specific embodiments.

[0287] In a seventh particular embodiment, the immunoconjugate provided herein has the following formula: [ka] or a pharma- ceutically acceptable salt thereof, [ka] is a biparatopic anti-FRα antibody or antigen-binding fragment thereof, connected to the L2 group via the amine group of Lys; [ka] is a biparatopic anti-FRα antibody or antigen-binding fragment thereof, connected to the L2 group via the thiol group of Cys; R 3 and R 4 are each independently H or Me; m1, m3, n1, r1, s1, and t1 each independently represent an integer of 1 to 6; m2, n2, r2, s2, and t2 each independently represents an integer of 1 to 7; t3 is an integer from 1 to 12; D1 is expressed by the following formula: [ka] q is an integer from 1 to 20. In some embodiments, q is an integer from 1 to 10. In some embodiments, q is an integer from 2 to 5. In some embodiments, q is an integer from 3 to 4. In more specific embodiments, D1 is represented by the formula: [ka]

[0288] In an eighth specific embodiment, the immunoconjugate provided herein is represented by the formula: [ka] During the ceremony: m1 and m3 each independently represent an integer of 2 to 4; m2 is an integer from 2 to 5; r1 is an integer from 2 to 6; r2 is an integer from 2 to 5; The remaining variables are as described in the seventh specific embodiment.

[0289] In a ninth specific embodiment, for the immunoconjugates described in the seventh or eighth specific embodiments, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly. In a more specific embodiment, for the immunoconjugates described in the seventh or eighth specific embodiments, A is L-Ala-D-Ala-L-Ala.

[0290] In a tenth particular embodiment, the immunoconjugate provided herein has the following formula: [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly; D1 is expressed by the following formula: [ka] The remaining variables are as described in the seventh, eighth, or ninth specific embodiments. In a more specific embodiment, A is L-Ala-D-Ala-L-Ala. In a more specific embodiment, D1 is represented by the formula: [ka]

[0291] In an eleventh particular embodiment, the immunoconjugate provided herein is represented by the following formula: [ka] In the formula, D1 is represented by the following formula: [ka] In a more specific embodiment, D1 is represented by the formula: [ka]

[0292] In a twelfth particular embodiment, the immunoconjugate provided herein is represented by the formula: [ka] During the ceremony: CBA is a biparatopic anti-FRα antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: (i) light chain complementarity determining regions L-CDR1, L-CDR2, and L-CDR3 having the sequences of SEQ ID NOs: 1 to 3, and heavy chain complementarity determining regions H-CDR1, H-CDR2, and H-CDR3 having the sequences of SEQ ID NOs: 7 to 9; and (ii) light chain complementarity determining regions L-CDR1, L-CDR2, and L-CDR3 having the sequences of SEQ ID NOs: 4 to 6, and heavy chain complementarity determining regions H-CDR1, H-CDR2, and H-CDR3 having the sequences of SEQ ID NOs: 10 to 12, respectively; q is 1 or 2; D1 is expressed by the following formula: [ka]

[0293] In a specific embodiment, for the immunoconjugate of formula (I-4) or (I-6), the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 18, a VH comprising the amino acid sequence of SEQ ID NO: 23, a VL comprising the amino acid sequence of SEQ ID NO: 19, and a VH comprising the amino acid sequence of SEQ ID NO: 24.

[0294] In a thirteenth particular embodiment, the immunoconjugate provided herein is represented by the formula: [ka] During the ceremony: CBA is a biparatopic anti-FRα antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: (i) light chain complementarity determining regions L-CDR1, L-CDR2, and L-CDR3 having the sequences of SEQ ID NOs: 1 to 3, and heavy chain complementarity determining regions H-CDR1, H-CDR2, and H-CDR3 having the sequences of SEQ ID NOs: 7 to 9; and (ii) light chain complementarity determining regions L-CDR1, L-CDR2, and L-CDR3 having the sequences of SEQ ID NOs: 4 to 6, and heavy chain complementarity determining regions H-CDR1, H-CDR2, and H-CDR3 having the sequences of SEQ ID NOs: 10 to 12, respectively; q is an integer from 1 to 10, for example, 1 or 10; D1 is expressed by the following formula: [ka]

[0295] In certain embodiments, for the immunoconjugate of formula (I-2), the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 18, a VH comprising the amino acid sequence of SEQ ID NO: 23, a VL comprising the amino acid sequence of SEQ ID NO: 19, and a VH comprising the amino acid sequence of SEQ ID NO: 24. In certain embodiments, for the immunoconjugate of formula (I-2), the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a polypeptide having the amino acid sequences of SEQ ID NOs: 41, 42, and 43.

[0296] In a fourteenth embodiment, the immunoconjugate provided herein comprises a biparatopic anti-FRα antibody conjugated to DM21C (also referred to as Mal-LDL-DM or MalC5-LDL-DM or compound 17A), a maytansinoid compound represented by the following structural formula: [ka] wherein the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises (i) a light chain variable region and a heavy chain variable region having the sequences of SEQ ID NO:18 and SEQ ID NO:23, respectively, and (ii) a light chain variable region and a heavy chain variable region having the sequences of SEQ ID NO:19 and SEQ ID NO:24, respectively, and D1 is represented by the formula: [ka]

[0297] In one embodiment, the immunoconjugate is represented by the following structural formula: [ka] During the ceremony: The CBA is a biparatopic anti-FRα antibody or antigen-binding fragment thereof, comprising (i) a light chain variable region and a heavy chain variable region having the sequences of SEQ ID NO:18 and SEQ ID NO:23, respectively, and (ii) a light chain variable region and a heavy chain variable region having the sequences of SEQ ID NO:19 and SEQ ID NO:24, respectively; q is 1 or 2.

[0298] In certain embodiments, for a composition (e.g., a pharmaceutical composition) comprising the immunoconjugate of the fourteenth particular embodiment, the DAR is in the range of 1.5 to 2.2, 1.7 to 2.2, or 1.9 to 2.1. In some embodiments, the DAR is 1.7, 1.8, 1.9, 2.0, or 2.1.

[0299] In a fifteenth particular embodiment, the immunoconjugate provided herein comprises a biparatopic anti-FRα antibody or antigen-binding fragment thereof linked via a γ-maleimidobutyric acid N-succinimidyl ester (GMBS) or N-(γ-maleimidobutryloxy)sulfosuccinimide ester (sulfo-GMBS or sGMBS) linker to DM21 (also referred to as DM21L, LDL-DM, or compound 14c), a maytansinoid compound represented by the following structural formula: [ka] The biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises (i) a light chain variable region and a heavy chain variable region having the sequences of SEQ ID NO: 18 and SEQ ID NO: 23, respectively, and (ii) a light chain variable region and a heavy chain variable region having the sequences of SEQ ID NO: 19 and SEQ ID NO: 24, respectively.

[0300] GMBS and sulfo-GMBS (or sGMBS) linkers are known in the art and can be represented by the following structural formula: [ka]

[0301] In one embodiment, the immunoconjugate is represented by the following structural formula: [ka] During the ceremony: The CBA is a biparatopic anti-FRα antibody or antigen-binding fragment thereof, comprising (i) a light chain variable region and a heavy chain variable region having the sequences of SEQ ID NO:18 and SEQ ID NO:23, respectively, and (ii) a light chain variable region and a heavy chain variable region having the sequences of SEQ ID NO:19 and SEQ ID NO:24, respectively; q is an integer from 1 to 10, for example, 1 or 10. In some embodiments, q is an integer from 2 to 5. In some embodiments, q is an integer from 3 to 4.

[0302] In a specific embodiment, for the immunoconjugate of the fifteenth specific embodiment, the biparatopic anti-FRα antibody or antigen-binding fragment thereof comprises a polypeptide having the amino acid sequence of SEQ ID NOs: 41, 42, and 43.

[0303] In certain embodiments, for a composition (e.g., a pharmaceutical composition) comprising the immunoconjugate of the fifteenth particular embodiment, the DAR is in the range of 3.0 to 4.0, 3.2 to 3.8, 3.1 to 3.7, or 3.4 to 3.7. In some embodiments, the DAR is 3.2, 3.3, 3.4, 3.5, 3.5, 3.7, or 3.8. In some embodiments, the DAR is 3.5.

[0304] In certain embodiments, for compositions comprising lysine conjugates, the DAR is in the range of 1.5 to 3.1, hi some embodiments, the DAR is about 2.0.

[0305] In certain embodiments, the first embodiment, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth specific embodiments. For compositions (e.g., pharmaceutical compositions) comprising an immunoconjugate of an embodiment, the average number of cytotoxic agents per antibody molecule (i.e., the average value of q), also known as the drug-antibody ratio (DAR), in the composition ranges from 1.0 to 8.0. In some embodiments, the DAR ranges from 1.0 to 5.0, 1.0 to 4.0, 1.5 to 4.0, 2.0 to 4.0, 2.5 to 4.0, 1.0 to 3.4, 1.0 to 3.0, 3.0 to 4.0, 3.1 to 3.5, 3.1 to 3.7, 3.4 to 3.6, 1.5 to 2.5, 2.0 to 2.5, 1.7 to 2.3, or 1.8 to 2.2. In some embodiments, the DAR is less than 4.0, less than 3.8, less than 3.6, less than 3.5, less than 3.0, or less than 2.5. In some embodiments, the DAR is in the range of 3.1 to 3.7. In some embodiments, the DAR is in the range of 3.1 to 3.4. In some embodiments, the DAR is in the range of 3.3 to 3.7. In some embodiments, the DAR is in the range of 3.5 to 3.9. In some embodiments, the DAR is 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, or 3.8. In some embodiments, the DAR is 3.5. In some embodiments, the DAR is in the range of 1.8 to 2.0. In some embodiments, the DAR is in the range of 1.7 to 1.9. In some embodiments, the DAR is in the range of 1.9 to 2.1. In some embodiments, the DAR is 1.9, 2.0, or 2.1. In some embodiments, for an immunoconjugate of the invention comprising a biparatopic anti-FRα antibody or antigen-binding fragment thereof conjugated to a maytansinoid compound via one or more cysteine ​​thiol groups, the DAR ranges from 1.5 to 2.5, 1.8 to 2.2, 1.1 to 1.9, or 1.9 to 2.1, hi some embodiments, the DAR is 1.8, 1.9, 2.0, or 2.1.

[0306] B. Linker Any suitable linker known in the art can be used in preparing the immunoconjugates of the present disclosure. In certain embodiments, the linker is a bifunctional linker. As used herein, the term "bifunctional linker" refers to a modifying agent having two reactive groups, one capable of reacting with a cell-binding agent and the other with a maytansinoid compound to link the two moieties together. Such bifunctional crosslinkers are well known in the art (see, for example, Isalm and Dent in Bioconjugation chapter 5, p218-363, Groves Dictionaries Inc. New York, 1999). For example, bifunctional crosslinkers that allow for linkage via a thioether bond include N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), which introduces a maleimide group, or N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), which introduces an iodoacetyl group. Other bifunctional crosslinkers that introduce maleimide or haloacetyl groups into cell-binding agents are known in the art (see U.S. Patent Publication Nos. 2008 / 0050310, 20050169933).Available from Pierce Biotechnology Inc. PO Box 117, Rockland, IL 61105, USA), bis-maleimide polyethylene glycol (BMPEO), BM(PEO)2, BM(PEO)3, N-(β-maleimidopropyloxy)succinimide ester (BMPS), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-aminopropyl)- docaproate), which is the "long chain" analogue of SMCC (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH), N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)butyric acid hydrazide or HCl salt (MPBH). Succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), Succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), Dithiobis-maleimidoethane (DTME), 1,4-Bis-maleimidobutane (BMB), 1,4-Bismaleimidyl-2,3-dihydroxybutane (BMDB), Bis-maleimidohexane (BMH), Bis-maleimidoethane (BMOE), Sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (Sulfo-SMCC), Sulfosuccinimidyl (4-iodo m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N-(γ-maleimidobutryloxy)sulfosuccinimide ester (sulfo-GMBS or sGMBS), N-(ε-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS), N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

[0307] Heterobifunctional crosslinkers are bifunctional crosslinkers with two different reactive groups. Heterobifunctional crosslinkers containing both amine-reactive N-hydroxysuccinimide groups (NHS groups) and carbonyl-reactive hydrazine groups can also be used to conjugate the cytotoxic compounds described herein with cell-binding agents (e.g., antibodies). Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazid terephthalate hydrochloride (SHTH), and succinimidyl hydrazinium nicotinic acid hydrochloride (SHNH). Conjugates with acid-labile linkages can also be prepared using the hydrazine-containing benzodiazepine derivatives of the present disclosure. Examples of bifunctional crosslinkers that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

[0308] Bifunctional crosslinkers that allow for the attachment of cell-binding agents to cytotoxic compounds via disulfide bonds are known in the art and include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB or sSPDB), which introduce a dithiopyridyl group. Other bifunctional crosslinkers that can be used to introduce disulfide groups are known in the art and are disclosed in U.S. Patent Nos. 6,913,748, 6,716,821, and U.S. Patent Publications 2009 / 0274713 and 2010 / 0129314, each of which is incorporated herein by reference in its entirety. Alternatively, crosslinkers such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetylsuccinic anhydride that introduce thiol groups can also be used.

[0309] C. Cytotoxic Agents In some embodiments, provided herein are cytotoxic agents that can be used to make the immunoconjugates of the present disclosure. The cytotoxic agents used in the immunoconjugates provided herein can be any compound that causes or induces cell death or reduces cell viability in any way, including, for example, maytansinoids and maytansinoid analogs, benzodiazepines, taxoids, CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes such as calicheamicin, dolastatins and dolastatin derivatives, including auristatins, tomaymycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, and the like. Examples of cytotoxic agents include maytansinoids, melphalan, mitomycin C, chlorambucil, and morpholinodoxorubicin. In certain embodiments, the cytotoxic agents are maytansinoids and maytansinoid analogs.

[0310] Examples of suitable maytansinoids include esters of maytansinol and maytansinol analogues, including any drug that inhibits microtubule formation and is highly toxic to mammalian cells, such as maytansinol and maytansinol analogues.

[0311] Exemplary cytotoxic agents have been previously described in WO2018 / 160539A1 and WO2011 / 106528, each of which is incorporated by reference in its entirety.

[0312] The immunoconjugates provided herein have the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein: L2 ’ is represented by the following structural formula: [ka] During the ceremony, R x , R y , R x’ , and R y’ is, for each occurrence, independently H, -OH, halogen, -O-(C 1-4 alkyl), -SO3H, -NR 40 R 41 R 42 + or optionally -OH, halogen, -SO3H, or -NR 40 R 41 R 42 + C replaced by 1-4 alkyl, where R 40 , R 41 , and R 42 are each independently H or C 1-4 is alkyl; l and k each independently represent an integer of 1 to 10; J CB ’ is -C(=O)OH or -COE, where -COE is a reactive ester; A is an amino acid or a peptide containing 2 to 20 amino acids; R 1 and R 2 are each independently H or C 1-3 is alkyl; L1 is expressed by the following formula: -CR 3 R 4 -(CH2) 1-8 -C(=O)-; In the formula, R 3 and R 4 are each independently H or Me, and the -C(=O)- moiety of L1 is connected to D; D is expressed by the following formula: [ka] q is an integer from 1 to 20. In some embodiments, q is an integer from 1 to 10. In some embodiments, q is an integer from 2 to 5. In some embodiments, q is an integer from 3 to 4.

[0313] In some embodiments, the maytansinoid of the present invention has the following formula: [ka] or a pharma- ceutically acceptable salt thereof, A' is an amino acid or a peptide containing 2 to 20 amino acids (i.e., A-NH2); R 1 and R 2 are each independently H or C 1-3 is alkyl; L1 is -CR 3 R 4 -(CH2) 1-8 -C(=O)-; R 3 and R 4 are each independently H or Me; D is expressed by the following formula: [ka] q is an integer from 1 to 20. In some embodiments, q is an integer from 1 to 10. In some embodiments, q is an integer from 2 to 5. In some embodiments, q is an integer from 3 to 4.

[0314] In some embodiments, the maytansinoid of the present invention has the following formula: [ka] or a pharma- ceutically acceptable salt thereof, R x’ and R y’ is, for each occurrence, independently H, -OH, halogen, -O-(C 1-4 alkyl), -SO3H, -NR 40 R 41 R42 + or optionally -OH, halogen, SO3H, or NR 40 R 41 R 42 + C replaced by 1-4 alkyl, where R 40 , R 41 , and R 42 are each independently H or C 1-4 is alkyl; k is an integer from 1 to 10. A is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1-3 is alkyl; L1 is -CR 3 R 4 -(CH2) 1-8 -C(=O)-; R 3 and R 4 are each independently H or Me; D is expressed as follows: [ka] q is an integer from 1 to 20. In some embodiments, q is an integer from 1 to 10. In some embodiments, q is an integer from 2 to 5. In some embodiments, q is an integer from 3 to 4.

[0315] In some embodiments, for a maytansinoid compound of formula (II), (III), or (IV), the variables are as described in the first embodiment, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh specific embodiment of the first embodiment.

[0316] In certain embodiments, the maytansinoid compound has the formula: [ka]

[0317] Additional examples of suitable maytansinol esters include those with modified aromatic rings and those with modifications at other positions. Such suitable maytansinoids are described in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; Nos. 4,450,254; 4,322,348; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497; and 7,473,796. Additionally, some descriptions for producing such antibody-maytansinoid conjugates are provided in U.S. Patent Nos. 6,333,410, 6,441,163, 6,716,821, and 7,368,565, each of which is incorporated herein by reference in its entirety.

[0318] In some embodiments, the immunoconjugate comprises 2’ -Deacetyl-N 2’ -(3-mercapto-1-oxopropyl)-maytansine (DM1), N 2’ -Deacetyl-N- 2’ (4-mercapto-1-oxopentyl)-maytansine (called DM3), N 2’ -Deacetyl-N 2’ -(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4), both of which have been previously described in PCT Application Publication No. WO2011 / 106528A1 and U.S. Patent No. 8,557,966 B2, each of which is incorporated by reference herein in its entirety.

[0319] D. Drug Conjugates Immunoconjugates comprising a biparatopic FRα-binding antibody or antigen-binding fragment thereof covalently linked to a cytotoxic agent (e.g., a maytansinoid) described herein can be prepared according to any suitable method known in the art.

[0320] In certain embodiments, the immunoconjugate of the first embodiment can be prepared by a first method comprising reacting a biparatopic FRα-binding antibody or antigen-binding fragment thereof with a maytansinoid compound of formula (II) described in the second embodiment.

[0321] In certain embodiments, the immunoconjugate of the first embodiment can be prepared by a second method comprising the steps of: (a) reacting a maytansinoid compound of Formula (III) or (IV) with a linker compound described herein to form a cytotoxic agent-maytansinoid compound (e.g., a compound of Formula (II)) having an amine-reactive group or a thiol-reactive group attached thereto, which can be covalently attached to a biparatopic FRα-binding antibody or antigen-binding fragment thereof; and (b) reacting a biparatopic FRα-binding antibody or antigen-binding fragment thereof with a maytansinoid-linker compound to form an immunoconjugate.

[0322] In certain embodiments, the immunoconjugate of the first embodiment can be prepared by a third method comprising the steps of: (a) reacting a biparatopic FRα-binding antibody, or antigen-binding fragment thereof, with a linker compound described herein to form an amine-reactive or thiol-reactive linked modified biparatopic FRα-binding antibody, or antigen-binding fragment thereof, which can be covalently attached to a maytansinoid compound of formula (III) or (IV); and (b) reacting the modified biparatopic FRα-binding antibody or antigen-binding fragment thereof with a maytansinoid compound of formula (III) or (IV) to form an immunoconjugate.

[0323] In certain embodiments, for the second, third, or fourth method described above, the linker compound is represented by any one of formulas (a1L)-(a10L), or a pharma- ceutically acceptable salt thereof: [ka] In the formula, X is a halogen; JD-SH, or -SSRd; R d is phenyl, nitrophenyl, dinitrophenyl, carboxynitrophenyl, pyridyl, or nitropyridyl; R g is alkyl; and U is -H or SO3H.

[0324] In one embodiment, the linker compound is GMBS or sulfo-GMBS (or sGMBS) represented by formula (a9L) or a pharma- ceutically acceptable salt thereof, where U is -H or SO3H.

[0325] In certain embodiments, the immunoconjugate of the invention is represented by the formula: [ka] The immunoconjugate can be prepared by the second, third, or fourth method described above, wherein the linker compound is GMBS or sulfo-GMBS represented by formula (a9L) or a pharma- ceutically acceptable salt thereof, where U is -H or SO3H. The maytansinoid compound is represented by formula (D-1) described above. In a more specific embodiment, the immunoconjugate of formula (I-1) is prepared by reacting a maytansinoid compound of formula (D-1) with the linker compound GMBS or sulfo-GMBS to form a maytansinoid-linker compound, and subsequently reacting a biparatopic FRα-binding antibody or antigen-binding fragment thereof with the maytansinoid-linker compound. In an even more specific embodiment, the maytansinoid linker compound is not purified prior to reacting with the biparatopic FRα-binding antibody or antigen-binding fragment thereof.

[0326] In another specific embodiment, the immunoconjugate is represented by the formula: [ka] The immunoconjugate can be prepared by the second, third, or fourth method described above, wherein the linker compound is GMBS or sulfo-GMBS represented by formula (a9L) or a pharma- ceutically acceptable salt, where U is -H or SO3H. The maytansinoid compound is represented by formula (D-2) described above. In a more specific embodiment, the immunoconjugate of formula (I-2) is prepared by reacting a maytansinoid compound of formula (D-2) with the linker compound GMBS or sulfo-GMBS to form a maytansinoid-linker compound, and subsequently reacting a biparatopic FRα-binding antibody or antigen-binding fragment thereof with the maytansinoid-linker compound. In an even more specific embodiment, the maytansinoid linker compound is not purified prior to reacting with the biparatopic FRα-binding antibody or antigen-binding fragment thereof.

[02] In another specific embodiment, the immunoconjugate is represented by the formula: [ka] The immunoconjugate is prepared by reacting a biparatopic FRα-binding antibody or antigen-binding fragment thereof with a maytansinoid compound of formula (D-3) above, according to the first method described above.

[0327] In another specific embodiment, the immunoconjugate is represented by the formula: [ka] The immunoconjugate is prepared by reacting a biparatopic FRα-binding antibody or antigen-binding fragment thereof with a maytansinoid compound of formula (D-4) above, according to the first method described above.

[0328] In another specific embodiment, the immunoconjugate is represented by the formula: [ka] The immunoconjugate is prepared by reacting an anti-FRα antibody or antigen-binding fragment thereof with a maytansinoid compound of formula (D-5) above, according to the first method described above.

[0329] In another specific embodiment, the immunoconjugate is represented by the formula: [ka] The immunoconjugate is prepared by reacting a biparatopic FRα-binding antibody or antigen-binding fragment thereof with a maytansinoid compound of formula (D-6) above, according to the first method described above.

[0330] In some embodiments, the immunoconjugates represented by Formulas I-3 to I-6 disclosed above are prepared according to the methods described in U.S. Provisional Application No. 62 / 821,707, filed March 21, 2019, and related U.S. application Ser. No. 16 / 825,127.

[0331] In some embodiments, the immunoconjugates prepared by any of the methods described above are subjected to a purification step. In this regard, the immunoconjugates can be purified from other components of the mixture using tangential flow filtration (TFF), non-adsorptive chromatography, adsorptive filtration, selective precipitation, or any other suitable purification process, as well as combinations thereof.

[0332] In some embodiments, the immunoconjugate is purified using a single purification step (e.g., TFF). Preferably, the conjugate is purified using a single purification step (e.g., TFF) and exchanged into a suitable formulation. In other embodiments of the present invention, the immunoconjugate is purified using two sequential purification steps. For example, the immunoconjugate can be first purified by selective precipitation, adsorptive filtration, absorbent chromatography, or non-absorbent chromatography, followed by TFF purification. Those skilled in the art will understand that purification of the immunoconjugate allows for the isolation of a stable conjugate that includes a cell-binding agent chemically linked to a cytotoxic agent.

[0333] Any suitable TFF system can be utilized for purification, including Pellicon-type systems (Millipore, Billerica, Mass.), Sartocon cassette systems (Sartorius AG, Edgewood, NY), and Centrasette-type systems (Pall Corp., East Hills, NY).

[0334] Any suitable adsorptive chromatographic resin can be utilized for purification. Preferred adsorptive chromatographic resins include hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye-ligand chromatography, affinity chromatography, reversed phase chromatography, and combinations thereof. Examples of suitable hydroxyapatite resins include ceramic hydroxyapatite (CHT type I and type II, Bio-Rad Laboratories, Hercules, Calif.), HA Ultrogel hydroxyapatite (Pall Corp., East Hills, NY), and ceramic fluoroapatite ( Examples of suitable HCIC resins include CFT Type I and Type II, Bio-Rad Laboratories, Hercules, Calif. An example of a suitable HCIC resin is MEP Hypercel resin (Pall Corp., East Hills, NY). Examples of suitable HIC resins include Butyl Sepharose, Hexyl Sepharose, Phenyl Sepharose, and Octyl Sepharose resins (all from GE Healthcare, Piscataway, NJ), as well as MacroPrep Methyl and MacroPrep t-Butyl resins (Biorad Laboratories, Hercules, Calif.). Examples of suitable ion exchange resins include SP-Sepharose, CM-Sepharose, and Q-Sepharose resins (all from GE Healthcare, Piscataway, NJ), and Unosphere S resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable mixed-mode ion exchangers include Bakerbond ABx resins (JT Baker, Phillipsburg NJ). Examples of suitable IMAC resins include chelating Sepharose resins (GE Examples of suitable dye-ligand resins include Blue Sepharose resin (GE Healthcare, Piscataway, NJ) and Affigel Blue resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable affinity resins include Protein A Sepharose resin (e.g., MabSelect, GE Healthcare, Piscataway, NJ) when the cell-binding agent is an antibody, and lectin affinity resins, e.g., Lentil Lectin Sepharose resin (GE Healthcare, Piscataway, NJ) when the cell-binding agent has a suitable lectin binding site. Alternatively, an antibody specific for the cell-binding agent can be used. Such an antibody can be immobilized, for example, on Sepharose 4 Fast Flow resin (GE Healthcare, Piscataway, NJ). Examples of suitable reverse phase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia, Calif.).

[0335] Any suitable non-adsorbent chromatographic resin can be utilized for purification. Examples of suitable non-adsorbent chromatographic resins include, but are not limited to, SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™ resins (e.g., S-200 and S-300), SUPERDEX™ resins (e.g., SUPERDEX™ 75 and SUPERDEX™ 200), BIO-GEL® resins (e.g., P-6, P-10, P-30, P-60, and P-100), and others known to those skilled in the art.

[0336] VII. Compositions and Kits Provided herein are compositions comprising the immunoconjugates, antibodies, or antigen-binding fragments thereof described herein, having the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.

[0337] Pharmaceutical compositions can be formulated for specific administration routes to subjects.For example, pharmaceutical compositions can be formulated for parenteral, for example, intravenous administration.Compositions used for in vivo administration can be sterilized.This can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0338] In one embodiment, the pharmaceutical compositions described herein are for use as medicines. The pharmaceutical compositions described herein may be useful for treating conditions such as cancer. Examples of cancers that can be treated as described herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include fallopian tube cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, small intestine cancer, endometrial or uterine carcinoma, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. The cancer may be a cancer that expresses FRα.

[0339] The pharmaceutical compositions provided herein can include an immunoconjugate, and the pharmaceutical composition (the immunoconjugate in the pharmaceutical composition) can have an average of 1-20 drugs per biparatopic antibody or antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition includes an average of 1-10 drugs per biparatopic antibody or antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition includes an average of 2-5 drugs per biparatopic antibody or antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition includes an average of 3-4 drugs per biparatopic antibody or antigen-binding fragment thereof.

[0340] VIII. METHODS AND USES The biparatopic anti-FRα antibodies, antigen-binding fragments thereof, and immunoconjugates of the present disclosure are useful for a variety of applications, including, but not limited to, therapeutic treatment methods, such as the treatment of cancer. In certain embodiments, the agents are useful for inhibiting tumor growth and / or reducing tumor volume. The method of use may be an in vitro or in vivo method.

[0341] The present disclosure provides a method of treating cancer, comprising administering a therapeutically effective amount of a biparatopic anti-FRα antibody, its antigen-binding fragment, or immunoconjugate to a subject (e.g., a subject in need of treatment). In certain embodiments, the cancer is a cancer including, but not limited to, fallopian tube cancer, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple-negative breast cancer (TNBC)), colon cancer, colorectal cancer, small intestine cancer, endometrial or uterine carcinoma, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer.

[0342] More specific examples of such cancers include ovarian cancer, epithelial ovarian cancer, ovarian primary peritoneal cancer, or fallopian tube cancer. In certain embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer (EOC). In certain embodiments, the ovarian cancer (e.g., EOC) is platinum-resistant, recurrent, or refractory. In certain embodiments, the cancer is peritoneal cancer. In certain embodiments, the peritoneal cancer is primary peritoneal cancer. In certain embodiments, the cancer is endometrial cancer. In certain embodiments, the endometrial cancer is serous endometrial cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In certain embodiments, the lung cancer is adenocarcinoma or bronchioloalveolar carcinoma. In certain embodiments, the cancer is uterine cancer.

[0343] In certain embodiments, the cancer is platinum refractory. In certain embodiments, the cancer is primary platinum refractory. In certain embodiments, the cancer is platinum sensitive.

[0344] In certain embodiments, the cancer is IMGN853 resistant.

[0345] In certain embodiments, the cancer is a metastatic or aggressive cancer.

[0346] In certain embodiments, the cancer expresses a folate receptor to which the FRα-binding agent or antibody binds. In certain embodiments, the cancer overexpresses human FRα.

[0347] In some embodiments, biparatopic anti-FRα antibodies, antigen-binding fragments thereof, immunoconjugates, or pharmaceutical compositions comprising the same are administered to patients with increased expression levels of FRα as described in U.S. Published Application No. 2012 / 0282175 or International Published Application No. WO2012 / 135675, both of which are incorporated herein by reference in their entirety. Exemplary antibodies, assays, and kits for the detection of FRα are provided in WO2014 / 036495 and WO2015 / 031815, both of which are incorporated herein by reference in their entirety. Thus, in some embodiments, the expression of FRα protein can be measured immunohistochemically (IHC) and can be given a staining intensity score and / or staining uniformity score compared to a control (e.g., a calibration control) that shows a defined score (e.g., a test sample is given an intensity score of 3 if the intensity is equivalent to a calibration control at level 3, and a test sample is given an intensity score of 2 (medium) if the intensity is equivalent to a calibration control at level 2). Staining uniformity that is "heterogeneous" (i.e., at least 25% and less than 75% of cells are stained) instead of "focal" (i.e., greater than 0% and less than 25% of cells are stained), or "uniform" (i.e., at least 75% of cells are stained) also indicates increased FRα expression. Staining intensity and staining uniformity scores can be used alone or in combination (e.g., 2 homo, 2 hetero, 3 homo, 3 hetero, etc.). In another example, increased FRα expression can be determined by detection of at least a 2-fold, at least a 3-fold, or at least a 5-fold increase compared to a control value (e.g., expression levels in tissues or cells from subjects without cancer or subjects with cancer that do not have elevated FRα values). In some embodiments, the staining uniformity score is based on the percent of cells stained.

[0348] In some embodiments, the cancer is a cancer that expresses FRα at 1 or more heteros by IHC. In some embodiments, the cancer is a cancer that expresses FRα at 2 or more heteros by IHC. In some embodiments, the cancer is a cancer that expresses FRα at 3 or more heteros by IHC. In some embodiments, the cancer is a lung cancer that expresses FRα at 2 or more heteros by IHC. In some embodiments, the cancer is a lung cancer that expresses FRα at 3 or more heteros by IHC. In some embodiments, the cancer is an ovarian cancer that expresses FRα at 2 or more heteros by IHC. In some embodiments, the cancer is an ovarian cancer that expresses FRα at 3 or more heteros by IHC. In some embodiments, the cancer is an endometrial cancer that expresses FRα at 2 or more heteros by IHC. In some embodiments, the cancer is an endometrial cancer that expresses FRα at 1 or more heteros by IHC.

[0349] In some embodiments, at least one cell in the sample obtained from the patient has an FRα score of at least 1. In some embodiments, at least one cell in the sample obtained from the patient has an FRα score of at least 2 (moderate). In some embodiments, at least one cell in the sample obtained from the patient has an FRα score of at least 3.

[0350] In some embodiments, at least 25% of cells in a sample obtained from a patient have an FRα IHC score of at least 1. In some embodiments, at least 33% of cells in a sample obtained from a patient have an FRα IHC score of at least 1. In some embodiments, at least 50% of the cells in the sample obtained from the patient have an FRα IHC score of at least 1. In some embodiments, at least 66% of the cells in the sample obtained from the patient have an FRα IHC score of at least 1. In some embodiments, at least 75% of the cells in the sample obtained from the patient have an FRα IHC score of at least 1.

[0351] In some embodiments, at least 25% of the cells in the sample obtained from the patient have an IHC score of at least 2 (moderate) for FRα. In some embodiments, at least 33% of the cells in the sample obtained from the patient have an IHC score of at least 2 (moderate) for FRα. In some embodiments, 25-75% of the cells in the sample obtained from the patient have an IHC score of at least 2 (moderate) for FRα. In some embodiments, at least 50% of the cells in the sample obtained from the patient have an IHC score of at least 2 (moderate) for FRα. In some embodiments, at least 66% of the cells in the sample obtained from the patient have an IHC score of at least 2 (moderate) for FRα. In some embodiments, at least 75% of the cells in the sample obtained from the patient have an IHC score of at least 2 (moderate) for FRα.

[0352] In some embodiments, at least 25% of the cells in the sample obtained from the patient have an IHC score for FRα of at least 3. In some embodiments, at least 33% of the cells in the sample obtained from the patient have an IHC score for FRα of at least 3. In some embodiments, at least 50% of the cells in the sample obtained from the patient have an IHC score for FRα of at least 3. In some embodiments, at least 66% of the cells in the sample obtained from the patient have an IHC score for FRα of at least 3. In some embodiments, at least 75% of the cells in the sample obtained from the patient have an IHC score for FRα of at least 3.

[0353] In some embodiments, FRα expression can be measured by immunohistochemistry and a visual score is given, with FRα positivity referring to 50% or more of the tumor cells having FRα membrane staining visible under a microscope objective of 10x or less. In some embodiments, FRα expression can be measured by immunohistochemistry and a visual score is given, with FRα positivity referring to 66% or more of the tumor cells having FRα membrane staining visible under a microscope objective of 10x or less. In some embodiments, FRα expression can be measured by immunohistochemistry and a visual score is given, with FRα positivity referring to 75% or more of the tumor cells having FRα membrane staining visible under a microscope objective of 10x or less.

[0354] In certain embodiments, the subject is a human.

[0355] The present disclosure further provides a method for inhibiting tumor growth using the biparatopic anti-FRα antibodies, their antigen-binding fragments, and immunoconjugates described herein. In certain embodiments, the method for inhibiting tumor growth comprises contacting a tumor in vitro with the biparatopic anti-FRα antibodies, their antigen-binding fragments, and immunoconjugates provided herein. For example, immortalized cell lines or cancer cell lines expressing FRα are cultured in a medium, to which biparatopic anti-FRα antibodies, their antigen-binding fragments, and immunoconjugates are added to inhibit tumor growth. In some embodiments, tumor cells are isolated from a patient sample, such as, for example, a tissue biopsy, pleural effusion, or blood sample, and cultured in a medium, to which biparatopic anti-FRα antibodies, their antigen-binding fragments, and immunoconjugates are added to inhibit tumor growth.

[0356] In some embodiments, the method of inhibiting tumor growth comprises administering to a tumor or tumor cells in vivo. The present invention includes contacting tumors or tumor cells with biparatopic anti-FRα antibodies, antigen-binding fragments thereof, and immunoconjugates. In certain embodiments, contacting tumors or tumor cells with biparatopic anti-FRα antibodies, antigen-binding fragments thereof, and immunoconjugates is carried out in an animal model. For example, biparatopic anti-FRα antibodies, antigen-binding fragments thereof, and immunoconjugates are administered to xenografts exhibiting one or more tumors grown in immunocompromised mice (e.g., NOD / SCID mice) to inhibit tumor growth. In some embodiments, cancer stem cells are isolated from patient samples, such as tissue biopsies, pleural effusions, or blood samples, and injected into immunocompromised mice, and then biparatopic anti-FRα antibodies, antigen-binding fragments thereof, and immunoconjugates are administered to inhibit tumor cell growth.

[0357] In certain embodiments, the method of inhibiting tumor growth comprises administering a therapeutically effective amount of a biparatopic anti-FRα antibody, antigen-binding fragment thereof, and immunoconjugate to a subject. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or has had a tumor removed.

[0358] Administration may be parenteral, including intravenous.

[0359] The amount of biparatopic immunoconjugate, antibody or antigen-binding fragment thereof, or composition that will be effective in treating a condition will depend on the nature of the disease. The precise dose to be employed in the composition will also depend on the route of administration, and the severity of the disease.

[0360] In some embodiments, provided herein is a biparatopic anti-FRα antibody, its antigen-binding fragment, immunoconjugate, or pharmaceutical composition comprising it for use as a medicament.In some aspects, provided herein is a biparatopic anti-FRα antibody, its antigen-binding fragment, immunoconjugate, or pharmaceutical composition for use in a method for treating cancer.In some aspects, provided herein is a biparatopic anti-FRα antibody, its antigen-binding fragment, immunoconjugate, or pharmaceutical composition for use in a method for treating cancer in a subject, comprising administering to the subject an effective amount of a biparatopic anti-FRα antibody, its antigen-binding fragment, immunoconjugate, or pharmaceutical composition provided herein.

[0361] In one aspect, the biparatopic anti-FRα antibodies, antigen-binding fragments thereof, and immunoconjugates of the present disclosure are useful for detecting the presence of FRα, for example, in biological samples. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In certain embodiments, such tissues include normal tissues and / or cancerous tissues that express FRα at higher levels compared to other tissues. In certain embodiments, FRα overexpression detects the presence of ovarian cancer, lung cancer, brain cancer, breast cancer, uterine cancer, kidney cancer, or pancreatic cancer.

[0362] In certain embodiments, a method for detecting the presence of FRα in a biological sample includes contacting the biological sample with a biparatopic anti-FRα antibody, its antigen-binding fragment, or immunoconjugate under conditions that allow binding of the biparatopic anti-FRα antibody, its antigen-binding fragment, or immunoconjugate, and detecting whether a complex is formed between the anti-FRα antibody, its antigen-binding fragment, or immunoconjugate and FRα.

[0363] In certain embodiments, the biparatopic anti-FRα antibody, antigen-binding fragment thereof, or immunoconjugate is labeled. Labels include directly detectable labels or moieties, such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels, as well as labels that are detectable by, for example, enzymes. These include, but are not limited to, moieties such as enzymes or ligands that are indirectly detected through elementary reactions or molecular interactions.

[0364]

[0023] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples which detail the preparation of particular antibodies of the present disclosure and methods for using the antibodies of the present disclosure. It will be apparent to those skilled in the art that numerous modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure. EXAMPLES

[0365] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in view thereof will be suggested to those skilled in the art and are within the spirit and scope of the present application.

[0366] Example 1. Generation of biparatopic antibodies Bispecific antibody expression As described above, a panel of mouse anti-FRα antibodies was generated by standard hybridoma technology and humanized using resurfacing techniques (see, e.g., WO2011 / 106528A1). Antibodies were classified into two bins depending on whether they competed with Mov19 for binding (Bin 1) or not (Bin 2; FRα antibody A, FRα antibody B, FRα antibody C, and FR57) using a FACS competition assay. Briefly, 1.5x10 -9 M9346A biotinylated antibody, typically 5 x 10 -8 M~5×10 -11The huM9346A antibody was mixed with FRα antibody A, FRα antibody B, FRα antibody C, and FR57 at concentrations ranging from 100 to 1000 M. As a control for complete binding competition, non-biotinylated M9346A antibody was used. The mixture was added to a 96-well plate containing 20,000 FRα-positive KB cells per well, and the plate was incubated on ice for 1 h. The cells were then washed with cold phosphate-buffered saline / 1% bovine serum albumin, and bound huM9346A-biotin was detected with streptavidin-PE reagent. The samples were analyzed using a FACSCalibur flow cytometer. As shown in Figure 1, only the control antibody M9346A competed with huM9346A-biotin for binding, and none of the four FR-antibodies analyzed interfered with the binding of huM9346A-biotin.

[0367] Using the variable regions (VH and VL) of Bin 1 and Bin 2 antibodies, several biparatopic molecules were constructed using two different formats: Morrison format and asymmetric Fc. Briefly, for the Morrison format-based molecules, sequences corresponding to the VH and VL regions of either Bin 1 or Bin 2 antibodies were connected with a (G4S)4 linker to generate single chain fragments (scFv), which were then fused to the C-terminus or N-terminus of the IgG1 heavy chain of Bin 2 or Bin 1 using a (G4S)3 linker. Asymmetric Fc-based biparatopic molecules were made with FR57scFv and Mov19 Fab using knobs-in-holes technology (Protein Eng.1996 Jul;9(7):617-21.'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Ridgway JB, Presta LC, Carter, P). Briefly, FR1-57scFv was fused to an engineered Fc region containing C220S (mutating the unpaired cysteine ​​to serine) and knob mutations (T366W), and Mov19 Fab region was fused to an engineered Fc containing hole mutations (T366S, L368A, and Y407V). All numbering is based on the EU system unless otherwise stated. Figure 2 shows the various antibody formats evaluated in the following experiments.

[0368] The sequences of the specific constructed molecules are shown in Tables 6-7. The genes were codon-optimized, synthesized, and cloned into plasmids using standard molecular biology techniques. The ratio of light chain to heavy chain plasmids for transfection was maintained at 1:3 for Morrison-based molecules and 9:3:1 for asymmetric Fc-based molecules (Mov19LC:Mov19 HC-Hole:FR57scFv-Knob). As shown in Figure 3, several heavy and light chain plasmid transfection ratios were explored to generate asymmetric Fc-based molecules, and a ratio of 9:3:1 showed minimal homodimerization.

[0369] All bispecific antibody molecules were transiently produced in 293T. Briefly, for 293T transfections, expression constructs were transiently produced in suspension-adapted HEK-293T cells using PEI as the transfection reagent in shake flasks. PEI transient transfections were performed as previously described (Durocher et al., Nucleic Acids Res. 30(2):E9 (2002)), except that HEK-293T cells were grown in Freestyle 293 and culture medium was left undiluted after addition of PEI-DNA complexes. Transfections were incubated for 1 week and harvested.

[0370] Antibody purification The filtered supernatant was purified using a scheme consisting essentially of two chromatographic steps: Protein A affinity and ceramic hydroxyapatite (CHT). Briefly, the filtered supernatant was loaded onto a Protein A column pre-equilibrated with 1x PBS (pH 7.3 ± 0.1). To reduce non-specific host cell proteins, the column was washed with 1x PBS (pH 7.3 ± 0.1). Bound antibodies were eluted using 25 mM acetic acid containing 50 mM sodium chloride (pH 3.2) and immediately neutralized to pH 7.0 ± 0.2 with 1 M Tris-base. The neutralized pool was diluted 1:10 in CHT binding buffer (15 mM sodium phosphate, pH 7.0 ± 0.1) and loaded onto a type II CHT column (40 μm particle size) pre-equilibrated with CHT binding buffer. Bound protein was eluted using a linear gradient (15 mM to 160 mM sodium phosphate in 10 column volumes) and fractions of interest (high percent monomer by size exclusion chromatography, SEC) were pooled, dialyzed against 1x PBS (pH 7.3 ± 0.1) and filter sterilized. The final antibody concentration was determined by absorbance at 280 nm and 1.44 mL mg -1 cm -1 was determined by measuring the extinction coefficient of

[0371] All purification experiments were performed on an AKTA purification system equipped with in-line UV, conductivity, and pH probes. SEC analysis was performed using an Agilent HPLC 1100 system by injecting 40 μg of sample onto a TSKgel G3000SWXL column (7.8 × 300 mm) also equipped with an in-line guard column (6.0 × 40 mm) to extend column life. The mobile phase contained 50 mM sodium phosphate buffer and 400 mM sodium perchlorate, the flow rate was 1.0 mL / min, and elution was isocratic.

[0372] Example 2. Effect of biparatopic antibody format on antibody production, stability, and functional activity Binding characteristics of parental (Bin 1 and Bin 2) antibodies Table 10 summarizes the kinetic parameters of binding of Bin 1 and Bin 2 antibodies to recombinant FRα antigen. KD values ​​were obtained via biolayer interferometry performed on an Octet96 system (Fortebio) essentially following the manufacturer's recommended procedure. Briefly, anti-human or anti-mouse-Fc sensors were pre-soaked in 1× Kinetic Buffer (Fortebio) for 10 min and incubated with 5 μg / mL of either Bin 1 or Bin 2 IgG for 5 min. The sensors were then incubated in 1× Kinetic Buffer (Fortebio) for 10 min and incubated with 5 μg / mL of either Bin 1 or Bin 2 IgG for 5 min. A baseline was determined by transferring to kinetic buffer for 5 min, followed by binding (10 min) with serial dilutions of antigen and dissociation (10 min) with 1x kinetic buffer. Raw data were collected, processed, and fitted to a simple 1:1 binding model using Fortebio analysis software to determine the kinetic parameters Kon and Koff. [Table 10]

[0373] Stability of biparatopic antibodies Biparatopic antibodies were created by combining Mov19 antibody with an antibody recognizing another non-overlapping epitope. In particular, IgGs based on the Morrison format were generated by fusing scFv from one of the Bin IgGs to the C-terminus or N-terminus of an IgG from another Bin. Table 11 shows all the combinations investigated. The scFv fused to the C-terminus was in the VH-VL orientation, and the one fused to the N-terminus was in the VL-VH orientation. Mov19 was explored only as a C-terminal scFv in both the VH-VL and VL-VH orientations, with or without the Brinkmann VH44-VL100 disulfide stabilizing mutation (PNAS 1993 August;90(16):7538-754.A recombinant immunotoxin containing a disulfide-stabilized Fv fragment.U Brinkmann,Y Reiter,SH Jung,B Lee,and I Pastan). [Table 11]

[0374] As shown in Table 9, a significant number of biparatopic molecules based on the Morrison format had low percent monomer after Protein A affinity purification. Because scalability or manufacturability may be a challenge for constructs with low percent monomer or titer, eight constructs (indicated with an asterisk in Table 9) that showed higher titers and monomer % above 70 were selected for further evaluation. These eight constructs were purified using ceramic hydroxyapatite chromatography for 9 h. They were further refined to a purity of >5% and further characterized. To account for the effect of the overall molecular conformation or potential structural changes of the scFv arms on the binding of the biparatopic arms, the binding efficiency of each arm of the eight Morrison constructs was assayed in a competitive FACS assay. Briefly, FRα-positive T47D cells were incubated with 0.8 nM of Morrison antibody mixed with the corresponding mouse parental antibody, usually in the concentration range of 50 nM to 0.2 nM. After 2 h of incubation on ice, cells were washed from unbound antibody and bound Morrison antibody was detected with a secondary anti-human FITC-labeled antibody. The decrease in binding of the Morrison antibody with increasing concentrations of the parental antibody indicated the effect on the binding of the second set of arms. As shown in Figures 4A-4H and Table 12, five out of eight of the Morrison antibodies were either completely inactive or had partially affected arms. Of the three Morrison antibodies with both sets of functional arms, two antibodies (FRα-Antibody-A-scFv2-Mov19-IgG1 and FRα-Antibody-C-scFv2-Mov19-IgG1) showed stability problems. Based on these data, FR57scFv2-Mov19-IgG1 ("tetravalent") was selected for further evaluation. [Table 12]

[0375] In a separate experiment, two biparatopic molecules based on the asymmetric Fc format (FR57scFv2-knob-Mov19-hole and FR57scFv3wt-knob-Mov19-hole) were also expressed. FR57scFv2-knob-Mov19-hole ("KIH") showed higher monomer % and titer and was selected for further evaluation. As shown in Figure 5, this molecule migrates as a single band (corresponding to approximately 125 kDa) in gel electrophoresis under non-reducing conditions and resolves into three bands (one corresponding to the light chain (approximately 25 kDa) and two corresponding to the heavy chains (FR57scFv-Fc-knob and Mov19-HC-hole) of similar size (approximately 50 kDa, respectively) under reducing conditions). These results suggest that FR57scFv2-knob-Mov19-hole assembles correctly in cell culture and does not degrade during purification.

[0376] The stability of the FR57scFv2-knob-Mov19-hole molecule was then assessed by heating the molecule at 40° C. (concentration: 10 mg / mL in 1× PBS) for 2 weeks and performing SEC analysis essentially using the procedure described in Example 1. Figure 6 shows the SEC overlay of the samples at day 0 and day 14. Notably, no aggregation or cleavage was observed, suggesting good stability of the molecule.

[0377] Antibody Binding and Processing The effect of antibody format on antibody binding and processing was evaluated in vitro using 3[H] antibodies. Briefly, FRα-positive KB cells were exposed to saturating concentrations of parental, KIH biparatopic, or Morrison antibodies for 30 min at 37°C, washed with PBS to remove unbound antibody, resuspended in fresh medium, and incubated for 22 h at 37°C in a humidified CO2 atmosphere at 6%. Following acetone extraction and liquid scintillation counting, the amount of protein-free radioactivity (processed antibody) and protein-associated radioactivity (unprocessed antibody) were assessed, and the data were used to calculate antibody binding sites (ABC) per cell, % processed antibody, and amount of processed antibody. Preliminary experiments showed that the processing of parental antibody M9346A and huFR57 was similar. Therefore, only one parent antibody (M9346A) was used in further experiments.

[0378] Both biparatopic formats (KIH and tetravalent) showed increased amounts of processed antibody compared to the parental antibody (Figures 7C and 7D). Interestingly, the mechanisms of improved delivery / processing of the two biparatopic formats were different. The KIH biparatopic antibody had higher ABC and similar internalization efficiency than the monospecific parental antibody (Figures 7A-7D), while the Morrison tetravalent antibody showed improved internalization efficiency and ABC values ​​comparable to the parental antibody (Figures 7C and 7D). The amount of degraded antibody for the two biparatopic formats was similar (Figures 7E and 7F).

[0379] Example 3. Preparation of biparatopic FRα-targeted immunoconjugates Preparation of FR57scfv-huMov19-sulfo-SPDB-DM4 conjugate The molar concentrations of FR57scfv-huMov19, sulfo-SPDB, and DM4 were calculated according to Beer's law using the UV / Vis absorbance values ​​and extinction coefficients at 280, 343, and 412 nm, respectively. Linker concentrations were determined by reacting the linker with 25 mM DTT in 50 mM potassium phosphate buffer, 50 mM sodium chloride, and 2 mM EDTA, pH 7.5, and measuring the thiopyridine release at 343 nm. Drug concentrations were determined by reacting DM4 with 10 mM DTNB [5,5-dithiobis-(2-nitrobenzoic acid)] in 50 mM potassium phosphate buffer, 50 mM sodium chloride, 2 mM EDTA, pH 7.5, and measuring the absorbance at 412 nm.

[0380] Prior to antibody conjugation, a sulfo-SPDB-DM4 in situ mixture was prepared by reacting 1.5 mM sulfo-SPDB with 1.95 mM DM4 and 70% organic [(NN-dimethylacetamide, DMA, SAFC)] in 30% aqueous [15 mM potassium phosphate pH 7.6] for 90 min at 25 °C. During the conjugation reaction, a 2.5 mg / mL antibody solution was reacted with an 8-8.5-fold molar excess of sulfo-SPDB-DM4 over antibody in 15 mM potassium phosphate pH 7.6 containing 10% DMA (v / v) for 15-20 h at 25 °C. The reaction was purified using a Sephadex 25 desalting column on an AKTA into formulation buffer of 10 mM acetate, 9% sucrose, 0.01% Tween 20, pH 5.0 and filtered through a syringe filter with a 0.22 μm PVDF membrane.

[0381] The molar ratio of conjugated DM4 to antibody (DAR) and the percentage of unconjugated maytansinoid species were determined as follows: The purified conjugate was found to be 3.4 moles DM4 / mole antibody by UV-Vis, 99.8% monomer by SEC, and <2% free drug by HPLC Hisep column analysis.

[0382] DAR was determined by measuring the UV / Vis absorbance at 252 and 280 nm and the The Ab concentration and DM4 concentration were determined by calculating the Ab concentration and DM4 concentration using a binomial equation to account for the contributions. The amount of unbound maytansinoid present in the final FR57scfv-huMov19-sulfo-SPDB-DM4 conjugate samples was calculated from the peak areas observed in samples analyzed on a HISEP column (25 cm×4.6 mm, 5 μm). The percent free maytansinoid (FM%) present in the conjugate samples was calculated using the following formula: Free maytansinoid %=(reversed phase PA 252 with DM4) / (reversed phase PA 252 with DM4+flow-through PA 252 with DM4)×100%.

[0383] Preparation of knob-in-hole (KIH)-FR57scfv-huMov19-sulfo-SPDB-DM4 conjugate Prior to antibody conjugation, a sulfo-SPDB-DM4 in situ mixture was prepared by reacting 1.5 mM sulfo-SPDB with 1.95 mM DM4 and 70% organic [(NN-dimethylacetamide, DMA, SAFC)] in 30% aqueous [15 mM potassium phosphate pH 7.6] for 90 min at 25 °C. During the conjugation reaction, a 3.0 mg / mL antibody solution was reacted with a 10-fold molar excess of sulfo-SPDB-DM4 over antibody in 15 mM potassium phosphate pH 7.6 containing 11% DMA (v / v) for 15-20 h at 25 °C. The reaction was purified twice using a NAP desalting column into formulation buffer of 10 mM succinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5 and filtered through a syringe filter with a 0.22 μm PVDF membrane. The purified conjugate was found to be 2.9 moles DM4 / mole antibody by UV-Vis, 90.6% monomer by SEC, and less than 1% free drug by HPLC Hisep column analysis.

[0384] Preparation of FR57scfv-huMov19-DM21 conjugate The molar concentrations of FR57scfv-huMov19, sulfo-GMBS, and DM21 were calculated according to Beer's law using the UV / Vis absorbance values ​​and extinction coefficients at 280, 343, and 412 nm, respectively. Linker concentrations were determined by reacting the linker with 50 mM potassium phosphate buffer, 50 mM sodium chloride, 2 mM EDTA, pH 7.5, 25 mM DTT, and measuring thiopyridine release at 343 nm. Drug concentrations were determined by reacting DM21 with 10 mM DTNB [5,5-dithiobis-(2-nitrobenzoic acid)] in 50 mM potassium phosphate buffer, 50 mM sodium chloride, 2 mM EDTA, pH 7.5, and measuring absorbance at 412 nm.

[0385] Prior to conjugation, an in situ mixture of sulfo-GMBS-DM21 was prepared by reacting 1.5 mM sulfo-GMBS with 1.95 mM DM21 in 60 / 40 (v / v) DMA and succinate buffer pH 5.0, respectively. Conjugation was performed with a 6.5 linker excess of sulfo-GMBS-DM21 to 2.5 mg / mL of antibody in 60 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) pH 8.0, containing 10% DMA (v / v). After incubation at 25 °C for 20-22 h, the reaction was purified using a NAP desalting column into 10 mM succinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5, and filtered through a 0.22 μm PVDF membrane filter.

[0386] The molar ratio of conjugated DM21 to antibody (DAR) and the percentage of unconjugated maytansinoid species were determined as follows: The purified conjugate was found to be 3.7 moles DM21 / mole antibody by UV-Vis, 98% monomer by SEC, and <2% free drug by HPLC Hisep column analysis.

[0387] The molar ratio of conjugated DM21 to antibody (DAR) was determined by measuring the UV / Vis absorbance at 252 and 280 nm and calculating the Ab and DM21 concentrations using a binomial equation accounting for the contribution of each component. The amount of unbound maytansinoid present in the final FR57scfv-huMov19-GMBS-DM21L conjugate samples was calculated from the peak area observed in samples analyzed on a HISEP column (25 cm x 4.6 mm, 5 μm). The percent free maytansinoid (FM%) present in the conjugate samples was calculated using the following formula: Free maytansinoid % = (Reversed phase PA 252 by DM21) / (Reversed phase PA 252 by DM21 + Flow-through PA with DM21 252) × 100%.

[0388] Preparation of knob-in-hole (KIH)-FR57scfv-huMov19-GMBS-DM21L conjugate The first batch of KIH-FR57scfv-huMov19-GMBS-DM21L was prepared using lower concentrations of drug and linker in the in situ mixture and lower concentrations of antibody during the conjugation process. Briefly, an in situ mixture of sulfo-GMBS-DM21 was prepared by reacting 1.5 mM sulfo-GMBS with 1.95 mM DM21 in 60 / 40 (v / v) DMA and succinate buffer pH 5.0, respectively, prior to conjugation. Conjugation was performed with a 7.5 linker excess of sulfo-GMBS-DM21 to 2.5 mg / mL antibody in 60 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) pH 8.0 with 10% DMA (v / v). After incubation at 25 °C for 20-22 h, the reaction was purified twice into 10 mM succinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5 using a NAP desalting column and filtered through a 0.22 μm PVDF membrane filter. The purified conjugate was found to be 3.1 moles DM21 / mole antibody by UV-Vis, 99.1% monomer by SEC, and less than 2% free drug by HPLC Hisep column analysis.

[0389] Subsequent batches of KIH-FR57scfv-huMov19-GMBS-DM21L for use in pharmacokinetic and efficacy studies were prepared using higher concentrations of drug and linker in the in situ mixture and higher concentrations of antibody during the conjugation process. Briefly, prior to conjugation, an in situ mixture of sulfo-GMBS-DM21 was prepared by reacting 3 mM sulfo-GMBS with 3.9 mM DM21 in 60 / 40 (v / v) DMA and succinate buffer pH 5.0, respectively. Conjugation was performed with a 6.5-7 linker excess of sulfo-GMBS-DM21 to 5.7-6 mg / mL antibody in 60 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) pH 8.0 with 10% DMA (v / v). After incubation at 25 °C for 20-22 h, the reaction was purified into 10 mM succinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5 using a Sephadex-25 desalting column on an AKTA and filtered through a 0.22 μm PVDF membrane filter. The purified conjugate was found to be 3.1 moles DM21 / mole antibody by UV-Vis, 98.7% monomer by SEC, and less than 2% free drug by HPLC Hisep column analysis.

[0390] The composition comprising the KIH-FR57scfv-huMov19-GMBS-DM21L construct with a DAR of 3.5 is referred to as "IMGN151."

[0391] Example 4. Effect of biparatopic antibody format on efficacy of immunoconjugates In vitro cytotoxicity of biparatopic immunoconjugates The effect of biparatopic antibody format on the cytotoxicity of immunoconjugates was evaluated in vitro using KB, Igrov-1, and T47D cells. Sulfo-SPDB-DM4 conjugates of parental, KIH, and Morrison antibodies were prepared according to the methods described in Example 3. Conjugates were diluted in the appropriate medium and incubated at 1 × 103 Cells / well were added to wells of a 96-well flat-bottom plate. The plates were incubated at 37 °C and 6% CO2 for 5 days. Cell viability was determined by WST-8 assay according to the manufacturer's protocol, and IC 50 was generated using a sigmoidal dose-response (variable slope) nonlinear regression curve fit (GraphPad Software Inc). [Table 13]

[0392] Both biparatopic sulfo-SPDB-DM4 conjugates were more active than the parent antibody conjugate against two of the three medium to low FRα expressing cell lines tested (Igrov-1 and T47D). The only cell line equally sensitive to the three conjugates was KB, which has a very high level of target expression. The KIH conjugate showed higher cytotoxic activity than the Morrison format conjugate against the T47D cell line (i.e., the cell with the lowest level of target expression). However, both the KIH and Morrison format conjugates were equally active against the other two cell lines analyzed.

[0393] In vivo antitumor activity of tetravalent biparatopic ADCs in SCID mice bearing OV-90 human ovarian cancer xenografts The effect of the tetravalent biparatopic antibody format on the therapeutic efficacy of the immunoconjugates was evaluated in vivo using an OV-90 xenograft model. Mice were randomized into groups (n=6 per group) according to tumor volume and then dosed 7 days post-inoculation. These groups included a control group that received formulation buffer, tetravalent-s-SPDB-DM4 at 2.5 and 5 mg / kg, and Ms-SPDB-DM4 at 2.5 and 5 mg / kg. All mice received a single intravenous dose of the above-mentioned compounds. The study was terminated 80 days post-inoculation.

[0394] Tumor volumes were measured twice weekly in three dimensions using calipers. Volumes were expressed in mm3 using the formula: volume = 1 / 2 (length x width x height). (Cancer Chemother. Pharmacol. 1989(24):148-154. Determination of subcutaneous tumor size in athymic (nude) mice. MM Tomayko and CP Reynolds). Body weights were measured twice weekly as a rough index of toxicity of the test agents. Activity was assessed according to the Cancer Res.1991 Sept.(51):4845-4852.Experimental Antitumor Activity of Taxotere(RP 56976,NSC 628503), a Taxol Analogue.M Bissery,D Guenard,F Gueritte-Voegelein,et al. It was evaluated to be included.

[0395] The results of the study are shown in Figure 8 and Table 14. The tetravalent-s-SPDB-DM4 conjugate was active at both the 2.5 and 5 mg / kg doses with T / C of 13% and 11%, respectively. At the 2.5 mg / kg dose, TC was 32 days, LCK was 1.25 (active), PR was 2 / 6, and CR was 0 / 6. At the 5 mg / kg dose, TC was 47 days, LCK was 1.84 (active), PR was 2 / 6, CR was 2 / 6, and TFS was 1 / 6. The Ms-SPDB-DM4 conjugate was active at the 2.5 mg / kg dose with T / C of 26% and highly active at the 5 mg / kg dose with T / C of 3%. At the 2.5 mg / kg dose, TC was 25 days, LCK was 0.98 (inactive), and there were no regressions. TC and LCK could not be determined for the 5 mg / kg group due to necrosis at low tumor volume. However, in this group, there were 4 / 6 PR, 3 / 6 CR, and 3 / 6 TFS. There was minimal weight loss of 2-5% in all groups in the study, with the nadir occurring on day 13 post-inoculation. The results of this study indicate that the tetravalent ADC format does not provide improved activity over the parent conjugate. [Table 14]

[0396] In vivo antitumor activity of tetravalent biparatopic ADCs in SCID mice bearing IGROV-1 human ovarian cancer xenografts The effect of the tetravalent biparatopic antibody format on the therapeutic efficacy of the immunoconjugates was evaluated in vivo using an IGROV-1 xenograft model. Mice were randomized into groups by tumor volume (n=8 per group) and then dosed on day 11 post-inoculation. These groups included a control group that received formulation buffer, 100 μg / kg tetravalent-s-SPDB-DM4, and 100 μg / kg Ms-SPDB-DM4. To account for the possibility of underdosing of the tetravalent conjugates due to the difference in molecular weight between the tetravalent and parental antibodies (i.e., a difference of 50 kDa), doses were normalized by payload in this and all future studies. All mice received a single intravenous dose of the compounds mentioned above. The study was terminated on day 81 post-inoculation. Tumor measurements and calculations were determined as described above in the subsection "In vivo antitumor activity of tetravalent biparatopic ADCs in SCID mice bearing OV-90 human ovarian cancer xenografts."

[0397] The results of the study are shown in Figure 9 and Table 15. Both the tetravalent-s-SPDB-DM4 and parental Ms-SPDB-DM4 conjugates were active at 100 μg / kg with T / C of 21% and 15%, respectively. TC and LCK could not be determined for either group due to necrosis in small tumor volumes. The tetravalent conjugates had 2 / 8 PR, 1 / 8 CR, and 0 / 8 TFS, while Ms-SPDB-DM4 had 3 / 8 PR, 1 / 8 CR, and 0 / 8 TFS. There was minimal weight loss in most groups in the study, except for the 100 μg / kg tetravalent ADC, which had a nadir of 8% weight loss on day 14 post-inoculation. Similarly, the results of this study indicate that the tetravalent ADC format does not provide improved activity over the parent conjugates. [Table 15]

[0398] In vivo antitumor activity of KIH biparatopic ADC in SCID mice bearing OV-90 human ovarian cancer xenografts The effect of the KIH biparatopic antibody format on the therapeutic efficacy of the immunoconjugates was evaluated in vivo using an OV-90 xenograft model. Mice were randomized into groups by tumor volume (n=6 per group) and then dosed 7 days after inoculation. These groups included a control group that received formulation buffer, KIH-s-SPDB-DM4 at 40, 20, and 10 μg / kg, and Ms-SPDB-DM4 at 20 μg / kg. All mice received a single intravenous dose of the above-mentioned compounds. The study was terminated 80 days after inoculation. Tumor measurements and calculations were determined as described in the subsection "In vivo antitumor activity of tetravalent biparatopic ADCs in SCID mice bearing OV-90 human ovarian cancer xenografts."

[0399] The results of the study are shown in Figure 10 and Table 16. The KIH-s-SPDB-DM4 conjugate was highly active at 40 μg / kg, active at 20 μg / kg, and inactive at 10 μg / kg with T / C of 6%, 12%, and 83%, respectively. In the 40 μg / kg dose group, TC was 29 days, LCK was 1.84 (active), PR was 3 / 6, CR was 2 / 6, and TFS was 0 / 6. In the 20 μg / kg dose group, TC was 38 days, LCK was 1.32 (active), and there were no regressions. In the 10 μg / kg group, TC was 2 days, LCK was 0.09 (inactive), and there were no regressions. The parent Ms-SPDB-DM4 conjugate was inactive at 20 μg / kg with T / C of 81% and no regressions. Due to necrosis in the small tumor volume, TC and LCK could not be determined in this group. No weight loss was observed in this study. In contrast to studies performed with tetravalent conjugates, KIH-s-SPDB-DM4 showed significantly higher activity than Ms-SPDB-DM4. Based on the results of these studies, the KIH format was selected over the tetravalent format for further evaluation. [Table 16]

[0400] Example 5. In vitro activity of knob-in-hole biparatopic antibodies conjugated to DM21 In vitro cytotoxicity of knob-in-hole biparatopic antibodies conjugated to DM21 In vitro cytotoxicity of KIH biparatopic antibodies conjugated to DM21 The KIH-DM21 activity was evaluated in multiple cell lines following the protocol described in Example 4 ("In vitro cytotoxicity of biparatopic immunoconjugates"). Previous studies showed that the activity of the two parental DM21 conjugates (M-DM21 and huFR57-DM21) was very similar (Figure 11). In all further studies, the activity of KIH-DM21 was compared to the parental M antibody conjugates (Ms-SPDB-DM4 and M-DM21) to evaluate the contribution of the biparatopic format and DM21 payload to the overall cytotoxicity of KIH-DM21.

[0401] KIH-DM21 was significantly more active than the two parental conjugates against three of the five cell lines tested (Igrov-1, T47D, and JHOS-4) (Figures 12A-12E). Furthermore, the two conjugates containing the DM21 linker / payload (KIH-DM21 and M-DM21) were similarly active against Jeg-3 cells, whereas the Ms-SPDB-DM4 conjugate showed lower activity against this cell line. The only cell line showing the highest FRα expression level (KB) was similarly sensitive to all three conjugates. Thus, these results indicate that KIH-DM21 shows increased activity against most of the tested cell lines compared to the parental antibody conjugates (Ms-SPDB-DM4 and M-DM21).

[0402] Furthermore, the binding, internalization, and processing of KIH-DM21 and the parent monospecific antibody were 3In tumor cells with intermediate (JHOS-4) and high (KB) FRα expression, KIH-DM21 boosted antibody binding events and processing by 100% and 170%, respectively.

[0403] Bystander killing activity of knob-in-hole biparatopic antibodies conjugated to DM21 The ability of KIH-DM21 to kill FRα cells in mixed cell cultures was evaluated in vitro using multiple cell lines. Consistent with other in vitro cytotoxicity experiments, the parent antibody conjugates M-DM21 and Ms-SPDB-DM4 were used as controls. Mixed cultures of target-positive cells (KB, Igrov-1, Jeg-3 or T47D) and target-negative cells Namalwa / luc (i.e., Namalwa cells expressing luciferase) were exposed to 0.5 nM of the conjugate. This concentration of the conjugate is not toxic to the target-negative cells when the cells are incubated alone. Various percentages of target-positive cells in the mixed cultures (9%-50%) were then tested. After 5 days of exposure, inhibition of cell proliferation of the target-negative cells in the mixture was determined by One Glo (Promega). [Table 17]

[0404] In all mixed cultures tested, KIH-DM21 showed the highest bystander activity, followed by M-DM21, and Ms-SPDB-DM4 was the least active conjugate, as shown in Figures 13A-13D and Table 17.

[0405] Taken together, these data indicate that the KIH biparatopic format combined with the DM21 linker / payload resulted in improved in vitro efficacy compared to parental antibodies conjugated to DM21 or s-SPDB-DM4.

[0406] Example 6. In vivo efficacy of KIH biparatopic immunoconjugates In vivo antitumor activity of KIH biparatopic ADC in SCID mice bearing OV-90 human ovarian cancer xenografts The in vivo efficacy of KIH-DM21 was evaluated and compared with KIH-s-SPDB-DM4 in an OV-90 xenograft model with low FRα expression (H-score 30). Mice were randomized into groups (n=6 per group) according to tumor volume and then dosed 7 days after inoculation. These groups included a control group administered formulation buffer, KIH-s-SPDB-DM4 at 40, 20, and 10 μg / kg, KIH-DM21 at 40, 20, and 10 μg / kg, M-DM21 at 20 μg / kg, and Ms-SPDB-DM4 at 20 μg / kg. All mice received a single intravenous dose of the above-mentioned compounds. The study was terminated 80 days after inoculation. Tumor measurements and calculations were determined as described in Example 4, subsection "In vivo antitumor activity of tetravalent biparatopic ADCs in SCID mice bearing OV-90 human ovarian cancer xenografts."

[0407] The results of the study are shown in Figures 14A-14B and Table 18. In the 40 μg / kg dose group of KIH-s-SPDB-DM4, TC was 29 days, LCK was 1.84 (active), PR was 3 / 6, CR was 2 / 6, and TFS was 0 / 6. In the 20 μg / kg dose group, TC was 38 days, LCK was 1.32 (active), and there were no regressions. In the 10 μg / kg group, TC was 2 days, LCK was 0.09 (inactive), and there were no regressions. The parent Ms-SPDB-DM4 conjugate was inactive at 20 μg / kg, with a T / C of 81% and no regressions. Due to necrosis at small tumor volumes, TC and LCK could not be determined for this group. The KIH-DM21 conjugate was highly active at all doses, with a T / C of 1% for the 40 μg / kg dose, 7% for the 20 μg / kg dose, and 9% for the 10 μg / kg dose. At the 40 μg / kg dose, TC was 53 days, LCK was 2.49 (active), PR was 5 / 6, CR was 5 / 6, and TFS was 3 / 6. At the 20 μg / kg dose, TC was 42 days, LCK was 1.98 (active), PR was 3 / 6, CR was 2 / 6, and TFS was 1 / 6. At the 10 μg / kg dose, PR was 2 / 6, and CR was 0 / 6. Due to necrosis in the small tumor volume, TC and LCK could not be determined in this group. The M-DM21 conjugate was active at 20 μg / kg with a T / C of 22%, TC at 30 days, LCK of 1.41 (active), and no regressions. No weight loss was observed in this study. In summary, the DM21 conjugate was more active than the s-SPDB-DM4 conjugate in both the parental and KIH biparatopic formats in this study. Furthermore, the biparatopic KIH conjugates were more active than their parental counterparts at a balanced 20 μg / kg dose within the same linker / payload format. Based on these results, KIH-DM21 was selected for evaluation in additional xenograft models. [Table 18]

[0408] In vivo antitumor activity of KIH biparatopic ADC in SCID mice bearing Ishikawa human endometrial adenocarcinoma xenografts The in vivo efficacy of KIH biparatopic antibodies conjugated to DM21 was evaluated in the Ishikawa xenograft model, which has moderate FRα expression (H-score 100).

[0409] Mice were randomized into groups by tumor volume (n=6 per group) and then dosed on day 11 post-inoculation. These groups included a control group that received formulation buffer, KIH-DM21 at 100, 50, and 25 μg / kg, M-DM21 at 100 and 50 μg / kg, and Ms-SPDB-DM4 at 100 μg / kg. All mice received a single intravenous dose of the above-mentioned compounds. The study was terminated on day 90 post-inoculation. Tumor measurements and calculations were determined as described in Example 4, subsection "In vivo antitumor activity of tetravalent biparatopic ADCs in SCID mice bearing OV-90 human ovarian cancer xenografts."

[0410] The results of the study are shown in Figure 15 and Table 19. The KIH-DM21 conjugate was highly active at 100 and 50 μg / kg, but inactive at 25 μg / kg, with T / C of 0%, 9%, and 78%, respectively. At the 100 μg / kg dose, TC was greater than 63 days, LCK greater than 3.33 (high activity), 6 / 6 PR, 5 / 6 CR, and 4 / 6 TFS. At the 50 μg / kg dose, PR was 3 / 6, and 0 / 6 CR. TC and LCK could not be determined in this group due to necrosis in small tumor volumes. At the 25 μg / kg dose, TC was 2 days, LCK was 0.11 (inactive), and there were no regressions. The M-DM21 conjugate was highly active at 130 μg / kg and active at 70 μg / kg, with T / C of 0% and 11%, respectively. At the 130 μg / kg dose, TC was >63 days, LCK >3.33 (high activity), PR 6 / 6, CR 6 / 6, and TFS 0 / 6. At the 50 μg / kg dose, TC was 27 days, LCK 1.43 (activity), PR 4 / 6, CR 2 / 6, and TFS 0 / 6. The Ms-SPDB-DM4 conjugate at 100 μg / kg was highly active, with a T / C of 1%, PR 6 / 6, and CR 3 / 6. TC and LCK could not be determined in this group due to necrosis in the small tumor volume. There was minimal weight loss of 1-5% in all groups in this study. In summary, KIH-DM21 was significantly more active than the parent conjugates M-DM21 and Ms-SPDB-DM4 when administered at 100 μg / kg. All three conjugates were highly active at 100 μg / kg, but the duration of response was much longer with the KIH biparatopic ADC than with the parent antibody conjugates. [Table 19]

[0411] In vivo antitumor activity of KIH biparatopic ADC in SCID mice bearing IGROV-1 human ovarian cancer xenografts The in vivo efficacy of KIH biparatopic antibodies conjugated to DM21 was evaluated in an IGROV-1 xenograft model with moderate FRα expression (H-score 140). Mice were randomized into groups (n=8 per group) by tumor volume and then dosed 10 days after inoculation. These groups included a control group administered formulation buffer, KIH-DM21 at 100 and 50 μg / kg, M-DM21 at 130 and 70 μg / kg, and Ms-SPDB-DM4 at 100 and 50 μg / kg. All mice received a single intravenous dose of the above-mentioned compounds. The study was terminated 120 days after inoculation. Tumor measurements and calculations were determined as described in Example 4, subsection "In vivo antitumor activity of tetravalent biparatopic ADCs in SCID mice bearing OV-90 human ovarian cancer xenografts."

[0412] The results of the study are shown in Figure 16 and Table 20. The KIH-DM21 conjugate was active at both 100 and 50 μg / kg with a T / C of 19% and 12%, respectively. At the 100 μg / kg dose, TC was >99 days, LCK >2.87 (high activity), PR 7 / 8, CR 6 / 8, and TFS 0 / 8. At the 50 μg / kg dose, TC was 53 days, LCK 1.53 (active), PR 5 / 8, CR 3 / 8, and TFS 0 / 8. The M-DM21 conjugate was active at both 130 μg / kg and 70 μg / kg with a T / C of 13% and 16%, respectively. At the 130 μg / kg dose, TC was >99 days, LCK >2.87 (high activity), PR 8 / 8, CR 7 / 8, and TFS 2 / 8. At the 70 μg / kg dose, TC was 36 days, LCK was 1.04 (active), PR was 3 / 8, CR was 2 / 8, and TFS was 0 / 8. The Ms-SPDB-DM4 conjugate was active at both the 100 and 50 μg / kg doses with T / C of 17% and 34%, respectively. At the 100 μg / kg dose, TC was 23 days, LCK was 0.67 (inactive), PR was 3 / 8, CR was 2 / 8, and TFS was 0 / 8. At the 50 μg / kg dose, TC was 17 days, LCK was 0.49 (inactive), and there was no regression. There was minimal weight loss of 1-5% in most groups, except for M-DM21 at 50 μg / kg (6%) and Ms-SPDB-DM4 at 50 μg / kg (7%) at the nadir 16 days postinoculation. In summary, the parental Ms-SPDB-DM4 conjugate was less effective in vivo than the DM21 conjugate. Moreover, KIH-DM21 and the parental M-DM21 were similarly active at the two doses tested. [Table 20]

[0413] In vivo antitumor activity of KIH biparatopic ADC in SCID mice bearing KB human cervical cancer xenografts The in vivo efficacy of KIH biparatopic antibodies conjugated to DM21 was evaluated in a KB xenograft model with high FRα expression (H-score 300). When tumors reached approximately 100 mm3, mice were randomized into groups by tumor volume (n=6 per group) and then dosed 6 days after inoculation. These groups included a control group administered formulation buffer, KIH-DM21 at 50 and 25 μg / kg, M-DM21 at 50 and 25 μg / kg, and Ms-SPDB-DM4 at 50 and 25 μg / kg. All mice received a single intravenous dose of the above-mentioned compounds. The study was terminated 120 days after inoculation. Tumor measurements and calculations were determined as described in Example 4, subsection "In vivo antitumor activity of tetravalent biparatopic ADCs in SCID mice bearing OV-90 human ovarian cancer xenografts."

[0414] The results of the study are shown in Figure 17 and Table 21. The KIH-DM21 conjugate was highly active at both 50 and 25 μg / kg, with a T / C of 0%, TC > 100 days, and LCK > 6.41 (high activity) at both doses. At the 100 μg / kg dose, there were 6 / 6 PR, 6 / 6 CR, and 6 / 6 TFS, whereas at the 25 μg / kg dose, there were 6 / 6 PR, 5 / 6 CR, and 5 / 6 TFS. The M-DM21 conjugate was highly active at both 50 and 25 μg / kg, with a T / C of 0% and 2%, respectively. At both doses, there were TC > 100 days, and LCK > 6.41 (high activity). At the 50 μg / kg dose, PR was 6 / 6, CR was 6 / 6, and TFS was 6 / 6, whereas at the 25 μg / kg dose, PR was 5 / 6, CR was 4 / 6, and TFS was 4 / 6. The Ms-SPDB-DM4 conjugate was highly active at both 50 and 25 μg / kg, with T / C of 0% and 8%, respectively. At the 50 μg / kg dose, TC was >100 days, LCK was >6.41 (high activity), PR was 6 / 6, CR was 6 / 6, and TFS was 5 / 6. At the 25 μg / kg dose, TC was 24 days, LCK was 1.54 (active), PR was 3 / 6, CR was 1 / 6, and TFS was 1 / 6. Minimal weight loss of 2-4% was seen in the M-DM21 and Ms-SPDB-DM4 groups at the nadir 8 days post-inoculation. In summary, the response of Ms-SPDB-DM4 at 25 μg / kg was transient, whereas administration of Ms-SPDB-DM4 at 50 μg / kg resulted in long-term complete regression in most mice. Administration of both 25 μg / kg and 50 μg / kg doses of M-DM21 and KIH-DM21 resulted in long-term complete regression in most mice.

[0415] Taken together, the in vivo efficacy studies described here indicate that KIH-DM21 was the most active conjugate in most xenograft models tested, followed by M-DM21 and Ms-SPDB-DM4. [Table 21]

[0416] In vivo antitumor activity of KIH biparatopic ADC in SCID mice bearing IMGN853-resistant KB human cervical cancer xenografts The in vivo efficacy of KIH biparatopic antibodies conjugated to DM21 was evaluated in an IMGN853-resistant KB xenograft model. Parental KB cells were grown in the presence of 1 nM DM1-Me. After stable growing cultures were established, cells were subcloned and clones were expanded, characterized, and frozen. Subclone 6A was selected for this study and inoculated subcutaneously into mice. When tumors reached approximately 100 mm 3 When tumor volume reached 100 μg / kg, mice were randomized into groups (n=6 per group) according to tumor volume and then dosed 5 days after inoculation. These groups included a control group that received formulation buffer, KIH-L-DM21 at 40 and 20 μg / kg, ML-DM21 at 40 and 20 μg / kg, and Ms-SPDB-DM4 at 40 and 20 μg / kg. All mice received a single intravenous dose of the above compounds. The study was terminated 78 days after inoculation. Tumor measurements and calculations were determined as described in the OV-90 human ovarian cancer xenograft experiment above.

[0417] The results of the study are shown in Figure 19 and Table 22. The KIH-L-DM21 conjugate was highly active at both 40 and 20 μg / kg with a T / C of 0%. In the 40 μg / kg dose group, TC was >60 days, LCK was >3.41 (high activity), PR was 6 / 6, CR was 6 / 6, and TFS was 6 / 6. In the 20 μg / kg dose group, TC was 46 days, LCK was 2.61 (active), PR was 6 / 6, CR was 4 / 6, and TFS was 1 / 6. The ML-DM21 conjugate was highly active at both 40 μg / kg and 20 μg / kg with a T / C of 0% and 2%, respectively. In the 40 μg / kg dose group, TC was >60 days, LCK was >3.41 (high activity), PR was 6 / 6, CR was 6 / 6, and TFS was 6 / 6. In the 20 μg / kg dose group, TC was 28 days, LCK was 1.59 (active), PR was 5 / 6, CR was 2 / 6, and TFS was 2 / 6. The Ms-SPDB-DM4 conjugate was highly active at 40 μg / kg but inactive at 20 μg / kg, with T / C of 0% and 63%, respectively. In the 40 μg / kg dose, TC was 41 days, LCK was 2.33 (active), PR was 6 / 6, CR was 6 / 6, and TFS was 0 / 6. At the 20 μg / kg dose, TC was 6 days, LCK was 0.34 (inactive), PR was 0 / 6, CR was 0 / 6, and TFS was 0 / 6. The 20 μg / kg KIH-L-DM21 group saw minimal weight loss of 3% at the nadir on day 8 post-inoculation. In summary, KIH-L-DM21 was as effective as ML-DM21 in this model, and both KIH-L-DM21 and ML-DM21 were more effective than Ms-SPDB-DM4. [Table 22]

[0418] Example 7. Pharmacokinetics and Tolerability of Biparatopic Immunoconjugates The toxicity and toxicokinetic profile of a biparatopic FRα-targeted immunoconjugate was evaluated in cynomolgus monkeys after a single dose administration. Briefly, KIH-DM21 was administered as a 10-minute slow bolus infusion at dose levels of 10 or 13 mg / kg to two male monkeys per dose level. Animals were observed up to 28 days after administration to evaluate recovery, persistence, or progression of effects. Body weight, clinical observations, and food consumption were assessed, and blood samples were collected for clinical pathology parameters (hematology, serum chemistry, and coagulation) and toxicokinetic parameters.

[0419] All animals survived to the end of the study. There were no KIH-DM21-related effects on body weight, hematology, or serum chemistry parameters. KIH-DM21-related clinical observations seen in animals in the single 10 Ab mg / kg group were hind paw redness on days 8 and 12, but no clinical findings were noted during the remainder of the off-treatment period. Higher fibrinogen values ​​associated with KIH L DM21 were seen on days 4 and 8 in both dose groups. Values ​​were similar to pretreatment values ​​by the end of the off-treatment period (day 29).

[0420] The KIH-DM21 ADC exhibits biphasic pharmacokinetics following a single intravenous dose in monkeys. The mean terminal t1 / 2 of the ADC was 156 hours at the 10 mg / kg dose. The mean t1 / 2 of the total antibody (TAb) was longer than that observed with the ADC (184 hours at the 10 mg / kg dose). Comparison of the concentration-time profiles of the ADC and TAb showed that the KIH-DM21 immunoconjugate was more stable than IMGN853 at the 10 mg / kg dose. KIH-DM21 has a longer terminal half-life and greater exposure metrics (AUC∞ values) than IMGN853 at the 10 mg / kg dose. [Table 23]

[0421] As shown in Figures 18A and 18B, both the FRα biparatopic immunoconjugate and IMGN853 were well tolerated at 10 mg / kg, and the biparatopic immunoconjugate was similarly well tolerated at 13 mg / kg. Furthermore, as shown in Table 23, the FRα biparatopic immunoconjugate was more stable than IMGN853 at the 10 mg / kg dose. Notably, the biparatopic immunoconjugate exhibited a terminal half-life approximately 60 hours longer than IMGN853 and a total exposure approximately 40% higher than IMGN853. In certain embodiments, for example, the following items are provided: (Item 1) A biparatopic antibody or antigen-binding fragment thereof that specifically binds to human folate receptor 1 (FRα), the antibody or antigen-binding fragment thereof comprising: (a) a first FRα-binding domain comprising a first variable heavy chain (VH) and a first variable light chain (VL) that bind to a first epitope of FRα; (b) a second FRα-binding domain comprising a second VH and a second VL that bind to a second epitope of FRα. (Item 2) the first FRα-binding domain specifically binds to the same FRα epitope as an antibody comprising a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, 26, and 57, and a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21; A biparatopic antibody or an antigen-binding fragment thereof described in 1. (Item 3) The biparatopic antibody or antigen-binding fragment thereof described in item 1, wherein the first FRα-binding domain competitively inhibits binding to the same epitope of FRα as an antibody comprising a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, 26, and 57, and a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21. (Item 4) A biparatopic antibody or an antigen-binding fragment thereof according to any one of items 1 to 3, wherein the second FRα-binding domain specifically binds to the same epitope of FRα as an antibody comprising a VH amino acid sequence of SEQ ID NO: 22 or 23 and a VL amino acid sequence of SEQ ID NO: 17 or 18. (Item 5) A biparatopic antibody or an antigen-binding fragment thereof according to any one of items 1 to 3, wherein the second FRα-binding domain competitively inhibits binding to the same epitope of FRα as an antibody comprising the VH amino acid sequence of SEQ ID NO: 22 or 23 and the VL amino acid sequence of SEQ ID NO: 17 or 18. (Item 6) 6. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the first VH comprises VH CDR1 to 3 comprising the amino acid sequences of (a) SEQ ID NOs: 10 to 12 or (b) SEQ ID NOs: 15, 16, and 12, respectively, and the first VL comprises VL CDR1 to 3 comprising the amino acid sequences of SEQ ID NOs: 4 to 6, respectively. (Item 7) 7. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 6, wherein the first VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, 26, and 57, and / or the first VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21. (Item 8) 8. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the second VH comprises VH CDR1 to 3 comprising the amino acid sequences of (a) SEQ ID NOs: 7 to 9 or (b) SEQ ID NOs: 13, 14, and 9, respectively, and the second VL comprises VL CDR1 to 3 comprising the amino acid sequences of SEQ ID NOs: 1 to 3, respectively. (Item 9) The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 8, wherein the second VH comprises the amino acid sequence of SEQ ID NO: 22 or 23, and / or the second VL comprises the amino acid sequence of SEQ ID NO: 17 or 18. (Item 10) 10. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 9, wherein the first VH and VL pair and / or the second VH and VL pair are murine, non-human, humanized, chimeric, resurfaced, or human. (Item 11) 11. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 10, wherein the antibody or antigen-binding fragment thereof binds to human FRα but does not bind to FOLR2 or FOLR3. (Item 12) 12. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 11, wherein the first FRα-binding domain is a single-chain variable fragment (scFv). (Item 13) 13. The biparatopic antibody or antigen-binding fragment thereof of item 12, wherein the scFv of the first FRα-binding domain has a VH-linker-VL peptide orientation. (Item 14) 13. The biparatopic antibody or antigen-binding fragment thereof of item 12, wherein the scFv of the first FRα-binding domain has a VL-linker-VH peptide orientation. (Item 15) 12. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 11, wherein the second FRα-binding domain is a single-chain variable fragment (scFv). (Item 16) 16. The biparatopic antibody or antigen-binding fragment thereof of item 15, wherein the scFv of the second FRα-binding domain has a VH-linker-VL peptide orientation. (Item 17) 16. The biparatopic antibody or antigen-binding fragment thereof of item 15, wherein the scFv of the second FRα-binding domain has a VL-linker-VH peptide orientation. (Item 18) 18. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 13 to 17, wherein the linker is a glycine-serine linker. (Item 19) 19. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 18, wherein the second FRα-binding domain comprises an amino acid sequence selected from SEQ ID NOs: 27 to 29. (Item 20) 19. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 18, wherein the first FRα-binding domain comprises an amino acid sequence selected from SEQ ID NOs: 30 to 32. (Item 21) 21. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 20, comprising the amino acid sequence of (i) SEQ ID NOs: 33 and 34, (ii) SEQ ID NOs: 35 and 36, (iii) SEQ ID NOs: 37 and 38, or (iv) SEQ ID NOs: 39 and 40. (Item 22) 21. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 20, comprising an amino acid sequence of SEQ ID NO: 41 to 43. (Item 23) 21. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 20, comprising an amino acid sequence of SEQ ID NO: 44 to 46. (Item 24) 24. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 23, wherein the biparatopic antibody or antigen-binding fragment thereof is a tetravalent biparatopic antibody or antigen-binding fragment thereof. (Item 25) 24. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 23, wherein the biparatopic antibody or antigen-binding fragment thereof is a divalent biparatopic antibody or antigen-binding fragment thereof. (Item 26) 26. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 25, wherein the bispecific antibody or antigen-binding fragment thereof comprises an FRα-binding domain selected from the group consisting of tandem scFv, diabody, triabody, tetrabody, and knob-in-hole structure. (Item 27) 27. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 20 and 22 to 26, having a knob-in-hole (KIH) structure. (Item 28) The FRα-binding domain including SEQ ID NOs: 1 to 3 and 7 to 9 is located on the knob side of the KIH structure. 28. The biparatopic antibody or antigen-binding fragment thereof according to item 27. (Item 29) 28. The biparatopic antibody or antigen-binding fragment thereof according to item 27, wherein the FRα-binding domain comprising SEQ ID NOs: 1 to 3 and 7 to 9 is located on the hole side of the KIH structure. (Item 30) 30. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 27 to 29, wherein the FRα-binding domain comprising SEQ ID NOs: 4 to 6 and 10 to 12 is located on the knob side of the KIH structure. (Item 31) 30. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 27 to 29, wherein the FRα-binding domain comprising SEQ ID NOs: 4 to 6 and 10 to 12 is located on the hole side of the KIH structure. (Item 32) 32. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 31, comprising a full-length antibody. (Item 33) 33. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 32, wherein the first FRα-binding domain is a full-length antibody. (Item 34) 34. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 33, wherein the second FRα-binding domain is a full-length antibody. (Item 35) 32. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 31, comprising an antigen-binding fragment thereof. (Item 36) 36. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 35, wherein the first FRα-binding domain is an antigen-binding fragment. (Item 37) 37. The biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 36, wherein the second FRα-binding domain is an antigen-binding fragment. (Item 38) A biparatopic antibody or an antigen-binding fragment thereof, comprising the amino acid sequence of SEQ ID NO:41 to 43. (Item 39) A combination of isolated nucleic acid molecules encoding biparatopic antibodies or antigen-binding fragments thereof according to items 1 to 38. (Item 40) 40. An isolated vector comprising one of the nucleic acid molecules described in item 39. (Item 41) A host cell comprising the isolated nucleic acid molecule of item 39 or the isolated vector combination of item 40. (Item 42) 42. The host cell according to item 41, selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture. (Item 43) A biparatopic antibody or antigen according to any one of items 1 to 38, a nucleic acid molecule according to item 39, a vector according to item 40, or a host cell according to item 41 or 42. A pharmaceutical composition comprising a combination of the cells and a pharma- ceutically acceptable carrier or excipient. (Item 44) 39. A pharmaceutical composition comprising the biparatopic antibody according to any one of items 1 to 38 and a pharmaceutical carrier or excipient. (Item 45) 24. A method for producing the biparatopic antibody according to any one of items 1 to 23, comprising: (a) culturing a cell expressing the antibody; and (b) isolating the antibody from the cultured cell. (Item 46) 46. ​​The method of claim 45, wherein the cell is a eukaryotic cell. (Item 47) The following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein: CB is the biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 38; L2 is represented by one of the following formulas: [ka] During the ceremony: R x , R y , R x’ , and R y’ is, for each occurrence, independently H, -OH, halogen, -O-(C 1-4 alkyl), -SO3H, -NR 40 R 41 R 42 + or optionally -OH, halogen, SO3H, or NR 40 R 41 R 42 + C replaced by 1-4 alkyl, where R 40 , R 41 , and R 42 are each independently H or C 1-4 is alkyl; l and k each independently represent an integer of 1 to 10; l1 is an integer from 2 to 5; k1 is an integer from 1 to 5; s1 indicates the site connected to the cell-binding agent CB, and s3 indicates the site connected to the A group; A is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1-3is alkyl; L1 is expressed by the following formula: -CR 3 R 4 -(CH2) 1-8 -C(=O)- In the formula, R 3 and R 4 are each independently H or Me, and the -C(=O)- moiety of L1 is connected to D; D is expressed by the following formula: [ka] The immunoconjugate, wherein q is an integer of 1 to 20. (Item 48) R x , R y , R x’ , and R y’ and l and K are each independently an integer number from 2 to 6. (Item 49) 49. The immunoconjugate according to item 47 or 48, wherein A is a peptide comprising 2 to 5 amino acid residues. (Item 50) A is Gly-Gly-Gly, Ala-Val, Val-Ala, D-Val-Ala, Val-Cit, D-Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Al a-Ala-D-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 54), β-Ala-Leu-Ala-Leu (SEQ ID NO: 55), Gly-Phe-Leu-Gly (SEQ ID NO: 56), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val- D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, D-Ala-Pro, and D-Ala-tBu-Gly, wherein the first amino acid of each peptide is connected to the L2 group and the last amino acid of each peptide is -NH-CR 1 R 2 - The immunoconjugate according to item 49, wherein the immunoconjugate is linked to S-L1-D. (Item 51) R 1 and R 2 and (Item 52) L1 is -(CH2) 4-6 52. The immunoconjugate according to any one of items 47 to 51, wherein -C(=O)-. (Item 53) 53. The immunoconjugate according to any one of items 47 to 52, wherein D is represented by the following formula: [ka] (Item 54) The immunoconjugate has the formula: [ka] or a pharma- ceutically acceptable salt thereof, [ka] is connected to group L2 via the amine group of Lys; [ka] is connected to group L2 via the thiol group of Cys; R 3 and R 4 are each independently H or Me; m1, m3, n1, r1, s1, and t1 each independently represent an integer of 1 to 6; m2, n2, r2, s2, and t2 are each independently an integer of 1 to 7; t3 is an integer from 1 to 12; 54. The immunoconjugate according to any one of items 47 to 53, wherein D1 is represented by the following formula: [ka] (Item 55) The immunoconjugate has the formula: [ka] During the ceremony: m1 and m3 each independently represent an integer of 2 to 4; m2 is an integer from 2 to 5; r1 is an integer from 2 to 6; 55. The immunoconjugate according to item 54, wherein r2 is an integer from 2 to 5. (Item 56) A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D- The immunoconjugate according to item 54 or 55, which is Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly. (Item 57) The immunoconjugate has the formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly; 55. The immunoconjugate according to item 54, wherein D1 is represented by the following formula: [ka] (Item 58) The immunoconjugate has the formula: [ka] 58. The immunoconjugate according to item 57, wherein D1 is represented by the following formula: [ka] (Item 59) The immunoconjugate has the formula: [ka] During the ceremony: The CBA is a biparatopic antibody or an antigen-binding fragment thereof according to any one of items 1 to 38; q is an integer from 1 to 10; 48. The immunoconjugate according to item 47, wherein D1 is represented by the following formula: [ka] (Item 60) The following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein: CBA is a biparatopic antibody or antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 41 to 43; D1 is expressed by the following formula: [ka] and The immunoconjugate, wherein q is an integer of 1 to 10. (Item 61) An immunoconjugate having the formula (A)-(L)-(C), wherein: (A) is the biparatopic antibody or antigen-binding fragment thereof according to any one of items 1 to 38; (L) is a linker; The immunoconjugate, wherein (C) is a cytotoxic drug and the linker (L) connects (A) to (C). (Item 62) 62. The immunoconjugate of item 61, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid-based linker. (Item 63) The linker may be selected from the group consisting of N-(gamma maleimidobutryloxy)sulfosuccinimide ester (sulfo-GMBS or sGMBS), gamma maleimidobutyric acid N-succinimidyl ester (GMBS), N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) or N-succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(maleimidomethyl)cyclohexane. 63. The immunoconjugate according to item 62, wherein the immunoconjugate is selected from the group consisting of N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC); N-succinimidyl-4-(iodoacetyl)-aminobenzoic acid (SIAB); and N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester (NHS-PEG4-maleimide). (Item 64) 64. The immunoconjugate according to any one of items 61 to 63, wherein the linker is sulfo-GMBS. (Item 65) 64. The immunoconjugate according to any one of items 61 to 63, wherein the linker is GMBS. (Item 66) 64. The immunoconjugate according to any one of items 61 to 63, wherein the linker is sulfo-SPDB. (Item 67) 67. The immunoconjugate of any one of items 61 to 66, wherein the cytotoxic agent is selected from the group consisting of maytansinoids, maytansinoid analogs, benzodiazepines, taxoids, CC-1065, CC-1065 analogs, duocarmycins, duocarmycin analogs, calicheamicins, dolastatins, dolastatin analogs, auristatins, tomaymycin derivatives, and leptomycin derivatives or prodrugs of the agents. (Item 68) 68. The immunoconjugate according to any one of items 61 to 67, wherein the cytotoxic agent is a maytansinoid. (Item 69) 69. The immunoconjugate of item 68, wherein the maytansinoid is DM4. (Item 70) 69. The immunoconjugate of item 68, wherein the maytansinoid is DM21. (Item 71) 71. The immunoconjugate according to any one of items 61 to 70, further comprising a second (C). (Item 72) 72. The immunoconjugate according to item 71, further comprising a third (C). (Item 73) 73. The immunoconjugate according to item 72, further comprising a fourth (C). (Item 74) 74. A composition comprising at least one immunoconjugate according to any one of items 61 to 73, wherein the immunoconjugate comprises on average 3 to 4 C per A. (Item 75) A pharmaceutical composition comprising the immunoconjugate according to any one of items 47 to 74 and a pharma- ceutically acceptable carrier. (Item 76) 76. The pharmaceutical composition according to item 75, wherein the pharmaceutical composition contains an average of 2 to 5 drugs per antibody or antigen-binding fragment thereof. (Item 77) 76. The pharmaceutical composition according to item 75, wherein the pharmaceutical composition contains an average of 3 to 4 drugs per antibody or antigen-binding fragment thereof. (Item 78) 1. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 38, the immunoconjugate according to any one of items 47 to 73, or the composition according to items 43, 44, or 74 to 77. (Item 79) 79. The method of claim 78, wherein the cancer is ovarian cancer, uterine cancer, peritoneal cancer, fallopian tube cancer, endometrial cancer, lung cancer, or brain cancer. (Item 80) 79. The method of claim 78, wherein the cancer is ovarian cancer. (Item 81) 81. The method of item 80, wherein the ovarian cancer is platinum-resistant epithelial ovarian cancer. (Item 82) 81. The method of item 80, wherein the ovarian cancer is recurrent epithelial ovarian cancer. (Item 83) 81. The method of item 80, wherein the ovarian cancer is platinum-refractory epithelial ovarian cancer. (Item 84) Item 79. The method of item 78, wherein the cancer is uterine cancer. (Item 85) 79. The method of claim 78, wherein the cancer is peritoneal cancer. (Item 86) Item 79. The method of item 78, wherein the cancer is fallopian tube cancer. (Item 87) 79. The method of claim 78, wherein the cancer is endometrial cancer. (Item 88) 79. The method of claim 78, wherein the cancer is lung cancer. (Item 89) 79. The method of claim 78, wherein the cancer is brain cancer. (Item 90) 90. The method of any one of items 78 to 89, wherein the cancer is IMGN853 resistant. (Item 91) 91. The method of any one of items 78 to 90, further comprising administration of a steroid.

Claims

1. 1. A pharmaceutical composition comprising an immunoconjugate for use in the treatment of cancer, the immunoconjugate comprising a biparatopic antibody or antigen-binding fragment thereof that specifically binds to human folate receptor 1 (FRα) and a cytotoxic agent, and having the formula: 【Chemistry 1】 During the ceremony: CBA is the biparatopic antibody or antigen-binding fragment thereof; q is an integer from 1 to 10; and D １ is of the formula: 【Chemistry 2】 is shown in The biparatopic antibody or antigen-binding fragment thereof (a) a first FRα-binding domain comprising a first variable heavy chain (VH) of SEQ ID NO: 24 and a first variable light chain (VL) of SEQ ID NO: 19; and (b) a second FRα-binding domain comprising a second VH of SEQ ID NO: 23 and a second VL of SEQ ID NO: 18; and The pharmaceutical composition comprises an average of 3 to 4 cytotoxic agents per biparatopic antibody or antigen-binding fragment thereof.

2. The cancer is ovarian cancer, uterine cancer, peritoneal cancer, fallopian tube cancer, endometrial cancer, lung cancer, or brain cancer. The pharmaceutical composition according to claim 1 .

3. The pharmaceutical composition of claim 2, wherein the cancer is platinum-sensitive.

4. The pharmaceutical composition of claim 1 , wherein the cancer is ovarian cancer.

5. The pharmaceutical composition of claim 4, wherein the ovarian cancer is platinum-resistant epithelial ovarian cancer.

6. The pharmaceutical composition of claim 4, wherein the ovarian cancer is recurrent epithelial ovarian cancer.

7. The pharmaceutical composition of claim 4, wherein the ovarian cancer is platinum-refractory epithelial ovarian cancer.

8. The pharmaceutical composition of claim 1 , wherein the cancer is uterine cancer.

9. The pharmaceutical composition according to claim 1, wherein the cancer is peritoneal cancer.

10. The pharmaceutical composition of claim 1 , wherein the cancer is fallopian tube cancer.

11. The pharmaceutical composition of claim 1 , wherein the cancer is endometrial cancer.

12. The pharmaceutical composition of claim 1 , wherein the cancer is lung cancer.

13. The pharmaceutical composition of claim 1 , wherein the cancer is brain cancer.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the cancer is IMGN853 resistant.

15. The pharmaceutical composition according to any one of claims 1 to 13, wherein the pharmaceutical composition is for administration in combination with a steroid.

16. 1. A pharmaceutical composition comprising an immunoconjugate for use in the treatment of cancer, the immunoconjugate comprising a biparatopic antibody that specifically binds to human folate receptor 1 (FRα) and a cytotoxic agent and having the formula: 【Chemistry 3】 During the ceremony: CBA is the biparatopic antibody; q is an integer from 1 to 10; and D １ is of the formula: 【Chemistry 4】 is shown in A pharmaceutical composition, wherein the biparatopic antibody comprises the amino acid sequence of SEQ ID NO: 41-43, and the pharmaceutical composition comprises an average of 3-4 cytotoxic agents per biparatopic antibody.

17. 17. The pharmaceutical composition of claim 16, wherein the cancer is ovarian cancer, uterine cancer, peritoneal cancer, fallopian tube cancer, endometrial cancer, lung cancer, or brain cancer.

18. The pharmaceutical composition of claim 17, wherein the cancer is platinum-sensitive.

19. The pharmaceutical composition of claim 16, wherein the cancer is ovarian cancer.

20. 20. The pharmaceutical composition of claim 19, wherein the ovarian cancer is platinum-resistant epithelial ovarian cancer.

21. The pharmaceutical composition of claim 19, wherein the ovarian cancer is recurrent epithelial ovarian cancer.

22. 20. The pharmaceutical composition of claim 19, wherein the ovarian cancer is platinum-refractory epithelial ovarian cancer.

23. The pharmaceutical composition of claim 16, wherein the cancer is uterine cancer.

24. The pharmaceutical composition of claim 16, wherein the cancer is peritoneal cancer.

25. The pharmaceutical composition of claim 16, wherein the cancer is fallopian tube cancer.

26. The pharmaceutical composition of claim 16, wherein the cancer is endometrial cancer.

27. The pharmaceutical composition of claim 16, wherein the cancer is lung cancer.

28. The pharmaceutical composition of claim 16, wherein the cancer is brain cancer.

29. The pharmaceutical composition of any one of claims 16 to 28, wherein the cancer is IMGN853 resistant.

30. The pharmaceutical composition according to any one of claims 16 to 28, wherein the pharmaceutical composition is for administration in combination with a steroid.

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