Treatment of cancer with therapeutic binding molecules

Abstract

A method of treating cancer in a subject in need thereof is provided herein, the method comprising administering to the subject an antibody-drug conjugate (ADC) in an amount from about 0.8 mg / kg to about 5.0 mg / kg, wherein the ADC comprises an anti-FRα antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor.

Description

[0001] TREATMENT OF CANCER WITH THERAPEUTIC BINDING MOLECULES

[0002] FIELD

[0003] The present disclosure relates to a method of treating cancer in a human subject in need thereof, comprising administering to the human subject an antibody-drug conjugate (ADC) in an amount from about 0.8 mg / kg to about 5.0 mg / kg, wherein the ADC comprises an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor.

[0004] BACKGROUND

[0005] Cancer remains one of the most leading diseases worldwide despite years of study into the mechanisms of cancer pathogenesis and the development of numerous potential anti-cancer drugs. In particular, lung cancer and ovarian cancer are the third- and fifth-most common cancer in women, respectively. Chemotherapy and radiotherapy are the most common cancer treatments. Nonetheless, these therapies are linked to various negative side effects, including fatigue, nausea, and hair loss. These problems are complicated by the fact that chemotherapy treatments are frequently administered over long periods of time. Over the last few decades, a number of antibody therapies for cancer have been developed and marketed, leading to a reduction in the need for conventional forms of chemotherapy for a number of cancer types. Although the availability of methodology for producing antibodies (e.g. monoclonal antibodies) has greatly improved over this time period, there are relatively few clinically available anticancer antibodies, and even fewer that may be used to target a variety of cancer types. Furthermore, there is a need to increase the potency of therapeutic antibodies, which is generally limited by the prevalence of the target antigen’s expression and the subsequent effects on the cancer cell following antibody binding. Conjugating monoclonal antibodies to cytotoxic molecules to generate antibody drug conjugates (ADCs) is a novel approach to deliver targeted therapy for malignancies. This approach has been successfully implemented for specific targets (HER2, CD30, and CD79b) leading to marketed ADCs such as fam-trastuzumab deruxtecan- nxki (breast and gastric cancer), brentuximab vedotin (Hodgkin lymphoma), and polatuzumab vedotin-piiq (Nonhodgkin lymphoma), respectively.

[0006] Folate receptors (FRs) are membrane-bound proteins present on the cell surface and thus may be exploited to develop new ADCs. The FR family includes FRa, FRP, FRy and FRS. FR binds folate molecules and transports them into cells, such that the folate molecules are delivered to the folate cycle to support metabolism of nucleotides. In particular, folate is important for DNA synthesis, methylation and repair (Cheung, et al., Oncotarget. 2016;7(32):52553-52574).

[0007] FRa is a glycosylphosphatidylinositol (GPI)-anchored membrane protein having high affinity to the active form of folate, 5-methyltetrahydrofolate (5-MTF). FRa is expressed by the F0LR1 gene. Previous studies have shown that FRa plays a crucial role in embryogenesis (Kelemen, Int J Cancer. 2006; 119(2):243-250). Folate transport in adults, however, is mainly driven by ubiquitous expression of reduced folate carriers and proton coupled folate transporters (Zhao, et al., Annu Rev Nutr. 2011;31 : 177-201). The distribution of FRa expression in adults is usually limited to the apical surfaces of polarised epithelia, such as choroid plexus, kidney, lung, and placenta.

[0008] Overexpression of FRa, which is also known as Folate Receptor 1 (FOLR1) or folate binding protein (FBP), is frequently observed in tumour cells such as ovarian, lung (e.g. nonsmall cell lung cancer (NSCLC)) and breast carcinomas (Shi, et al., Drug Des Devel Ther. 2015;9:4989-4996). In particular, a previous study has found that the level of soluble FRa in the blood of ovarian cancer patients is elevated, supporting the potential application of FRa as a biomarker of early ovarian cancer (Basal, et al., PLoS One. 2009;4(7):e6292). Pre-clinical ovarian cancer models have also revealed that overexpression of FRa is associated with tumour progression, and the binding of folate to FRa could mediate activation of the pro-oncogene STAT3 (Hansen, et al., Cell Signal. 2015;27(7): 1356-1368).

[0009] Antibodies against FRa in the art suffer from deficiencies, such as poor internalisation, short half-life, and insufficient cytotoxicity. Moreover, the FRa-targeting ADCs in the art employ microtubule inhibitors, which have been associated with specific toxicities in clinical trials, such as corneal inflammation (mirvetuximab soravtansine, which consists of an anti-FRa antibody M9346A conjugated via a sulfo-SPBD linker with the maytansinoid warhead DM4) (Moore etal. (2017) Cancer 123:3080-7), interstitial lung disease (MORAb-202, which consists of the humanised antibody farletuzumab derived from LK26 conjugated to the eribulin warhead) (Sato, et al. (2020) ESMO Abstract https: / / doi.Org / 10.1016 / j.annonc.2020.01.026), and neuropathy and neutropenia (STRO-002, which consists of an anti-FRa antibody SP8166 conjugated to the hemiasterlin warhead) (Naumann, et al. (2021) J Clin Oncol 39 (Suppl 15 / abstr 5550) https: / / doil0.1200 / JCO.2021.39.15_suppl.5550).

[0010] AZD5335 (AB1370049-SG3932 DAR8) is a specific, FRa-targeted ADC comprising an anti-FRa human IgGlK monoclonal antibody conjugated via a cleavable maleimide-PEG8- valine-alanine linker to a topoisomerase 1 inhibitor (TOPOi) cytotoxin (SG3932 payload) with a drug-to-antibody ratio (DAR) of 8 (Figure 1). Upon binding of the ADC to FRa on tumour cells, the complex is internalised and trafficked to the lysosome where the linker is cleaved by cathepsin B, resulting in the release of the TOPOi payload into the cytoplasm. The mechanism of action (MoA) of TOPOi is the trapping of topoisomerase I on replicating DNA and subsequent induction of DNA double strand breaks that lead to apoptosis (Chowdhuri and Das (2021) NAR Cancer 3(l):zcab003). The toxin can also diffuse through cell membranes and target neighbouring tumour cells that do not express FRa (bystander killing). This property becomes essential when the receptor expression is low or heterogenous in the tumour. AZD5335 is efficacious in pre-clinical models of ovarian and lung cancer and is currently being investigated in the clinic as a treatment option for these tumour types.

[0011] There is a need to develop new cancer therapies based on FRa-targeting ADCs.

[0012] SUMMARY OF THE DISCLOSURE

[0013] Aspects and embodiments of the disclosure are set out in the appended claims. These and other aspects and embodiments of the disclosure are also described herein.

[0014] In one aspect, there is provided a method of treating cancer in a human subject in need thereof, comprising administering to the human subject an antibody-drug conjugate (ADC) in an amount from about 0.8 mg / kg to about 5.0 mg / kg , wherein the ADC comprises an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor.

[0015] In some embodiments of any aspect of the disclosure, the amount of ADC administered is about 0.8 mg / kg, about 1.2 mg / kg, about 1.6 mg / kg, about 2.0 mg / kg, about 2.4 mg / kg, about 2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg, about 4.8 mg / kg or about 5.0 mg / kg.

[0016] In some embodiments of any aspect of the disclosure, the amount of ADC administered is about 0.8 mg / kg to about 4.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg or about 1.6 mg / kg to about 2.4 mg / kg.

[0017] In particular embodiments, the amount of ADC administered is about 1.6 mg / kg. In particular embodiments, the amount of ADC administered is about 2.0 mg / kg. In particular embodiments, the amount of ADC administered is about 2.4 mg / kg.

[0018] In some embodiments of any aspect of the disclosure, the ADC is administered to the subject once every week, once every two weeks, once every three weeks or once every four weeks. In particular embodiments, the ADC is administered to the subject once every three weeks.

[0019] In particular embodiments, about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0020] In some embodiments of any aspect of the disclosure, the ADC is administered intravenously.

[0021] In some embodiments of any aspect of the disclosure, at least about 75% of the cells in said cancer are FRa-positive cells. In some embodiments, about 25% to about 75% of cancer cells in said cancer are FRa-positive cells. In some embodiments, about 1% to about 25% of cancer cells in said cancer are FRa-positive cells. In some embodiments, at least about 1% of cancer cells in said cancer are FRa-positive cells.

[0022] In some embodiments of any aspect of the disclosure, the method further comprises a step of determining the percentage of FRa-positive cells in a sample obtained from the cancer before administering the ADC to the human subject.

[0023] In some embodiments of any aspect of the disclosure, the method further comprises a step of determining the percentage of FRa-positive cells in a sample obtained from the cancer before administering the ADC to the human subject, and selecting the human subject as suitable for said treatment with the ADC based on:

[0024] (a) at least about 75% of the cells in said sample are FRa-positive cells;

[0025] (b) at least about 25% of the cells in said sample are FRa-positive cells; or

[0026] (c) at least about 1% of the cells in said sample are FRa-positive cells.

[0027] In another aspect, there is provided a method of producing a unit dose of an ADC for the treatment of cancer in a human subject, wherein the ADC comprises an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor, the method comprising:

[0028] (a) determining the percentage of FRa-positive cells in a sample from the cancer; and

[0029] (b) producing a unit dose of the ADC by formulating the ADC in an amount determined based on the percentage of FRa-positive cells determined in the subject.

[0030] In some embodiments, the amount of the ADC in said unit dose is:

[0031] (a) about 0.8 mg / kg, about 1.2 mg / kg, about 1.6 mg / kg, about 2.0 mg / kg, about 2.4 mg / kg, about 2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg, about 4.8 mg / kg or about 5.0 mg / kg, based on the body weight of the human subject;

[0032] (b) about 0.8 mg / kg to about 5.0 mg / kg, about 0.8 mg / kg to about 4.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg or about 1.6 mg / kg to about 2.4 mg / kg, based on the body weight of the human subject;

[0033] (c) about 1.6 mg / kg, based on the body weight of the human subject;

[0034] (d) about 2.0 mg / kg, based on the body weight of the human subject; or

[0035] (e) about 2.4 mg / kg, based on the body weight of the human subject.

[0036] In particular embodiments, the amount of the ADC in said unit dose is about 2.0 mg / kg, based on the body weight of the human subject.

[0037] In some embodiments of any aspect of the disclosure, the percentage of FRa-positive cells is assayed using immunohistochemistry (IHC) or the Quantitative Continuous Scoring (QCS) assay. In some embodiments, the IHC is performed with an antibody reagent.

[0038] In some embodiments of any aspect of the disclosure, the cancer is a solid tumour and / or an epithelial tumour.

[0039] In some embodiments of any aspect of the disclosure, the cancer is ovarian cancer, lung cancer (e.g. lung adenocarcinoma), endometrial cancer, pancreatic cancer, gastric cancer, renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinomas (HNSCC), breast cancer (e.g. TNBC), cervical cancer, malignant pleural mesothelioma, peritoneal cancer or fallopian tube cancer.

[0040] In some embodiments of any aspect of the disclosure, the cancer is ovarian cancer. In particular embodiments, the ovarian cancer is platinum-resistant ovarian cancer.

[0041] In some embodiments of any aspect of the disclosure, the cancer is lung cancer. In particular embodiments, the lung cancer is a non-small-cell lung cancer (NSCLC). In particular embodiments, the NSCLC is selected from squamous NSCLC, adenocarcinoma NSCLC, or a combination thereof.

[0042] In some embodiments of any aspect of the disclosure, the anti-FRa antibody or antigenbinding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT).

[0043] In some embodiments of any aspect of the disclosure, the anti-FRa antibody or antigenbinding fragment thereof comprises a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments of any aspect of the disclosure, the anti-FRa antibody or antigenbinding fragment thereof comprises:

[0044] L at the N-terminus (e.g. position 1) of the VH;

[0045] E at the N-terminus (e.g. position 1) of the VH; or

[0046] Q at the N-terminus (e.g. position 1) of the VH.

[0047] In some embodiments of any aspect of the disclosure, the anti-FRa antibody or antigenbinding fragment thereof comprises a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8.

[0048] In some embodiments of any aspect of the disclosure, the anti-FRa antibody comprises a constant heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19 or 21 and a constant light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

[0049] In some embodiments of any aspect of the disclosure, the anti-FRa antibody comprises a constant heavy chain amino acid sequence of SEQ ID NO: 19 or 21 and a constant light chain amino acid sequence of SEQ ID NO: 20.

[0050] In some embodiments of any aspect of the disclosure, the antigen-binding fragment comprises a constant heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19 and a constant light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

[0051] In some embodiments of any aspect of the disclosure, the antigen-binding fragment comprises a constant heavy chain amino acid sequence of SEQ ID NO: 19 and a constant light chain amino acid sequence of SEQ ID NO: 20.

[0052] In some embodiments of any aspect of the disclosure, the anti-FRa antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9 and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10.

[0053] In some embodiments of any aspect of the disclosure, the anti-FRa antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10.

[0054] In some embodiments of any aspect of the disclosure, the antigen-binding fragment is a Fab fragment, a Fab’ fragment, or a F(ab’)2 fragment.

[0055] In some embodiments of any aspect of the disclosure, the anti-FRa antibody or antigenbinding fragment thereof is fully human. In some embodiments of any aspect of the disclosure, the anti-FRa antibody or antigenbinding fragment thereof is monoclonal, polyclonal, recombinant, or multispecific.

[0056] In some embodiments of any aspect of the disclosure, the anti-FRa antibody or antigenbinding fragment thereof is of the IgGl, IgG2, IgG3 or IgG4 type. In particular embodiments, the anti-FRa antibody or antigen-binding fragment thereof is of the IgGl type.

[0057] In some embodiments of any aspect of the disclosure, the topoisomerase I inhibitor is represented by formula A*: and salts and solvates thereof.

[0058] In some embodiments of any aspect of the disclosure, the topoisomerase I inhibitor is linked to the anti-FRa antibody or antigen-binding fragment thereof via a linker, wherein the linker + topoisomerase I inhibitor is:

[0059]

[0060] In particular embodiments, the linker + topoisomerase I inhibitor is:

[0061] In some embodiments of any aspect of the disclosure, the drug to antibody ratio (DAR) of the ADC is in the range of about 1 to 20. In particular embodiments, the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In some embodiments of any aspect of the disclosure, the DAR of the ADC is about 8 or about 4. In particular embodiments, the DAR is about 8.

[0062] In some embodiments of any aspect of the disclosure:

[0063] (i) the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigenbinding fragment thereof has a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8, optionally wherein the anti-FRa antibody has a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10;

[0064] (ii) the anti-FRa antibody or antigen-binding fragment thereof is conjugated to SG3932

[0065] (iii) the DAR of the ADC is about 8.

[0066] In another aspect, there is provided a method of treating cancer in a human subject in need thereof, comprising administering to the human subject an antibody-drug conjugate (ADC) in an amount from about 0.8 mg / kg to about 5.0 mg / kg, wherein the ADC comprises an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor, wherein:

[0067] (i) the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti -FRa antibody or antigenbinding fragment thereof has a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8, optionally wherein the anti-FRa antibody has a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID

[0068] NO: 10;

[0069] (ii) the anti-FRa antibody or antigen-binding fragment thereof is conjugated to

[0070] SG3932

[0071] (iii) the DAR of the ADC is about 8.

[0072] In particular embodiments, the amount of ADC administered is about 1.6 mg / kg. In particular embodiments, the amount of ADC administered is about 2.0 mg / kg. In particular embodiments, the amount of ADC administered is about 2.4 mg / kg.

[0073] In particular embodiments, about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0074] BRIEF DESCRIPTION OF FIGURES

[0075] The present disclosure will now be described in more detail with reference to the attached Figures, in which: Figure 1 shows the schematic structure of AZD5335. The FRa monoclonal antibody

[0076] AB 1370049 is conjugated to the TOPOi using a cleavable peptide linker (linker + TOPOi = SG3932 payload). AZD5335 has an average drug-antibody ratio of 8. TOPOi, topoisomerase 1 inhibitor.

[0077] Figure 2 shows the results of preclinical studies of AZD5335 in a xenograft model of OC resistant to a surrogate of mirvetuximab soravtansine (FRa-MTI), an FRa ADC with a microtubule inhibitor payload, demonstrating the ability of AZD5335 to significantly reduce tumour volume in this model. FRa, folate receptor a; MTI, microtubule inhibitor; TOPOi, topoisomerase 1 inhibitor.

[0078] Figure 3 shows the study design of the dose escalation of AZD5335 monotherapy in human subjects. ECOG PS, Eastern Cooperative Oncology Group performance status; FRa, folate receptor a; IV, intravenous; mTPI-2, modified toxicity probability interval-2; PRROC, platinum-resistant recurrent ovarian cancer; Q3W, once every 3 weeks.

[0079] Figure 4 shows Cycle 1 plasma concentration of AZD5335, total antibody, and the total unconjugated payload in patient samples from the Phase l / 2a FONTANA clinical trial (data represented as geometric means and 95% confidence intervals).

[0080] Figure 5 shows Cycle 1 dose-normalised Cmax and AUC of AZD5335 and the total unconjugated payload in patient samples from the Phase l / 2a FONTANA clinical trial. ADC, antibody-drug conjugate; AUC, area under curve; Cmax, maximum serum concentration.

[0081] Figure 6 shows the best percentage change in target lesion size from baseline in patients treated with AZD5335 in the Phase l / 2a FONTANA clinical trial. Three patients were non- evaluable for radiological response and were therefore not included. FRa, folate receptor a; PD, progressive disease.

[0082] Figure 7 shows the best objective response and treatment status for individual patients treated with AZD5335 in the Phase l / 2a FONTANA clinical trial. cPR, confirmed partial response; NE, not evaluable; PD, progressive disease; SD, stable disease; uPR, unconfirmed partial response.

[0083] Figure 8 shows the pharmacodynamic biomarker data for (A) circulating tumour DNA (ctDNA) and (B) CA-125 at C2D1 (week 3), C3D1 (week 6) and C4D1 (week 9) for the combined M1A+M1B1 dataset with dose levels of 1.6 mg / kg, 2.0 mg / kg and 2.4 mg / kg. BOR: best overall response; CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease.

[0084] Figure 9 shows the dose adjusted observed ADC (AZD5335) (A), total antibody (B) and total unconjugated payload (C) exposure by FRa expression status. The dots represent the PK parameter in individual participant. The box presents the 25th to 75th quantile of the tumour reduction changes. The horizontal black line is the median. The upper and lower whisker are 1.5xIQR (inter-quartile range) from the upper and lower bound of the box. FRa-high is defined as > 75% TC with > 2+ membrane intensity. FRa-low is defined as > 25% tumour cells with > 1+ membrane intensity excluding > 75% tumour cells > 2+ membrane intensity.

[0085] Figure 10 shows the best overall change of tumour reduction from baseline based on PK (ADC AUC1) exposure quantiles. The dots represent the sum of lesion diameter change from baseline in individual participant. The box presents the 25th to 75th quantile of the tumour reduction changes. The horizontal black line is the median. The upper and lower whisker are 1.5*IQR (inter-quartile range) from the upper and lower bound of the box. FRa-high is defined as > 75% TC with > 2+ membrane intensity. FRa-low is defined as > 25% tumour cells with > 1+ membrane intensity excluding > 75% tumour cells > 2+ membrane intensity.

[0086] Figure 11 shows tumour growth inhibition as a function of time (cycles) based on PK (ADC AUC1) exposure quantiles (QI : 45 to 135 pg / mL*day; Q2: 135 to 180 pg / mL*day; Q3: 180 to 216 pg / mL*day; Q4: 216 to 414 pg / mL*day) for (A) all participants, (B) participants with FRa-high expression and (C) participants with FRa-low expression. Points represent median change from baseline in the sum of lesion diameters as a function of time expressed as the number of cycles on treatment. Rectangles denote the number of participants contributing to each data point. Cycle information was derived based on dosing information for each participant and date of tumour scan assessment. FRa-high is defined as > 75% TC with > 2+ membrane intensity. FRa-low is defined as > 25% tumour cells with > 1+ membrane intensity excluding > 75% tumour cells > 2+ membrane intensity.

[0087] DETAILED DESCRIPTION

[0088] General definitions

[0089] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure.

[0090] Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0091] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” includes a plurality of such agents and reference to “the agent” includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth. “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. In particular embodiments, the term “about” shall be understood herein as plus or minus (±) 5%, ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, of the numerical value of the number with which it is being used. Embodiments described herein as "comprising" one or more features may also be considered as disclosure of the corresponding embodiments "consisting of' such features.

[0092] Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term “protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and / or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3 -letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0093] Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0094] Antibody formats and definitions

[0095] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen.

[0096] The antibodies of the present disclosure are generally isolated or recombinant. “Isolated”, when used herein refers to a polypeptide, e.g. an antibody, that has been identified and separated and / or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated antibody will be prepared by at least one purification step. Thus, an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. For instance, an isolated antibody that specifically binds to FRa is substantially free of antibodies that specifically bind antigens other than FRa.

[0097] Generally, an antibody comprises at least two “light chains” (LC) and two “heavy chains” (HC). The light chains and heavy chains of such antibodies are polypeptides consisting of several domains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as “VH”) and a heavy chain constant region (abbreviated herein as “CH”). The heavy chain constant region comprises the heavy chain constant domains CHI, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain (abbreviated herein as “VL”) and a light chain constant domain (abbreviated herein as “CL”).

[0098] In some embodiments, the antibody is a full-length antibody. An “intact” or “full- length” antibody, as used herein, refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulphide bonds.

[0099] A “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 variable regions VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) (also known as hypervariable regions), interspersed with regions that are more conserved, termed framework regions (FRs). In particular embodiments, each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region.

[0100] Binding between an antibody and its target antigen or epitope is mediated by the CDRs. The term “epitope” refers to a target protein region (e.g. polypeptide) capable of binding to (e.g. being bound by) an antibody or antigen-binding fragment of the disclosure. The CDRs are the main determinants of antigen specificity. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e. Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273 :927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0101] The sequence of a CDR may be identified by reference to any number system known in the art, for example, the Kabat system (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); the Chothia system (Chothia &, Lesk, “Canonical Structures for the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196, 901-917 (1987)); or the IMGT system (Lefranc el al., “IMGT Unique Numbering for Immunoglobulin and Cell Receptor Variable Domains and Ig superfamily V-like domains,” Dev. Comp. Immunol. 27, 55-77 (2003)) (see

[0102] Table 1)

[0103] Table 1: CDR definitions

[0104] The “constant domains” (or “constant regions”) of the heavy chain and of the light chain are not involved directly in binding of an antibody to a target, but exhibit various effector functions. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system.

[0105] There are five major classes of heavy chain constant region, classified as IgA, IgG, IgD, IgE and IgM, each with characteristic effector functions designated by isotype. Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and FcyRIII. Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody- coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production. The important sequences for the binding of IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains.

[0106] In particular embodiments, the antibody or antigen-binding fragment thereof is based on an IgG isotype. The antibody or antigen-binding fragment can be any IgG subclass, for example IgGl, IgG2, IgG3, or IgG4 isotype. In particular embodiments, the antibody or antigen-binding fragment thereof is based on an IgGl isotype. The use of a wildtype human IgGl molecule that is close to a natural IgG could reduce developability and other risks. For example, the present inventors have devised anti-FRa ADCs using a human IgGl mAh structure, which it is believed, without being bound by theory, will be less immunogenic than other anti-FRa ADCs being developed such as IMGN151.

[0107] For heavy chain constant region amino acid positions discussed in the antibodies of the disclosure, numbering is according to the EU index first described in Edelman, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85). The EU numbering of Edelman is also set forth in Kabat et al. (1991) (supra.). Thus, the terms “EU index as set forth in Kabat”, “EU Index”. “EU index of Kabat” or “EU numbering” in the context of the heavy chain refers to the residue numbering system based on the human IgGl EU antibody of Edelman etal. as set forth in Kabat et al. (1991). The numbering system used for the light chain constant region amino acid sequence is similarly set forth in Kabat et al. (supra ). Thus, as used herein, “numbered according to Kabat” refers to the Kabat numbering system set forth in Kabat et al. (supra.).

[0108] The terms “Fc region”, “Fc part” and “Fc” are used interchangeably herein and refer to the portion of a native immunoglobulin that is formed by two Fc chains. Each “Fc chain” comprises a constant domain CH2 and a constant domain CH3. Each Fc chain may also comprise a hinge region. A native Fc region is homodimeric. In some embodiments, the Fc region may be heterodimeric because it may contain modifications to enforce Fc heterodimerisation. The Fc region contains the carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor), and has no antigen binding activity. Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been found in a number of Fc domain sites, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, resulting in minor variations between the sequences described in the instant application and sequences known in the art. As a result, every naturally occurring IgG Fc region is referred to as a “wild type IgG Fc domain” or “WT IgG Fc domain” (i.e. any allele). Human IgGl, IgG2, IgG3, and IgG4 heavy chain sequences can be obtained in a variety of sequence databases, including the UniProt database (www.uniprot.org) under accession numbers P01857 (IGHG1 HUMAN), P01859 (IGHG2 HUMAN), P01860 (IGHG3 HUMAN), and P01861 (IGHG4 HUMAN) respectively.

[0109] In some embodiments, the antibody is a monoclonal antibody. A “monoclonal antibody” (mAb) refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” can encompass both full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals. In particular embodiments, the antibody is an isolated monoclonal antibody. In more particular embodiments, the antibody is a fully human monoclonal antibody. In alternative embodiments, methods of the disclosure may employ ADCs having polyclonal antibodies.

[0110] The antibodies and antigen-binding fragments thereof of the disclosure may be derived from any species by recombinant means. For example, the antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. For use in administration to humans, non-human derived antibodies or antigen-binding fragments may be genetically or structurally altered to be less immunogenic upon administration to the human patient. In particular embodiments, the antibodies and antigen-binding fragments thereof of the disclosure are human or humanised antibodies, especially as recombinant human or humanised antibodies. In some embodiments, the anti-FRa antibody or antigen-binding fragment thereof is a human antibody.

[0111] The term “human antibody” means an antibody produced in a human or an antibody having an amino acid sequence corresponding to an antibody produced in a human made using any technique known in the art. A human antibody may include intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides. A human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. mutations introduced by random or site-specific mutagenesis in vitro or during gene rearrangement or by somatic mutation in vivo). A human antibody can be made in a human cell (through recombinant expression), a non-human animal, or a prokaryotic or eukaryotic cell that can express functionally rearranged human immunoglobulin (such as heavy and light chain) genes. A linker peptide that is not found in native human antibodies can be included in a single chain human antibody. For example, an Fv may have a linker peptide, such as two to about eight glycine or other amino acid residues, that joins the heavy chain’s variable region and the light chain’s variable region. These linker peptides are considered to be of human origin. Human antibodies can be produced using a variety of techniques, including phage display techniques that use antibody libraries derived from human immunoglobulin sequences. Transgenic mice that are unable to express functional indigenous immunoglobulins but can express human immunoglobulin genes can also be used to make human antibodies (see, for example, PCT Publication Nos. WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, each of which is incorporated by reference herein). Human antibodies can also be directly prepared using various techniques known in the art. Immortalised human B lymphocytes immunised in vitro or isolated from an immunised individual that produce an antibody directed against a target antigen can be generated. See, e.g. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147 (l):86-95 (1991); U.S. Patent 5,750,373.

[0112] The term “humanised antibody” refers to an antibody in which the framework or CDRs have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. For example, a murine CDR may be grafted into the framework region of a human antibody to prepare the “humanised antibody.” See, e.g. Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270. In some embodiments, “humanised antibodies” are those in which the constant region has been additionally modified or changed from that of the original antibody to generate desirable properties.

[0113] Humanised antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanised and engineered products using three-dimensional models of the parental, engineered, and humanised sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen, such as FRa . In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

[0114] Humanised antibodies can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimise antibody specificity, affinity, and / or capability. In general, humanised antibodies will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Humanised antibodies can 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 humanised antibodies are described in U.S. Pat. Nos. 5,225,539 or 5,639,641, each of which is incorporated by reference herein.

[0115] The term “chimeric antibody” refers to an antibody comprising a variable region, i.e. binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. In particular embodiments, the chimeric antibody may comprise a murine variable region and a human constant region. Alternatively or in addition to, “chimeric antibodies” encompassed by the present disclosure may be those in which the constant region has been modified or changed from that of the original antibody to generate desirable properties. Such chimeric antibodies are also referred to as “cl ass- switched antibodies”. Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involving conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g. Morrison, S.L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US Patent Nos. 5,202,238 and 5,204,244, each of which is incorporated by reference herein.

[0116] In some embodiments, the antibody of the disclosure is a full-length antibody described above. Alternatively, the antibody can be an antigen-binding fragment. The term “antigenbinding fragment” as used herein includes any naturally-occurring or artificially-constructed configuration of an antigen-binding polypeptide comprising one, two or three light chain CDRs, and / or one, two or three heavy chain CDRs, wherein the polypeptide is capable of binding to the antigen. In some embodiments, the anti-FRa antibody is a full-length antibody.

[0117] In some embodiments, the antibody of the disclosure is a multispecific antibody. In some embodiments, the antibody of the disclosure is a bispecific antibody.

[0118] In some embodiments, the antigen-binding fragment of the disclosure is a Fab fragment. The antibody according to the disclosure can also be a Fab', an Fv, an scFv, an Fd, a V NAR domain, an IgNAR, an intrabody, an IgG CH2, a minibody, a single-domain antibody, an Fcab, an scFv-Fc, F(ab')2, a di-scFv, a bi-specific T-cell engager (BiTE®), a F(ab')3, a tetrabody, a triabody, a diabody, a DVD-Ig, an (scFv)2, a mAb2 or a DARPin. The terms “Fab fragment” and “Fab” are used interchangeably herein and contain a single light chain (e.g. a constant domain CL and a VL) and a single heavy chain (e.g. a constant domain CHI and a VH). The heavy chain of a Fab fragment is not capable of forming a disulphide bond with another heavy chain.

[0119] A “Fab1fragment” contains a single light chain and a single heavy chain but in addition to the CHI and the VH, a “Fab1fragment” contains the region of the heavy chain between the CHI and CH2 domains that is required for the formation of an inter-chain disulphide bond. Thus, two “Fab1fragments” can associate via the formation of a disulphide bond to form a F(ab')2 molecule.

[0120] A “F(ab')2 fragment” contains two light chains and two heavy chains. Each chain includes a portion of the constant region necessary for the formation of an inter-chain disulphide bond between two heavy chains.

[0121] An “Fv fragment” contains only the variable regions of the heavy and light chain. It contains no constant regions.

[0122] A “single-domain antibody” is an antibody fragment containing a single antibody domain unit (e.g. VH or VL).

[0123] A “single-chain Fv” (“scFv”) is an antibody fragment containing the VH and VL domain of an antibody, linked together to form a single chain. A polypeptide linker is commonly used to connect the VH and VL domains of the scFv.

[0124] A “tandem scFv”, also known as a TandAb®, is a single-chain Fv molecule formed by covalent bonding of two scFvs in a tandem orientation with a flexible peptide linker.

[0125] A “bi-specific T cell engager” (BiTE®) is a fusion protein consisting of two single-chain variable fragments (scFvs) on a single peptide chain. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumour cell antigen.

[0126] A “diabody” is a small bivalent and bispecific antibody fragment comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites.

[0127] A “DARPin” is a bispecific ankyrin repeat molecule. DARPins are derived from natural ankyrin proteins, which can be found in the human genome and are one of the most abundant types of binding proteins. A DARPin library module is defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin libraries. The library modules resemble human genome sequences. A DARPin is composed of 4 to 6 modules. Because each module is approx. 3.5 kDa, the size of an average DARPin is 16-21 kDa. Selection of binders is done by ribosome display, which is completely cell-free and is described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof can be further modified to contain additional chemical moieties not normally part of a protein. Those derivatised moieties can improve the solubility, the biological half-life or absorption of the antibody or antigen-binding fragment thereof. The moieties can also reduce or eliminate any undesirable side effects of the antibodies or antigen-binding fragments thereof. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0129] Antibody sequences

[0130] The present disclosure encompasses the antibodies or antigen-binding fragments defined herein having the recited CDR sequences or variable heavy and variable light chain sequences (reference antibodies), as well as functional variants thereof. A functional variant binds to the same target antigen as the reference antibody, and may exhibit the same antigen cross-reactivity as the reference antibody. The functional variants may have a different affinity for the target antigen when compared to the reference antibody, or it may be substantially the same affinity.

[0131] In some embodiments, functional variants of a reference antibody show sequence variation at one or more CDRs when compared to corresponding reference CDR sequences. Thus, a functional antibody variant may comprise a functional variant of a CDR. Where the term “functional variant” is used in the context of a CDR sequence, this means that the CDR has at most 2 amino acid differences, or at most 1 amino acid difference when compared to a corresponding reference CDR sequence, and when combined with the remaining 5 CDRs (or variants thereof) enables the variant antibody to bind to the same target antigen as the reference antibody, and in particular embodiments exhibit the same antigen cross-reactivity as the reference antibody. A functional variant may be referred to as a “variant antibody”.

[0132] Minor variations in the amino acid sequences of the antibody are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence(s) maintain at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to the antibody or antigen-binding fragment thereof as defined anywhere herein. In particular embodiments, the variations in the amino acid sequence(s) maintain at least 99% sequence identity to the antibody of the disclosure or antigen-binding fragment thereof as defined anywhere herein.

[0133] The antibody may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In some embodiments, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. In particular, conservative amino acid replacements are contemplated.

[0134] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, or histidine), acidic side chains (e.g. aspartic acid or glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. The inclusion of conservatively modified variants in the antibody does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.

[0135] “Non-conservative amino acid substitutions” include those in which (i) a residue having an electropositive side chain (e.g. Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g. Glu or Asp), (ii) a hydrophilic residue (e.g. Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g. Ala, Leu, He, Phe or Vai), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g. Vai, His, He or Trp) is substituted for, or by, one having a smaller side chain (e.g. Ala or Ser) or no side chain (e.g. Gly).

[0136] In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of the antibody. A limited number of non- conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. The antibody can also comprise non- naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3 -methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl -threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesising amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al.. J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271 : 19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g. phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g. 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0137] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of the antibody.

[0138] Essential amino acids in the antibody can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the antibody.

[0139] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomising two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenised polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g. Lowman et al., Biochem. 30: 10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988).

[0140] The “percent sequence identity” between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimise alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

[0141] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g. segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g. CLUSTAL W, see, e.g. Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g. Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g. Match-box, see, e.g. Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g. C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g. Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428- 1435 (2004).

[0142] Percent sequence identity can be determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-19, 1992. Briefly, two amino acid sequences are aligned to optimise the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).

[0143] In some embodiments, the variable domains in both the heavy and light chains of an antibody or antigen-binding fragment thereof are altered by at least partial replacement of one or more CDRs and / or by partial framework region replacement and sequence changing. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and in certain embodiments from an antibody from a different species. It is not necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen-binding capacity of one variable domain to another. Rather, it is only necessary to transfer those residues that are necessary to maintain the activity of the antigen-binding site. Given the explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, each of which is incorporated by reference herein, it will be well within the competence of those skilled in the art to carry out routine experimentation to obtain a functional antibody with reduced immunogenicity.

[0144] In some embodiments, the antibody or antigen-binding fragment thereof can include, in addition to a VH and a VL, a heavy chain constant region or fragment thereof. In some embodiments, the heavy chain constant region is a human heavy chain constant region, e.g. a human IgG constant region, e.g. a human IgGl constant region.

[0145] In some embodiments, a residue is inserted to the heavy chain constant region of the antibody for site-specific conjugation e.g. for conjugating the cytotoxin. For example, a cysteine residue may be inserted between amino acid S239 and V240 in the CH2 region of IgGl, which may be referred to as “a 239 insertion” or “239i ” In some embodiments, the antibody can be modified to comprise alterations or modifications to one or more of the three heavy chain constant domains (CHI, CH2 or CH3) and / or to the light chain constant domain (CL). In some embodiments, a modified constant region wherein one or more domains are partially or entirely deleted are contemplated. In some embodiments, a modified antibody will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ACH2 constructs). In some embodiments, the omitted constant region domain can be replaced by a short amino acid spacer (e.g. 10 residues) that provides some of the molecular flexibility typically imparted by the absent constant region. The deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody. In other cases, it can be that the constant region modifications moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region can be used to eliminate disulphide linkages or oligosaccharide moieties that allow for enhanced localisation due to increased antigen specificity or antibody flexibility. In some embodiments, the antibody or antigen-binding fragment thereof has no antibody-dependent cellular cytotoxicity (ADCC) activity and / or no complement-dependent cytotoxicity (CDC) activity.

[0146] In some embodiments, the antibody or antigen-binding fragment thereof can be engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibodies or fragments thereof. In other constructs a peptide spacer can be inserted between the hinge region and the modified CH2 and / or CH3 domains. For example, compatible constructs can be expressed in which the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such a spacer can be added, for instance, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. Amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. In some embodiments, any spacer added to the construct can be relatively non- immunogenic, or even omitted altogether, so as to maintain the desired biochemical qualities of the modified antibodies.

[0147] Besides the deletion of whole constant region domains, an antibody or antigen-binding fragment thereof provided herein can be modified by the partial deletion or substitution of a few or even a single amino acid in a constant region. For example, the mutation of a single amino acid in selected areas of the CH2 domain can be enough to substantially reduce Fc binding and thereby increase tumour localisation. Similarly one or more constant region domains that control the effector function (e.g. complement C1Q binding) can be fully or partially deleted. Such partial deletions of the constant regions can improve selected characteristics of the antibody or antigen-binding fragment thereof (e.g. serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, the constant regions of the antibody and antigen-binding fragment thereof can be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it is possible to disrupt the activity provided by a conserved binding site (e.g. Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody or antigen-binding fragment thereof. In some embodiments, there may be an addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function or provide for more cytotoxin or carbohydrate attachment. In some embodiments, it can be desirable to insert or replicate specific sequences derived from selected constant region domains. In some embodiments, a heavy chain constant region or fragment thereof, e.g. a human IgG constant region or fragment thereof, can include one or more amino acid substitutions relative to a wildtype IgG constant domain wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type IgG constant domain. For example, the IgG constant domain can contain one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, wherein the amino acid position numbering is according to the EU index as set forth in Kabat. In some embodiments the IgG constant domain can contain one or more of a substitution of the amino acid at Kabat position 252 with Tyrosine (Y), Phenylalanine (F), Tryptophan (W), or Threonine (T), a substitution of the amino acid at Kabat position 254 with Threonine (T), a substitution of the amino acid at Kabat position 256 with Serine (S), Arginine (R), Glutamine (Q), Glutamic acid (E), Aspartic acid (D), or Threonine (T), a substitution of the amino acid at Kabat position 257 with Leucine (L), a substitution of the amino acid at Kabat position 309 with Proline (P), a substitution of the amino acid at Kabat position 311 with Serine (S), a substitution of the amino acid at Kabat position 428 with Threonine (T), Leucine (L), Phenylalanine (F), or Serine (S), a substitution of the amino acid at Kabat position 433 with Arginine (R), Serine (S), Isoleucine (I), Proline (P), or Glutamine (Q), or a substitution of the amino acid at Kabat position 434 with Tryptophan (W), Methionine (M), Serine (S), Histidine (H), Phenylalanine (F), or Tyrosine. In some embodiments, the antibody or antigen-binding fragment thereof comprises a YTE mutant. The terms “YTE” or “YTE mutant” refer to a mutation in IgGl Fc that results in an increase in the binding to human FcRn and improves the serum half-life of the antibody having the mutation. A YTE mutant comprises a combination of three mutations, M252Y / S254T / T256E (EU numbering Kabat et al. (1991) Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, D.C.), introduced into the heavy chain of an IgGl. See U.S. Patent No. 7,658,921, which is incorporated by reference herein. The YTE mutant has been shown to increase the serum half-life of antibodies approximately four-times as compared to wild-type versions of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281 :23514-24 (2006); Robbie et al., (2013) Antimicrob. Agents Chemother. 57, 6147- 6153). See also U.S. Patent No. 7,083,784, which is hereby incorporated by reference in its entirety.

[0148] Antibody preparation

[0149] The antibody or antigen-binding fragment thereof of the disclosure can be produced by transfecting a host cell with one or more vectors comprising polynucleotides encoding the respective antibodies or fragments, culturing the host cell under conditions that allow synthesis of said antibody or antigen-binding fragment molecule; and recovering said antibody or antigen-binding fragment molecule from said culture.

[0150] The antibody or antigen-binding fragment thereof (e.g. as monoclonal antibodies) can be made using recombinant DNA methods as described in U.S. Patent No. 4,816,567, which is incorporated by reference herein. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Also, recombinant monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described in McCafferty et al., Nature 348:552-554 (1990); Clackson etal., Nature, 352:624-628 (1991); and Marks etal., J. Mol. Biol. 222:581-597 (1991).

[0151] Affinity maturation strategies and chain shuffling strategies are known in the art and can be employed to generate high affinity human antibodies or antigen-binding fragments thereof. See Marks et al., BioTechnology 10:779-783 (1992), incorporated by reference in its entirety. Various techniques are known for the production of antibody fragments. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies, as described, for example, by Morimoto et al., J. Biochem. Biophys. Meth. 24: 107-117 (1993) and Brennan et al., Science 229:81 (1985). In some embodiments, an antibody fragment is produced recombinantly. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coll or other host cells, thus allowing the production of large amounts of these fragments. Such antibody fragments can also be isolated from the antibody phage libraries discussed above. The antibody fragment can also be linear antibodies as described in U.S. Patent No. 5,641,870, which is incorporated by reference herein. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0152] Techniques can be adapted for the production of single-chain antibodies specific to a certain target (e.g. FRa). (see, e.g. U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for FRa, or derivatives, fragments, analogs or homologs thereof. See, e.g. Huse et al., Science 246: 1275-1281 (1989). Antibody fragments can be produced by techniques known in the art including, but not limited to: F(ab')2 fragment produced by pepsin digestion of an antibody molecule; Fab fragment generated by reducing the disulphide bridges of an F(ab')2 fragment; Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent; or Fv fragments.

[0153] Anti-FRa antibodies

[0154] An exemplary array of anti-FRa antibodies were developed with a high affinity and specific binding to FRa on cancer cells (e.g. does not specifically bind to other FR family members such as FRP and FRy), as described in WO 2023 / 169896. A thorough assessment of antibody developability, checking propensity for reversible-self association, internalisation, non-specific binding and hydrophobicity and the stability of the mAbs to thermal and photo stressors was carried out. Additionally, an in vivo mouse PK study was employed for a focused panel of mAbs to remove any that exhibited poor in vivo half-life and increased clearance. In order to reduce the likelihood of developing an anti-drug antibody (ADA) response in humans, an in silico immunogenicity assessment was conducted to remove any antibodies from consideration with increased predicted risk of ADA response. Through the above screening strategy, a panel of 6 antibodies (AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 and AB1370117) with similar, beneficial properties was identified, as described in WO 2023 / 169896. In particular, Example 1 of WO 2023 / 169896 describes the generation of anti-FRa antibodies. Examples 2, 3 and 5-9 describe the screening of anti-FRa antibodies through assessment of species cross-reactivity, internalisation, binding affinities, physiochemical properties and pharmacokinetics in mice. Example 4 describes the epitope binning of the generated anti-FRa antibodies. Thus, Examples 1-9 of WO 2023 / 169896 are incorporated by reference herein. In some embodiments, these antibodies are included in the ADCs described in the present disclosure. In a particular embodiment, the construct AB 1370049 described in WO 2023 / 169896 is included in the ADCs of the present disclosure.

[0155] Antibody sequences

[0156] Tables 2-6 show the CDR sequences, the VH and VL sequences, the heavy chain and light chain sequences, the FR sequences and the constant domain sequences, respectively, of the construct AB1370049. In the event of any discrepancy, the sequences in the Tables take precedence. The sequences of the constructs AB1370026, AB1370035, AB1370083, AB1370095 and AB1370117 are described in Tables 1-5 of WO 2023 / 169896, which are incorporated by reference herein.

[0157] In some embodiments, the anti-FRa antibody or antigen-binding fragment thereof comprises the 6 CDRs of the construct AB1370049 of Table 2 of the present disclosure, or the 6 CDRs of any one of the constructs AB1370026, AB1370035, AB1370083, AB1370095 and AB 1370117 as described in Table 2 of WO 2023 / 169896.

[0158] In some embodiments, the anti-FRa antibody, or antigen-binding fragment, comprises: a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); wherein any one or more of said CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to said sequences.

[0159] In some embodiments, the anti-FRa antibody, or antigen-binding fragment, comprises: a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT).

[0160] In some embodiments, the anti-FRa antibody, or antigen-binding fragment thereof, has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 7 and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-FRa antibody, or antigen-binding fragment thereof, comprises a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8.

[0161] In some embodiments, the anti-FRa antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 9 and a light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-FRa antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10.

[0162] In some embodiments of any of the aspects described herein, the anti-FRa antibody, or antigen-binding fragment thereof, comprises heavy chain VH FR1, VH FR2, VH FR3, and / or VH FR4 that is at least 80%, 85%, 90% or 95% identical, or identical to reference heavy chain VH FR1, VH FR2, VH FR3, and / or VH FR4, respectively of the construct AB1370049 described in Table 5 of the present disclosure, or any one of the constructs AB 1370026, AB1370035, AB1370083, AB1370095 and AB1370117 as described in Table 4 of WO 2023 / 169896, wherein the antibody or fragment is capable of binding FRa alone (e.g. in the form of a single chain antibody fragment).

[0163] In some embodiments of any of the aspects described herein, the anti-FRa antibody, or antigen-binding fragment thereof, comprises light chain VL FR1, VL FR2, VL FR3, and / or VL FR4 that are at least 80%, 85%, 90% or 95% identical, or identical to reference light chain VL FR1, VL FR2, VL FR3, and / or VL FR4, respectively of the construct AB 1370049 described in Table 5 of the present disclosure, or any one of the constructs AB1370026, AB1370035, AB1370083, AB1370095 and AB1370117 as described in Table 4 of WO 2023 / 169896, wherein the antibody or fragment is capable of binding FRa alone (e.g. in the form of a single chain antibody fragment). In some embodiments of any of the aspects described herein, the anti-FRa antibody, or antigen-binding fragment thereof, comprises (a) light chain VL FR1, VL FR2, VL FR3, and VL FR4 that are at least 80%, 85%, 90% or 95% identical, or identical to reference light chain VL FR1, VL FR2, VL FR3, and VL FR4, respectively, of the construct AB 1370049 as described in Table 5 of the present disclosure, or any one of the constructs AB 1370026, AB1370035, AB1370083, AB1370095 and AB1370117 as described in Table 4 of WO 2023 / 169896; and (b) heavy chain VH FR1, VH FR2, VH FR3, and VH FR4 that are at least 80%, 85%, 90% or 95% identical, or identical to reference heavy chain VH FR1, VH FR2, VH FR3, and VH FR4, respectively, of the construct AB 1370049 as described in Table 5 of the present disclosure, or any one of the constructs AB1370026, AB1370035, AB1370083, AB1370095 and AB1370117 as described in Table 4 of WO 2023 / 169896.

[0164] In some embodiments of any of the aspects described herein, the anti-FRa antibody comprises a constant heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 19 and a constant light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-FRa antibody comprises a constant heavy chain amino acid sequence of SEQ ID NO: 19 and a constant light chain amino acid sequence of SEQ ID NO: 20.

[0165] In some embodiments of any of the antigen-binding fragment aspects described herein, the anti-FRa antigen-binding fragment comprises a constant heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 21 and a constant light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-FRa antigen-binding fragment comprises a constant heavy chain amino acid sequence of SEQ ID NO: 21 and a constant light chain amino acid sequence of SEQ ID NO: 20. Table 2: FRa antibody AB1370049 CDR sequences

[0166] Table 3: FRa antibody AB1370049 VH and VL sequences Table 4: FRa antibody AB1370049 heavy chain and light chain sequences Table 5: FRa antibody AB1370049 FR regions

[0167] Table 6: FRa antibody constant domain sequences In some embodiments, the anti -FRa antibody or antigen-binding fragment thereof in the

[0168] ADC comprises:

[0169] L at the N-terminus (e.g. position 1) of the VH;

[0170] E at the N-terminus (e.g. position 1) of the VH; or

[0171] Q at the N-terminus (e.g. position 1) of the VH. Polynucleotide sequences of the antibody

[0172] In some aspects, there is provided polynucleotides encoding the anti-FRa antibodies or antigen-binding fragments thereof of the ADCs of the disclosure. The polynucleotides encoding the anti-FRa antibodies or antigen-binding fragments thereof in the ADCs of the disclosure may be the nucleotide sequences of the construct AB1370049 in Tables 7-8 of the present disclosure, or the nucleotide sequences of any one of the constructs AB1370026, AB1370035, AB1370083, AB1370095 and AB1370117 as described in Tables 6-7 of WO 2023 / 169896, which are incorporated by reference herein. In the event of any discrepancy, the sequences in the Tables take precedence.

[0173] In another aspect, the polynucleotide encoding the anti-FRa antibodies or antigenbinding fragments thereof in the ADCs of the disclosure comprises a sequence encoding (a) VL that is at least 80%, 85%, 90% or 95% identical, or identical to reference VL nucleotide sequence of the construct AB 1370049 as described in Table 7 of the present disclosure, or any one of the constructs AB 1370026, AB 1370035, AB 1370083, AB 1370095 and AB 1370117 as described in Table 6 of WO 2023 / 169896; and (b) VH that is at least 80%, 85%, 90% or 95% identical, or identical to reference VH nucleotide sequence of the construct AB1370049 as described in Table 7 of the present disclosure, or any one of the constructs AB 1370026, AB1370035, AB1370083, AB1370095 and AB1370117 as described in Table 6 of WO 2023 / 169896.

[0174] In another aspect, the polynucleotide encoding the anti-FRa antibodies in the ADCs of the disclosure comprises a sequence encoding (a) a light chain that is at least 80%, 85%, 90% or 95% identical, or identical to reference light chain nucleotide sequence of the construct AB 1370049 as described in Table 8 of the present disclosure, or any one of the constructs AB1370026, AB1370035, AB1370083, AB1370095 and AB1370117 as described in Table 7 of WO 2023 / 169896; and (b) a heavy chain that is at least 80%, 85%, 90% or 95% identical, or identical to reference heavy chain nucleotide sequence of the construct AB1370049 as described in Table 8 of the present disclosure, or any one of the constructs AB 1370026, AB1370035, AB1370083, AB1370095 and AB1370117 as described in Table 7 of WO 2023 / 169896. Table 7: FRa antibody AB1370049 VH and VL nucleotide sequences

[0175] Table 8: FRa antibody AB1370049 heavy and light chain nucleotide sequences

[0176] The polynucleotide sequence(s) include sequences that have been removed from their naturally occurring environment, recombinant or cloned (e.g. DNA) isolates, and chemically synthesised analogues or analogues biologically synthesised by heterologous systems. The polynucleotide sequence(s) may be prepared by any means known in the art. For example, large amounts of the sequence(s) may be produced by replication and / or expression in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will typically be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured bacterial, insect, mammalian, plant or other eukaryotic cell lines.

[0177] The polynucleotide sequence(s) may also be produced by chemical synthesis, e.g. a polynucleotide by the phosphoramidite method or the tri -ester method and may be performed on commercial automated oligonucleotide synthesisers. A double-stranded (e.g. DNA) fragment may be obtained from the single stranded product of chemical synthesis either by synthesising the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

[0178] Variants of a polynucleotide are also described herein. Polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant comprises an alteration that produces silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced by a silent substitution due to the degeneracy of the genetic code. A polynucleotide variant can be produced for a variety of reasons, e.g. to optimise codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coif).

[0179] FRa binding

[0180] In particular embodiments, the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure specifically bind to FRa. The term “specifically binding to FRa” refers to an antibody that is capable of binding to the defined target with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting FRa. In some embodiments, an antibody specifically binding to FRa does not bind to other antigens, or does not bind to other antigens with sufficient affinity to produce a physiological effect. In some embodiments, the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure specifically bind to human FRa (UniProt ID: P15328) and / or cynomolgus monkey FRa (UniProt ID: A0A2K5U044). In particular embodiments, the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure specifically bind to human FRa. In particular embodiments, the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure specifically bind to human FRa and cynomolgus monkey FRa.

[0181] In some embodiments of any aspect of the disclosure, the FRa has a sequence of SEQ ID NO: 26 or SEQ ID NO: 27. In particular embodiments, the FRa has a sequence of SEQ ID NO: 26.

[0182] SEQ ID NO: 26: Human FRa protein (predicted mature, secreted polypeptide)

[0183] RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYL YRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKED CEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTH SYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMS

[0184] SEQ ID NO: 27: Cyno FRa protein (predicted mature, secreted polypeptide)

[0185] RTARARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWKKNACCSTNTSQEAHKDVSYLY RFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCE QWWEDCRTSYTCKSNWHKGWNWTSGFNKCPVGAACQPFHFYFPTPTVLCNEIWTYSYK VSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMS

[0186] In some embodiments, the antibody or antigen-binding fragment thereof in the ADC does not bind to one or more selected from a mouse FRa (UniProt ID: P35846), rat FRa (UniProt ID: G3V8M6), human FRp (UniProt ID: P14207), human FRy (UniProt ID: P41439), or a combination thereof.

[0187] The term “does not bind” means that the antibody or antigen-binding fragment thereof of the disclosure does not substantially bind to one of more of said molecules (e.g. mouse FRa, rat FRa, human FRP, human FRy, or a combination thereof). The term “substantially no” when used in the context of binding herein may mean less than 5%, 2%, 1%, 0.5% or 0.1% of cells expressing one or more of said molecules in a cell culture become bound by the antibody or antigen-binding fragment thereof in the ADC of the disclosure (upon contact therewith). Suitably, the term “substantially no” when used in the context of binding herein may mean no such cells become bound.

[0188] Binding affinity

[0189] “Binding affinity” generally refers to the strength of the sum total 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 indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.

[0190] Suitably, the antibody or antigen-binding fragment in the ADC of the disclosure binds to FRa molecule with sufficient affinity such that the antibody is useful as a therapeutic agent or a diagnostic reagent in targeting FRa.

[0191] In some embodiments, the anti -FRa antibody or antigen-binding fragment thereof in the ADC binds to human FRa with a KD of about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, about 1 nM or less.

[0192] In some embodiments, the anti -FRa antibody or antigen-binding fragment thereof in the ADC binds to human FRa with a KD of about 0.5 to about 50 nM, about 0.5 to about 40 nM, about 0.5 to about 30 nM, about 0.5 to about 20 nM, about 1 to about 50 nM, about 1 to about 40 nM, about 1 to about 30 nM, about 1 to about 20 nM, about 2 to about 50 nM, about 2 to about 40 nM, about 2 to about 30 nM, about 2 to about 20 nM, about 5 to about 50 nM, about 5 to about 40 nM, about 5 to about 30 nM, about 5 to about 20 nM, about 10 to about 50 nM, about 10 to about 40 nM, about 10 to about 30 nM or about 10 to about 20 nM.

[0193] In some embodiments, the anti -FRa antibody or antigen-binding fragment thereof in the ADC binds to cyno FRa with a KD of about 100 nM or less, about 80 nM or less, about 60 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less.

[0194] In some embodiments, the anti -FRa antibody or antigen-binding fragment thereof in the ADC binds to cyno FRa with a KD of about 1 to about 100 nM, about 1 to about 80 nM, about 1 to about 60 nM, about 1 to about 40 nM, about 2 to about 100 nM, about 2 to about 80 nM, about 2 to about 60 nM, about 2 to about 40 nM, about 5 to about 100 nM, about 5 to about 80 nM, about 5 to about 60 nM, about 5 to about 40 nM, about 10 to about 100 nM, about 10 to about 80 nM, about 10 to about 60 nM, about 10 to about 40 nM, about 20 to about 100 nM, about 20 to about 80 nM, about 20 to about 60 nM, about 20 to about 40 nM, about 30 to about 100 nM, about 30 to about 80 nM, about 30 to about 60 nM, or about 30 to about 40 nM.

[0195] The affinity or avidity of an antibody or antigen-binding fragment thereof for an antigen can be determined experimentally using any suitable method well known in the art, e.g. flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g. KINEXA® or BIACORE™ analysis). Direct binding assays as well as competitive binding assay formats can be readily employed. (See, e.g. Berzofsky et al., Antibody-Antigen Interactions, In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and methods described herein.)

[0196] The binding affinity of the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure may be determined using a FRa binding affinity assay as described herein. In some embodiments, the binding affinity of the anti-FRa antibody, or antigen-binding fragment thereof, in the ADC of the disclosure is determined by Biacore, e.g. Biacore T200 at 25°C. For example, the affinity of the recombinant human FRa ECD for the anti-FRa antibody, or antigen-binding fragment thereof, may be measured using the Biacore T200 at 25°C, for example using the following protocol. Protein A is covalently immobilised to a CM5 chip surface using standard amine coupling techniques at a concentration of 50 pg / ml in 10 mM Sodium acetate pH 4.0. The antibody, or antigen-binding fragment thereof, is captured onto the Protein A surface in HBS-EP+ buffer pH 7.4 at 10 pl / min to enable FRa ECD binding. The FRa ECD is serially diluted (0.4 nM-100 nM human FRa ECD; 0.8 nM-200 nM cyno FRa ECD; 30 nM-4000 nM mouse FRa ECD and rat FRa ECD) in HBS-EP+ buffer pH 7.4 and flowed over the chip at 50 pl / min, with 2 minutes association and 8 minutes dissociation. The chip surface is fully regenerated with pulses of 3 M MgCh to remove captured antibody, or antigen-binding fragment thereof, together with any bound FRa ECD. Multiple buffer-only injections are made under the same conditions to allow for double reference subtraction of the final sensorgram sets, which are analysed using Biacore T200 Evaluation Software.

[0197] Alternatively, the binding affinity of the anti-FRa antibody, or antigen-binding fragment thereof, in the ADC of the disclosure may be determined by Octet, e.g. Octet red. For example, the binding affinity of the anti-FRa antibody may be assayed by Octet red at 25 °C , for example using the following protocol. The binding assays are performed on Octet RED384 (ForteBio) at 25 °C in assay buffer containing PBS, 0.1% v / v BSA (Sigma, A9576), 0.01% v / v Tween-20 (Sigma, P9416) (pH 7.4) using tilted bottom black 384-well plates (ForteBio, 18-5076). Assays are set up using either protein A or anti human capture biosensors (AHC) (ForteBio, 18-5089) according to the manufacturer’s instructions. 10 pg / ml of anti-rat FRa IgG (Sino Biological, 81073-RP01) is coated onto protein A biosensors (ForteBio, NC9490476) and 10 pg / ml of test human IgG is loaded onto anti human capture biosensors (AHC) (ForteBio, 18-5089) for 180 seconds. Association is measured by incubating loaded biosensors with 500 nM human FRa (in house) or 500 nM rat FRa (Sino Biological, 81073-R08H). Dissociation is measured following transfer into assay buffer. Data are analysed using the Octet data analysis software version 7.0.

[0198] Antibody-drug conjugates (ADC)

[0199] The “antibody-drug conjugate” (ADC) of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof as described herein, wherein the anti-FRa antibody or antigen-binding fragment thereof is conjugated to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor (TOPOi).

[0200] Cytotoxin

[0201] A cytotoxin (also referred to as a cytotoxic agent) can be any molecule known in the art that inhibits or prevents the function of cells and / or causes destruction of cells (cell death), and / or exerts anti-neoplastic / anti-proliferative effects. A number of classes of cytotoxic agents are known to have potential utility in ADC molecules. In the present disclosure, the cytotoxic agent is a topoisomerase I inhibitor. The present disclosure demonstrates, using in vivo models, the efficacy of anti-FRa mAbs when conjugated to a TOPOi payload and deployed as ADCs.

[0202] The cytotoxic agent is typically linked to, or “loaded onto” the antibody or antigenbinding fragment. The agent loading (p) is the average number of agent(s) per antibody or antigen-binding fragment. It will be understood by the person skilled in the art that more than one of said agent(s) may be conjugated to the antibody or antigen-binding fragment thereof.

[0203] In some embodiments, the average number of cytotoxic agents per antibody (or antigenbinding fragment thereof) is in the range of about 1 to 20. In some embodiments the range is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In some embodiments, there is one agent per antibody (or antigen-binding fragment thereof). In some embodiments, the number of agents per antibody (or antigen-binding fragment thereof) can be expressed as a ratio of agent (i.e. drug) to antibody. This ratio is referred to as the Drug to Antibody Ratio (DAR). The DAR is the average number of drugs (i.e. cytotoxic agents) linked to each antibody. In some embodiments of the present disclosure, the DAR is in the range of about 1 to 20. In some embodiments the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4 (e.g. 3.8-4.2) or about 8 (e.g. 7.6-8.4). In particular embodiments, the DAR is about 8 (e.g. 7.6-8.4).

[0204] In some embodiments, the antibody or antigen-binding fragment thereof may be conjugated to one or more of SG3932 (also known as AZ14170133), SG4010, SG4057 or SG4052 (the structures of which are provided below), or a combination thereof. SG3932, SG4010, SG4057 and SG4052 are molecules including a topoisomerase I inhibitor and a linker (also referred to herein as “Drug Linker units” or as the “payload”). In particular embodiments, the antibody or antigen-binding fragment thereof may be conjugated to SG3932.

[0205] In certain embodiments, the antibody or antigen-binding fragment thereof in the ADC of the disclosure is not conjugated to, or the anti-FRa ADC of the disclosure does not comprise, a microtubule inhibitor such as a tubulin inhibitor (e.g. maytansinoids, auristatins). Microtubule inhibitor class of molecules suffer from potentially difficult-to-treat toxicities that limit dosing.

[0206] Topoisomerase I inhibitor

[0207] Topoisomerase inhibitors are chemical compounds that block the action of topoisomerase (topoisomerase I and II), which is a type of enzyme that controls the changes in DNA structure by catalysing the breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. Topoisomerase I inhibitors are advantageous as they mediate highly effective tumour cell killing with fewer toxicities to the patient. In particular, alternative payloads such as microtubule inhibitor that have generally been used to-date for the development of anti-FRa ADCs are known to have toxicity problems (Hinrichs, et al. AAPS J. 2015 Sep; 17(5): 1055-1064). Moreover, the use of a less hydrophobic linker with a less potent warhead (e.g. TOPOi) would facilitate bystander killing in heterogeneous tumours. Although bystander activity may be achieved by increasing the potency and / or improving warhead permeability through increased hydrophobicity, this may result in increased toxicity due to nonspecific uptake.

[0208] A general example of a suitable topoisomerase I inhibitor is represented by the following compound:

[0209] A*

[0210] Said compound is denoted as A*, and may be referred to as a “Drug Unit” herein.

[0211] The compound (e.g. A*) may be provided with a linker for connecting (particularly, conjugating) to an antibody or antigen-binding fragment in the ADC of the disclosure. In particular embodiments, the linker is attached (e.g. conjugated) in a cleavable manner to an amino residue, for example, an amino acid of an antibody or antigen-binding fragment in the ADC of the disclosure.

[0212] More particularly, an example of a suitable topoisomerase I inhibitor is represented by the following compound, with the formula “I”: and salts and solvates thereof, wherein RLis as defined herein.

[0213] Accordingly, for an ADC of the disclosure having the general formula IV:

[0214] L - (DL)P(IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L and p are defined above, DLis a topoisomerase I inhibitor having a linker (e.g. Drug Linker unit) that is of formula III: and salts and solvates thereof, wherein RLLis as defined herein. In some embodiments, the compound of formula I is of the formula Ip: and salts and solvates thereof, wherein RLPis a linker for connection to an antibody or antigenbinding fragment thereof in the ADC of the disclosure, wherein said linker is selected from: (Iap): u wherein

[0215] Qpis:

[0216] , where Qxpis such that Qpis an amino-acid residue, a dipeptide residue or a tripeptide residue;

[0217] Xpis: where aP = 0 to 5, bP = 0 to 16, cP = 0 or 1, dP = 0 to 5;

[0218] GLis defined above; (lb): where RL1and RL2are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group; and e is 0 or 1. aP may be 0, 1, 2, 3, 4 or 5. In some embodiments, aP is 0 to 3. In some of these embodiments, aP is 0 or 1. In further embodiments, aP is 0. bP may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b is 0 to 12. In some of these embodiments, bP is 0 to 8, and may be 0, 2, 4 or 8. cP may be 0 or 1. dP may be 0, 1, 2, 3, 4 or 5. In some embodiments, dP is 0 to 3. In some of these embodiments, dP is 1 or 2. In further embodiments, dP is 2.

[0219] In some embodiments of Xp, aP is 0, cP is 1 and dP is 2, and bP may be from 0 to 8. In some of these embodiments, bP is 0, 4 or 8.

[0220] The options for Qxabove for compounds of Formula I may apply to Qxp(for example, where appropriate).

[0221] The options for GL, RL1, RL2and e above for compounds of Formula I may apply to compounds of Formula Ip.

[0222] In some embodiments, the ADC of formula IV is of the formula IVP:

[0223] L - (DLP)P(IVP) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigenbinding fragment thereof, DLPis a topoisomerase I inhibitor (e.g. Drug Linker unit) that is of formula IIIP:

[0224] RLLPis a linker connected to the antibody or antigen-binding fragment thereof, wherein said linker is selected from

[0225] (Iap’): where Qp, Xpand GLLare as defined above; and (lb’): lb' where RL1and RL2are as defined above; and p is an integer of from 1 to 20.

[0226] In some embodiments, the compound of formula I is of the formula IP2: and salts and solvates thereof, wherein RLP2is a linker for connection to an antibody or antigenbinding fragment thereof in the ADC of the disclosure, wherein said linker is selected from: (IaP2): la2

[0227] , where Qxis such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; v P2 : I „S .. where aP2 = 0 to 5, blP2 = 0 to 16, b2P2 = 0 to 16, cP2 = 0 or 1, dP2 = 0 to 5, wherein at least blP2 or b2P2 = 0 (z.e. only one of bl and b2 may not be 0);

[0228] GL is a linker for connecting to an antibody or antigen-binding fragment thereof in the ADC of the disclosure;

[0229] (lb): where RL1and RL2are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group; and e is 0 or 1. aP2 may be 0, 1, 2, 3, 4 or 5. In some embodiments, aP2 is 0 to 3. In some of these embodiments, aP2 is 0 or 1. In further embodiments, aP2 is 0. blP2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, blP2 is 0 to 12. In some of these embodiments, blP2 is 0 to 8, and may be 0, 2, 3, 4, 5 or 8. b2P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b2P2 is 0 to 12. In some of these embodiments, b2P2 is 0 to 8, and may be 0, 2, 3, 4, 5 or 8.

[0230] In particular embodiments, only one of blP2 and b2P2 may not be 0. cP2 may be 0 or 1. dP2 may be 0, 1, 2, 3, 4 or 5. In some embodiments, dP2 is 0 to 3. In some of these embodiments, dP2 is 1 or 2. In further embodiments, dP2 is 2. In further embodiments, dP2 is 5.

[0231] In some embodiments of XP2, aP2 is 0, blP2 is 0, cP2 is 1 and dP2 is 2, and b2P2 may be from 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5 or 8. In some embodiments of XP2, aP2 is 1, b2P2 is 0, cP2 is 0 and dP2 is 0, and blP2 may be from 0 to 8. In some of these embodiments, blP2 is 0, 2, 3, 4, 5 or 8. In some embodiments of XP2, aP2 is 0, blP2 is 0, cP2 is 0 and dP2 is 1, and b2P2 may be from 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5 or 8. In some embodiments of XP2, blP2 is 0, b2P2 is 0, cP2 is 0 and one of aP2 and dP2 is 0. The other of aP2 and d is from 1 to 5. In some of these embodiments, the other of aP2 and d is 1. In other of these embodiments, the other of aP2 and dP2 is 5.

[0232] The options for Qxabove for compounds of Formula I may apply to Qxin Formula IaP2(e.g. where appropriate).

[0233] The options for G1, RL1, RL2and e above for compounds of Formula I may apply to compounds of Formula IP2.

[0234] In some embodiments, the ADC of formula IV is of the formula IVP2:

[0235] L - (DLP2)P(IVP2) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigenbinding fragment thereof, DLP2is a topoisomerase I inhibitor e.g. Drug Linker unit) that is of formula IIIP2:

[0236] RLLP2is a linker connected to the antibody or antigen-binding fragment thereof, wherein said linker is selected from

[0237] (IaP2’): o where Q and XP2are as defined above and G, J' is a linker connected to the antibody or antigen- binding fragment thereof; and (lb’): where RL1and RL2are as defined above; and p is an integer of from 1 to 20.

[0238] Particularly suitable topoisomerase I inhibitors comprising a linker (“Drug Linker unit”) for conjugation with the antibody component of the ADC include those having the following formulas:

[0239] ; and / or

[0240] In particular embodiments, an antibody or antigen-binding fragment thereof in the ADC of the disclosure is conjugated to SG3932 having the following formula:

[0241] Synthetic methods of making topoisomerase I inhibitors are described in, for example, WO 2020 / 200880, which is incorporated by reference herein.

[0242] It should be noted that any suitable agent (e.g. drug / cytotoxin) may be further linked to an antibody or antigen-binding fragment thereof in the ADC comprising a topoisomerase I inhibitor of the disclosure.

[0243] Linkers

[0244] In the ADC of the disclosure, the antibody or antigen-binding fragment may be conjugated to the cytotoxin by a linker.

[0245] The term “Linker" or “Spacer” as used herein means a divalent chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody or antigenbinding fragment thereof to a cytotoxin to form an ADC. In some embodiments, the linker or spacer is a peptide spacer. In some embodiments, the linker or spacer is a non-peptide (e.g. chemical) spacer. Suitable linkers have two reactive termini, one for antibody conjugation and the other for cytotoxin conjugation. Because of the formation of bonds between the linker and / or the cytotoxin, and between the linker and / or the antibody or antigen-binding fragment thereof, one or both of the reactive termini will be absent or incomplete (such as being only the carbonyl of the carboxylic acid). These conjugation reactions are discussed in more detail below.

[0246] In particular embodiments, the linker is attached (e.g. conjugated) in a cleavable manner to an amino residue, for example, an amino acid of an antibody or antigen-binding fragment in the ADC described herein.

[0247] In some embodiments, the linker is cleavable under intracellular circumstances, allowing the drug unit to be released from the antibody in the intracellular environment.

[0248] Alternatively, the linker unit may not be cleavable. In such embodiments the drug is released, for example, by antibody degradation. However, non-cleavable payloads require complete mAb digestion in the lysosome and the resulting drug-containing product may be too polar, e.g. for achieving bystander effect.

[0249] The ADC is typically stable and intact before being transported or delivered into a cell, i.e. the antibody should be attached to the drug moiety. Outside the target cell, the linkers are stable, but inside the cell, they can be cleaved at a high rate. An effective linker will: (i) maintain the antibody's specific binding properties; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e. not cleaved, until the conjugate has been delivered or transported to its targeted site; and (iv) maintain the cytotoxic moiety's cell-killing or cytostatic effect. Standard analytical methods such as mass spectroscopy, HPLC, and the separation / analysis technique LC / MS can be used to assess the stability of the ADC.

[0250] The linkers may be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulphide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0251] Linkers hydrolysable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, or the like. (See, e.g. U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661). Linkers cleavable under reducing conditions include, for example, a disulphide. A variety of disulphide linkers are known in the art, including, for example, those that can be formed using SATA (N- succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl- alpha-methyl-alpha-(2-pyridyl-dithio)toluene) (See, e.g. Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987)). In particular embodiments, the linker is susceptible to enzymatic hydrolysis. Such linkers may be advantageous in particular embodiments over pH sensitive cleavable linkers, which may not be stable enough and cleave prematurely before reaching the target cell, and thus potential off-target toxicity may be observed. The enzymatically cleavable linker can be, e.g. a peptide-containing linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. One benefit of employing intracellular proteolytic release of the therapeutic drug is that the agent is usually attenuated when conjugated, and the conjugates' serum stabilities are usually high. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Exemplary amino acid linkers include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Peptides comprising the amino acids valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine are examples of appropriate peptides. Natural amino acids, minor amino acids, and non- naturally occurring amino acid analogs, such as citrulline, are all examples of amino acid residues that make up an amino acid linker component. Exemplary dipeptides include valinecitrulline (VC or Val-Cit) and alanine-phenylalanine (AF or Ala-Phe). Exemplary tripeptides include glycine-valine-citrulline (Gly-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly). In some embodiments, the linker includes a dipeptide such as Val-Cit, Ala-Vai, or Phe-Lys, Val- Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit.

[0252] In some embodiments, the linker comprises PEG. A stable protease-cleavable linker containing PEG can limit payload hydrophobicity and be able to selectively cleave and release the free drug inside target cancer cells. A less hydrophobic nature of the linker as described herein can enable high loading of the drug onto the antibody or antigen-binding fragment (e.g. DAR8) without aggregation, which would be significantly higher than mirvetuximab soravtansine (DAR3-4) or derivatives thereof, such as IMGN151 (DAR3.5). This could allow the ADC to deliver a significantly higher concentration of cytotoxin payload to the target cancer cells via binding to FRa on the cancer cells.

[0253] In some embodiments, the linker comprises maleimide. The use of maleimide in the linker may allow the generation of DAR8 and DAR4 ADCs by making use of the native interchain disulphides in the antibodies. This is advantageous over the conjugation of surface amines from lysine residues which could result in a mixture of DAR species and batch-to-batch variability. There may also be reproducibility issues that affect ADC efficacy if conjugation sites interfere with antigen binding. Moreover, other conjugation methods e.g. azide-alkyne click chemistry involving an engineered antibody may not easily achieve a DAR of more than 4. In certain embodiments, the anti-FRa antibody or antigen-binding fragment thereof in the ADC of the disclosure is linked to a cytotoxin via a linker RLselected from: (la): wherein

[0254] Q is:

[0255] , where Qxis such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;

[0256] X is: where a = 0 to 5, bl = 0 to 16, b2 = 0 to 16, cl = 0 or 1, c2 = 0 or 1, d = 0 to 5, wherein at least bl or b2 = 0 (z.e. only one of bl and b2 may not be 0) and at least cl or c2 = 0 (z.e. only one of cl and c2 may not be 0);

[0257] GLis a linker for connecting to an antibody or antigen-binding fragment thereof in the ADC of the disclosure;

[0258] (lb): where RL1and RL2are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group; and e is 0 or 1; or

[0259] (lb’) lb' where RL1and RL2are as defined above.

[0260] By way of example, particular embodiments of GL, X, Qx(e.g. within the linker of la described above) and the linker of lb will be outlined. The following options may apply to all aspects of the disclosure as described herein, or may relate to a single aspect. The options may be combined together in any combination.

[0261] Various definitions which pertain to certain terms in this section are provided under the heading “Chemical Definitions” provided below. GLmay be selected from: where Ar represents a C5-6 arylene group, e.g. phenylene, and X represents Cl-4 alkyl.

[0262] In some embodiments, GLis selected from GL land GL1'2. In some of these embodiments, GLis GL1-1.

[0263] X

[0264] X may be: where a = 0 to 5, bl = 0 to 16, b2 = 0 to 16, c = 0 or 1, d = 0 to 5, wherein at least bl or b2 = 0 and at least cl or c2 = 0. a may be 0, 1, 2, 3, 4 or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0. bl may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, bl is 0 to 12. In some of these embodiments, bl is 0 to 8, and may be 0, 2, 3, 4, 5 or 8. b2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b2 is 0 to 12. In some of these embodiments, b2 is 0 to 8, and may be 0, 2, 3, 4, 5 or 8. Optionally, only one of bl and b2 may not be 0. cl may be 0 or 1. c2 may be 0 or 1. Optionally, only one of cl and c2 may not be 0. d may be 0, 1, 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2. In further embodiments, d is 5.

[0265] In some embodiments of X, a is 0, bl is 0, cl is 1, c2 is 0 and d is 2, and b2 may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5 or 8. In some embodiments of X, a is 1, b2 is 0, cl is 0, c2 is 0 and d is 0, and bl may be from 0 to 8. In some of these embodiments, bl is 0, 2, 3, 4, 5 or 8. In some embodiments of X, a is 0, bl is 0, cl is 0, c2 is 0 and d is 1, and b2 may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5 or 8. In some embodiments of X, bl is 0, b2 is 0, cl is 0, c2 is 0 and one of a and d is 0. The other of a and d is from 1 to 5. In some of these embodiments, the other of a and d is 1. In other of these embodiments, the other of a and d is 5. In some embodiments of X, a is 1, b2 is 0, cl is 0, c2 is 1, d is 2, and bl may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5 or 8.

[0266] Qx

[0267] In some embodiments, Q is an amino acid residue. The amino acid may be a natural amino acid or a non-natural amino acid. For example, Q may be selected from: Phe, Lys, Vai, Ala, Cit, Leu, He, Arg, and Trp, where Cit is citrulline.

[0268] In some embodiments, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.

[0269] In some embodiments, Q is selected from:

[0270] NH-Phe-Lys-C=o, n -Val-Ala-c <),xn-Val-Lys-c <),

[0271] XII-Ala-Lys-c

[0272] XII-Val-Cit-c

[0273] NH-Phe-Cit-c=o,

[0274] XII-Leu-Cit-cx”-He-Cit-c

[0275] NH-Phe-Arg-c=o,

[0276] NH-Trp-Cit-c=o, and

[0277] NH-Gly-Val-C=o; where Cit is citrulline.

[0278] In particular embodiments, Q is selected from:

[0279] NH-Phe-Lys-c=o,

[0280] XII-Val-Ala-cxn-Val-Lys-c <),

[0281] NH-Ala-Lys-c=o, and

[0282] NH-Val-Cit-c=o.

[0283] In specific embodiments, Q is selected fromNH-Phe-Lys-c=o,NH-Val-Cit-c=oorNH-Val- Ala-C°

[0284] Other suitable dipeptide combinations include:

[0285] NH-Gly-Gly-c=o,

[0286] X" -Gly-Val-C°

[0287] NH-Pro-Pro-c=o, and

[0288] XII-Val-Glu-c°

[0289] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.

[0290] In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin. Tnpeptide linkers of particular interest are:

[0291] NH-Glu-Val-Ala-C=o

[0292] NH-Glu-Val-Cit-c=o

[0293] NH-aGlu- V al - Al a-c=o

[0294] NH-aGlu-Val-Cit-c=o

[0295] In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. The tetrapeptide then is a recognition site for cathepsin. Tetrapeptide linkers of particular interest are:

[0296] NH-Gly-Gly-Phe-Glyc=o; and

[0297] NH-Gly-Phe-Gly-Glyc=o.

[0298] In some embodiments, the tetrapeptide is:

[0299] NH-Gly-Gly-Phe-Glyc=o.

[0300] In the above representations of peptide residues,NH- represents the N-terminus, and -c=orepresents the C-terminus of the residue. The C-terminus binds to the NH of the “Drug Unit” (e.g. A* as discussed below).

[0301] Glu represents the residue of glutamic acid, i.e. : aGlu represents the residue of glutamic acid when bound via the a-chain, i.e. :

[0302] In some embodiments, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed above. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin. Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and as described above.

[0303] Linker lb

[0304] RL1and RL2may be independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group.

[0305] In some embodiments, both RL1and RL2are H. In some embodiments, RL1is H and RL2is methyl. In some embodiments, both RL1and RL2are methyl.

[0306] In some embodiments, RL1and RL2together with the carbon atom to which they are bound form a cyclopropylene group. In some embodiments, RL1and RL2together with the carbon atom to which they are bound form a cyclobutylene group.

[0307] In the group lb, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group may be in any available position of the ring. In some of these embodiments, it is in the ortho position. In others of these embodiments, it is in the para position.

[0308] RL

[0309] In some embodiments, RLis selected from:

[0310]

[0311] In particular embodiments, R is

[0312] For example, an antibody-drug conjugate of the disclosure may be of the general formula IV:

[0313] L - (DL)P(IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigenbinding fragment thereof in the ADC of the disclosure, DLis a “Drug Unit” (e.g. cytotoxin such as TOPOi) having a linker RLLconnected to the antibody or antigen-binding fragment thereof in the ADC of the disclosure, wherein the linker is optionally selected from

[0314] (la’): where Q and X are as defined above and GLLis a linker connected to an antibody or antigenbinding fragment thereof in the ADC of the disclosure; and (lb’): lb' where RL1and RL2are as defined above; and p is an integer of from 1 to 20. The drug loading is represented by p, the number of “Drug Units” (e.g. cytotoxin such as TOPOi) per antibody or antigen-binding fragment thereof. Drug loading may range from 1 to 20 Drug units (D) per antibody or antigen-binding fragment thereof. For compositions, p represents the average drug loading of the conjugates in the composition, and p ranges from 1 to 20. In some embodiments, the range of p is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10; optionally wherein p is 8.

[0315] GLLmay be selected from: where Ar represents a C5-6 arylene group, e.g. phenylene and X represents Ci-4 alkyl.

[0316] In some embodiments, GLLis selected from GLL land GLL1'2. In some of these embodiments, G1 1' is G1^1’1.

[0317] In some embodiments, RLLis a group derived from the RLgroups above. It will be recognised by one of skill in the art that any one or more of the chemical groups, moieties and features disclosed herein may be combined in multiple ways to form linkers useful for conjugation of the antibodies and cytotoxins as disclosed herein.

[0318] In some embodiments where compounds described herein are provided in a single enantiomer or in an enantiomerically enriched form, the enantiomerically enriched form has an enantiomeric ratio greater than 60:40, 70:30; 80:20 or 90: 10. In further embodiments, the enantiomeric ratio is greater than 95:5, 97:3 or 99: 1. Specific ADC embodiments

[0319] In some embodiments, the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor, represented by the following compound with the formula “I”: , wherein RLis defined above; wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8. In other embodiments, the DAR is about 4. In some embodiments, the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to SG3932 wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8. In other embodiments, the DAR is about 4.

[0320] In some embodiments,

[0321] (i) the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 7 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 8, optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 10;

[0322] (ii) the cytotoxin is a topoisomerase I inhibitor represented by the following compound, with the formula “I”:

[0323] , wherein RLis defined above; and

[0324] (iii) the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8. In other embodiments, the DAR is about 4.

[0325] In some embodiments,

[0326] (i) the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 7 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 8, optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 10;

[0327] (ii) the anti-FRa antibody or antigen-binding fragment thereof is conjugated to SG3932

[0328] (iii) the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8. In other embodiments, the DAR is about 4.

[0329] In particular embodiments according to all aspects of the disclosure, the anti-FRa ADC is “AZD5335”, comprising an anti-FRa antibody with a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10, wherein the anti-FRa antibody is conjugated to SG3932

[0330] wherein the DAR of the ADC is 8, or about 8.

[0331] Internalisation

[0332] Internalisation can be a useful property of an ADC. For example, internalisation allows the delivery of payloads to a cell. The inventors have shown that antibodies and ADCs described herein demonstrated rapid internalisation and lysosome trafficking.

[0333] In some embodiments, the anti-FRa ADC of the disclosure binds to FRa on the surface of a cell, and is internalised into the cell. In some embodiments, the internalisation of the anti- FRa ADC of the disclosure into a FRa-expressing cell is saturated within about 4 hours or less, about 5 hours or less, about 6 hours or less, about 7 hours or less, about 8 hours or less, about 9 hours or less, about 10 hours or less, about 11 hours or less, or about 12 hours or less.

[0334] Cytotoxicity

[0335] In some embodiments, the anti-FRa ADC of the present disclosure inhibits or suppresses proliferation (e.g. of a tumour) by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or about 100% (in particular embodiments, at least 40%) relative to a level of inhibition or suppression in the absence of the anti-FRa ADC. Cellular proliferation can be assayed using art-recognised techniques which measure rate of cell division, and / or the fraction of cells within a cell population undergoing cell division, and / or rate of cell loss from a cell population due to terminal differentiation or cell death (e.g. thymidine incorporation).

[0336] In some embodiments, the anti-FRa ADC of the present disclosure exerts cytotoxicity towards a cell expressing FRa at an EC50 value of about 1000 ng / ml or less, about 500 ng / ml or less, about 400 ng / ml or less, about 300 ng / ml or less, about 290 ng / ml or less, about 280 ng / ml or less, about 270 ng / ml or less, about 260 ng / ml or less, or about 250 ng / ml or less.

[0337] In some embodiments, the anti-FRa ADC of the present disclosure exerts cytotoxicity towards a cell expressing FRa at an IC50 value of about 100 pg / ml or less, about 50 pg / ml or less, about 25 pg / ml or less, about 10 pg / ml or less, about 5 pg / ml or less, about 2.5 pg / ml or less, about 1 pg / ml or less, about 0.75 pg / ml or less, about 0.5 pg / ml or less, about 0.25 pg / ml or less, about 0.1 pg / ml or less, about 0.075 pg / ml or less, about 0.05 pg / ml or less, about 0.025 pg / ml or less, about 0.01 pg / ml or less.

[0338] In some embodiments, the anti-FRa ADC of the present disclosure inhibits or suppresses proliferation of a cell population having heterogeneous expression of FRa and / or a low expression of FRa. In some embodiments, the anti-FRa ADC of the present disclosure inhibits or suppresses proliferation of a cell population having a medium expression of FRa (e.g. Jeg-3, OVCAR-3 cell line or cells with a similar or equivalent level of FRa expression), medium-high expression of FRa (e.g. Igrov-1 cell line or cells with a similar or equivalent level of FRa expression), high expression of FRa (e.g. KB cell line or cells with a similar or equivalent level of FRa expression).

[0339] Preparation of ADC

[0340] The ADCs of the present disclosure can be made in a variety of ways, using known organic chemistry reactions, conditions, and reagents, such as: (1) reacting a reactive substituent of an antibody or antigen-binding fragment with a bivalent linker reagent, then reacting with a cytotoxin, such as a topoisomerase I inhibitor; or (2) reacting a reactive substituent of a cytotoxin, such as a topoisomerase I inhibitor, with a bivalent linker reagent, then reacting with a reactive substituent of an antibody or antigen-binding fragment thereof.

[0341] Reactive substituents that may be present within an antibody, or antigen-binding fragment thereof, as disclosed herein include, without limitation, nucleophilic groups such as (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Reactive substituents that may be present within an antibody, or antigen-binding fragment thereof, as disclosed herein include, without limitation, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl (e.g. fluoroaryl), haloheteroaryl (e.g. fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids. In some embodiments, the reactive substituents present within an antibody, or antigen-binding fragment thereof as disclosed herein include amine or thiol moieties. Certain antibodies have cysteine bridges, which are reducible interchain disulphides. By treating antibodies with a reducing agent (such as DL-dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP)), they can be made reactive for conjugation with linker reagents. Each cysteine bridge will theoretically result in the formation of two reactive thiol nucleophiles. The reaction of lysines with 2-iminothiolane (Trauf s reagent), which results in the conversion of an amine to a thiol, can be used to introduce additional nucleophilic groups into antibodies. One, two, three, four, or more cysteine residues can be used to insert reactive thiol groups into an antibody (or fragment thereof) (e.g. preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). Engineering antibodies with reactive cysteine amino acids is described in U.S. Pat. No. 7,521,541, which is incorporated by reference herein.

[0342] In another aspect, the antibody or antigen-binding fragment thereof can have one or more carbohydrate groups that can be chemically changed to contain one or more sulphydryl groups. The ADC is then formed by conjugation through the sulphur atom of the sulphydryl group.

[0343] In yet another aspect, the antibody may contain one or more carbohydrate groups that can be oxidised to produce an aldehyde (-CHO) group (see, for example, Laguzza etal., J. Med. Chem. 1989, 32(3), 548-55). Conjugation through the corresponding aldehyde results in the formation of the ADC. Further protocols for the modification of proteins for the attachment or association of cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002). Methods for the conjugation of linker-drug moieties to cell- targeted proteins such as antibodies, immunoglobulins or fragments thereof are found, for example, in U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,441,163; W02005 / 037992; W02005 / 081711; and W02006 / 034488, each of which is incorporated by reference herein.

[0344] Conventional conjugation strategies for antibodies or antigen-binding fragments thereof rely on randomly or stochastically conjugating the payload to the antibody or fragment through lysines or cysteines. In some embodiments, the antibody or antigen-binding fragment thereof is stochastically conjugated to a cytotoxin, such as a topoisomerase I inhibitor, for example, by partial reduction of the antibody or fragment, followed by reaction with a desired agent, with or without a linker moiety attached. The antibody or fragment may be reduced using DTT or other reducing agent to perform a similar reduction e.g. TCEP. The agent with or without a linker moiety attached can then be added at a molar excess to the reduced antibody or fragment in the presence of DMSO. After conjugation, a quenching agent such as N-acetyl-L-cysteine may be added to quench unreacted agent. The reaction mixture may then be purified (by e.g. TFF, SEC-FPLC, CHT, spin filter centrifugation) and buffer-exchanged into PBS or other relevant formulation buffer.

[0345] In some embodiments, a cytotoxin is conjugated to an antibody or antigen-binding fragment thereof by site-specific conjugation. In some embodiments, site-specific conjugation of therapeutic moieties to antibodies using reactive amino acid residues at specific positions yields homogeneous preparations of an ADC with uniform stoichiometry.

[0346] The site-specific conjugation can be through a cysteine residue or a non-natural amino acid. In a particular embodiment, the cytotoxin is conjugated to the antibody or antigen-binding fragment thereof through at least one cysteine residue. Cysteine amino acids may be engineered at reactive sites in an antibody (or antigen-binding fragment thereof) and which preferably do not form intrachain or intermolecular disulphide linkages (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Doman et al. (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; W02009 / 052249). In some embodiments, the cytotoxin is conjugated to the antibody or antigen-binding fragment thereof through a cysteine substitution of at least one of positions 239, 248, 254, 273, 279, 282, 284, 286, 287, 289, 297, 298, 312, 324, 326, 330, 335, 337, 339,

[0347] 350, 355, 356, 359, 360, 361, 375, 383, 384, 389, 398, 400, 413, 415, 418, 422, 440, 441, 442,

[0348] 443 and 446, wherein the numbering corresponds to the EU index in Kabat. In some embodiments, the specific Kabat positions are 239, 442, or both. In some embodiments, the specific positions are Kabat position 442, an amino acid insertion between Kabat positions 239 and 240, or both. In some embodiments, the cytotoxin is conjugated to the antibody or antigenbinding fragment thereof through a thiol-maleimide linkage. In some aspects, the amino acid side chain is a sulphydryl side chain.

[0349] Where more than one nucleophilic or electrophilic group of the antibody or antigenbinding fragment thereof reacts with a cytotoxic agent, then the resulting product may be a mixture of ADCs with a distribution of agent units attached to an antibody, e.g. 1, 2, 3, etc. Liquid chromatography methods such as hydrophobic interaction (HIC) may separate compounds in the mixture by agent loading value. Preparations of an ADC with a single agent loading value (p) may be isolated.

[0350] The average number of cytotoxic agents per antibody (or antigen-binding fragment) in preparations of ADCs from conjugation reactions may be characterised by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis. The quantitative distribution of ADC in terms of p may also be determined. By ELISA, the averaged value of p in a particular preparation of an ADC may be determined (Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al. (2005) Clin. Cancer Res. 11 :843-852). In some instances, separation, purification, and characterisation of homogeneous ADC, where p is a certain value from antibody with other agents, may be achieved by means such as reverse phase HPLC, electrophoresis, TFF, SEC-FPLC, CHT, spin filter centrifugation. Such techniques are also applicable to other types of conjugates.

[0351] The preparation of specific anti-FRa antibodies is described in WO 2023 / 169896, which is incorporated by reference. In particular, Examples 10 and 11 of WO 2023 / 169896 describe the generation of anti-FRa ADCs in DAR8 and DAR4 formats (including AZD5335, which is referred to in WO 2023 / 169896 as AB1370049-SG3932 DAR8). Examples 12-24 of WO 2023 / 169896 also describe the screening of ADCs through in vitro serum stability, in vivo serum stability, pharmacokinetics, in vitro cell-killing activity, bystander killing, in vivo anticancer activity in cell derived and patient-derived xenograft models, in vitro safety studies and in vivo pharmacokinetic studies.

[0352] Chemical Definitions

[0353] The following definitions pertain, in particular, to the description of topoisomerase I inhibitors above.

[0354] C5-6 arylene: The term “C5-6 arylene”, as used herein, pertains to a divalent moiety obtained by removing two hydrogen atoms from an aromatic ring atom of an aromatic compound.

[0355] In this context, the prefixes (e.g. C5-6) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms.

[0356] The ring atoms may be all carbon atoms, as in “carboarylene groups”, in which case the group is phenylene (Ce).

[0357] Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroarylene groups”. Examples of heteroarylene groups include, but are not limited to, those derived from: Ni: pyrrole (azole) (C5), pyridine (azine) (Ce);

[0358] Oi : furan (oxole) (C5);

[0359] Si: thiophene (thiole) (C5);

[0360] N1O1: oxazole (C5), isoxazole (C5), isoxazine (Ce);

[0361] N2O1: oxadiazole (furazan) (C5);

[0362] N3O1: oxatriazole (Cs);

[0363] N1S1: thiazole (C5), isothiazole (Cs); N2: imidazole (1,3-diazole) (Cs), pyrazole (1,2-diazole) (Cs), pyridazine (1,2-diazine) (Ce), pyrimidine (1,3 -diazine) (Ce) (e.g. cytosine, thymine, uracil), pyrazine (1,4-diazine) (Ce); and Ns: triazole (Cs), triazine (Ce).

[0364] Ci-4 alkyl: The term “Ci-4 alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). The term “Ci-n alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to n carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.

[0365] Examples of saturated alkyl groups include, but are not limited to, methyl (Ci), ethyl (C2), propyl (C3) and butyl (C4).

[0366] Examples of saturated linear alkyl groups include, but are not limited to, methyl (Ci), ethyl (C2), n-propyl (C3) and n-butyl (C4).

[0367] Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4) and tert-butyl (C4).

[0368] C2-4 Alkenyl: The term “C2-4 alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.

[0369] Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2), isopropenyl (1- methylvinyl, -C(CH3)=CH2) and butenyl (C4).

[0370] C2-4 alkynyl: The term “C2-4 alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.

[0371] Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C=CH) and 2-propynyl (propargyl, -CEh-OCEl).

[0372] C3-4 cycloalkyl: The term “C3-4 cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

[0373] Examples of cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C3) and cyclobutane (C4); and unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3) and cyclobutene (C4).

[0374] Connection labels: In the formula , the superscripted labelsC(=O)andNHindicate the group to which the atoms are bound. For example, the NH group is shown as being bound to a carbonyl (which is not part of the moiety illustrated), and the carbonyl is shown as being bound to a NH group (which is not part of the moiety illustrated).

[0375] Salts

[0376] It may be convenient or desirable to prepare, purify, and / or handle a corresponding salt of the active compound / agent, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, etal.,J Pharm. Set., 66, 1-19 (1977).

[0377] For example, if the compound is anionic, or has a functional group which may be anionic (e.g. -COOH may be -COO'), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+and K+, alkaline earth cations such as Ca2+and Mg2+, and other cations such as Al+3. Examples of suitable organic cations include, but are not limited to, ammonium ion (z.e. NHC) and substituted ammonium ions (e.g. NH3R , NH2R2+, NHR3+, NRC). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.

[0378] If the compound is cationic, or has a functional group which may be cationic (e.g. -NH2 may be -NH3+), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulphuric, sulphurous, nitric, nitrous, phosphoric, and phosphorous.

[0379] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acety oxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulphonic, cinnamic, citric, edetic, ethanedisulphonic, ethanesulphonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulphonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulphonic, propionic, pyruvic, salicylic, stearic, succinic, sulphanilic, tartaric, toluenesulphonic, trifluoroacetic acid and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0380] Solvates

[0381] It may be convenient or desirable to prepare, purify, and / or handle a corresponding solvate of the active compound. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a monohydrate, a di-hydrate, a tri-hydrate, etc.

[0382] Isomers

[0383] Certain compounds / agents of the disclosure may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and P-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

[0384] Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

[0385] Pharmaceutical compositions

[0386] The ADC of the disclosure can be administered to the subject as a pharmaceutical composition.

[0387] Accordingly, in one aspect, the present disclosure provides a pharmaceutical composition comprising: (i) the ADC of the disclosure; and (ii) a pharmaceutically acceptable excipient.

[0388] In one aspect, the present disclosure provides a pharmaceutical composition comprising: (a) an ADC comprising an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein:

[0389] (i) the anti-FRa antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8, optionally wherein the anti-FRa antibody has a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10;

[0390] (ii) the anti-FRa antibody or antigen-binding fragment is conjugated to SG3932

[0391] (iii) the DAR of the ADC is about 8; and

[0392] (b) a pharmaceutically acceptable excipient.

[0393] In another aspect, the present disclosure provides a pharmaceutical composition comprising the ADC of the disclosure in an amount from about 0.8 mg / kg to about 5.0 mg / kg based on the body weight of the subject to be treated. For example, provided herein is a unit dose comprising the ADC of the disclosure in an amount from about 0.8 mg / kg to about 5.0 mg / kg based on the body weight of the subject to be treated.

[0394] In another aspect, the present disclosure provides a pharmaceutical composition, for example a unit dose, comprising the ADC of the disclosure in an amount from about 0.8 mg / kg to about 5.0 mg / kg based on the body weight of the subject to be treated, wherein:

[0395] (i) the anti-FRa antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8, optionally wherein the anti-FRa antibody has a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10;

[0396] (ii) the anti-FRa antibody or antigen-binding fragment is conjugated to SG3932

[0397] (iii) the DAR of the ADC is about 8.

[0398] In some embodiments, the amount of the ADC in the composition, such as the amount of the ADC in the unit dose, is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about

[0399] 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg.

[0400] In some embodiments, the amount of the ADC in the composition, such as the amount of the ADC in the unit dose, is about 0.8 mg / kg to about 4.9 mg / kg, is about 0.8 mg / kg to about

[0401] 4.8 mg / kg, about 0.8 mg / kg to about 4.6 mg / kg, about 0.8 mg / kg to about 4.4 mg / kg, about 0.8 mg / kg to about 4.2 mg / kg, about 0.8 mg / kg to about 4.0 mg / kg, about 0.8 mg / kg to about 3.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.4 mg / kg, about 0.8 mg / kg to about 3.2 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.0 mg / kg to about 2.6 mg / kg, about 1.0 mg / kg to about 2.4 mg / kg, about 1.0 mg / kg to about 2.2 mg / kg, about 1.0 mg / kg to about 2.0 mg / kg, about 1.0 mg / kg to about 1.8 mg / kg, about 1.0 mg / kg to about 1.6 mg / kg, about 1.0 mg / kg to about 1.4 mg / kg, about 1.0 mg / kg to about 1.2 mg / kg, about 1.2 mg / kg to about 3.0 mg / kg, about 1.2 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.2 mg / kg to about 2.4 mg / kg, about 1.2 mg / kg to about 2.2 mg / kg, about 1.2 mg / kg to about 2.0 mg / kg, about 1.2 mg / kg to about 1.8 mg / kg, about 1.2 mg / kg to about 1.6 mg / kg, about 1.2 mg / kg to about 1.4 mg / kg, about 1.4 mg / kg to about 3.0 mg / kg, about 1.4 mg / kg to about 2.8 mg / kg, about 1.4 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg, about 1.4 mg / kg to about 2.2 mg / kg, about 1.4 mg / kg to about 2.0 mg / kg, about 1.4 mg / kg to about 1.8 mg / kg, about 1.4 mg / kg to about 1.6 mg / kg, about 1.6 mg / kg to about 3.0 mg / kg, about 1.6 mg / kg to about 2.8 mg / kg, about 1.6 mg / kg to about 2.6 mg / kg, about 1.6 mg / kg to about 2.4 mg / kg, about 1.6 mg / kg to about 2.2 mg / kg, about 1.6 mg / kg to about 2.0 mg / kg, about 1.6 mg / kg to about 1.8 mg / kg, about 1.8 mg / kg to about 3.0 mg / kg, about 1.8 mg / kg to about 2.8 mg / kg, about 1.8 mg / kg to about 2.6 mg / kg, about 1.8 mg / kg to about 2.4 mg / kg, about 1.8 mg / kg to about 2.2 mg / kg, about 1.8 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 3.0 mg / kg, about 2.0 mg / kg to about 2.8 mg / kg, about 2.0 mg / kg to about 2.6 mg / kg, about 2.0 mg / kg to about 2.4 mg / kg, about 2.0 mg / kg to about 2.2 mg / kg, about 2.2 mg / kg to about 3.0 mg / kg, about 2.2 mg / kg to about 2.8 mg / kg, about 2.2 mg / kg to about 2.6 mg / kg, about 2.2 mg / kg to about 2.4 mg / kg, about 2.4 mg / kg to about 3.0 mg / kg, about 2.4 mg / kg to about 2.8 mg / kg, about 2.4 mg / kg to about 2.6 mg / kg, about 2.6 mg / kg to about 3.0 mg / kg, about 2.6 mg / kg to about 2.8 mg / kg or about 2.8 mg / kg to about 3.0 mg / kg.

[0402] In one embodiment, the amount of the ADC in the composition, such as the amount of the ADC in the unit dose, is about 0.8 mg / kg to about 4.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg or about 1.6 mg / kg to about 2.4 mg / kg. In particular embodiments, the amount of the ADC in the composition, such as the amount of the ADC in the unit dose, is about 1.6 mg / kg to about 2.4 mg / kg.

[0403] In one embodiment, the amount of the ADC in the composition, such as the amount of the ADC in the unit dose, is about 0.8 mg / kg, about 1.2 mg / kg, about 1.6 mg / kg, about 2.0 mg / kg, about 2.4 mg / kg, about 2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg or about 4.8 mg / kg.

[0404] In one embodiment, the amount of the ADC in the composition, such as the amount of the ADC in the unit dose, is about 1.6 mg / kg.

[0405] In one embodiment, the amount of the ADC in the composition, such as the amount of the ADC in the unit dose, is about 2.0 mg / kg. In one embodiment, the amount of the ADC in the composition, such as the amount of the ADC in the unit dose, is about 2.4 mg / kg.

[0406] The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile, and can comprise a pharmaceutically acceptable carrier.

[0407] The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognised pharmacopeia for use in animals, and more particularly in humans.

[0408] In some embodiments, pharmaceutical compositions according to the present disclosure, and for use in accordance with the present disclosure, may comprise, in addition to the active ingredient (the ADC of the disclosure), a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.

[0409] In some embodiments, a pharmaceutical composition of the disclosure can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0410] A person skilled in the art would understand that the appropriate choice of excipient or excipients for use with the ADC of the disclosure, would depend on the desired properties of the pharmaceutical composition.

[0411] In some embodiments, a pharmaceutical composition of the disclosure may be comprised within one or more formulation selected from a capsule, a tablet, an aqueous suspension, a solution, a nasal aerosol, a lyophilised powder which can be reconstituted to make a suspension or solution before use, or a combination thereof.

[0412] In some embodiments, the pharmaceutical composition comprises more than one type of ADC of the disclosure.

[0413] The pharmaceutical compositions disclosed herein are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, in preventing, treating, managing, or ameliorating a disorder or one or more symptoms thereof, and / or in research. The pharmaceutical compositions disclosed herein may be suitable for veterinary uses or pharmaceutical uses in humans.

[0414] Kits, Articles of Manufacture, Methods for Manufacturing a Medicament

[0415] In one aspect, there is provided a kit comprising the ADC of the disclosure. In another aspect, there is provided a kit comprising the pharmaceutical composition of the disclosure, such as a unit dose of the disclosure. There is further embraced use of said kit in the methods of the present disclosure.

[0416] In some embodiments, the kit comprises an isolated (e.g. purified) ADC as described herein. In some embodiments, the kit comprises one or more containers. In some embodiments, the kit comprises all of the components necessary and / or sufficient to administer the ADC to a subject. In some embodiments, the kit comprises all of the instructions necessary to administer the ADC to a subject.

[0417] In some embodiments, the kit comprises one or more containers filled with one or more of the ADCs of the disclosure, such as filled with a pharmaceutical composition of the disclosure, such as a unit dose of the disclosure. In some embodiments, the kit comprises one or more containers comprising one or more of the ADCs of the disclosure, such as comprising a pharmaceutical composition of the disclosure, such as a unit dose of the disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. For example, instructions on how to employ the provided pharmaceutical composition in the treatment of cancer, such as ovarian cancer, lung cancer (e.g. lung adenocarcinoma or NSCLC), endometrial cancer, breast cancer (e.g. TNBC), cervical cancer, pancreatic cancer, gastric cancer, renal cell carcinoma, colorectal cancer, head and neck squamous cell carcinomas (HNSCC), malignant pleural mesothelioma, peritoneal cancer or fallopian tube cancer, may also be included or be made available to a patient or a medical service provider. In some embodiments, the cancer is a solid tumour and / or an epithelial tumour. In one embodiment, the ovarian cancer is platinum-resistant ovarian cancer. In particular embodiments, the ovarian cancer is platinum-resistant refractory ovarian cancer. In other embodiments, the ovarian cancer is platinum-sensitive ovarian cancer.

[0418] In some embodiments, the kit may provide the antigen or antigen-binding fragment and a cytotoxin (i.e. topoisomerase I inhibitor) that is not conjugated to the antibody or antigen- binding fragment, but is in a form suitable for conjugation thereto; optionally wherein the kit is further provided with instructions and / or reagents for conjugating the cytotoxin to the antibody or antigen-binding fragment. In some embodiments, the kit comprises all of the components necessary and / or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.

[0419] In one aspect, provided herein is an article of manufacture comprising:

[0420] (i) the ADC of the disclosure; and

[0421] (ii) instructions for administering said ADC to a human subject in need of a treatment for cancer.

[0422] In one aspect, provided herein is an article of manufacture comprising:

[0423] (i) the pharmaceutical composition of the disclosure, such as a unit dose of the disclosure; and

[0424] (ii) instructions for administering the pharmaceutical composition of the disclosure to a human subject in need of a treatment for cancer.

[0425] In one aspect, provided herein is a method for manufacturing a medicament, wherein the method comprises:

[0426] (a) using the ADC of the disclosure; and

[0427] (b) combining the ADC with a pharmaceutically acceptable carrier.

[0428] In one aspect, provided herein is a method for manufacturing a pharmaceutical composition, such as a unit dose, wherein the method comprises:

[0429] (a) using the ADC of the disclosure; and

[0430] (b) combining the ADC with a pharmaceutically acceptable carrier.

[0431] For example, provided is a method for manufacturing a pharmaceutical composition of the disclosure, such as a unit dose of the disclosure, wherein the method comprises:

[0432] (a) using the ADC of the disclosure; and

[0433] (b) combining the ADC with a pharmaceutically acceptable carrier.

[0434] In one aspect, provided herein is a method of producing a unit dose of an ADC for the treatment of cancer in a human subject, wherein the ADC is an ADC of the disclosure, such as an ADC that comprises an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor, the method comprising:

[0435] (a) determining the percentage of FRa-positive cells in a sample from the cancer; and

[0436] (b) producing a unit dose of the ADC by formulating the ADC in an amount determined based on the percentage of FRa-positive cells determined in the subject. In one aspect, a method of producing a unit dose of an ADC for the treatment of cancer in a human subject, comprises formulating an ADC of the disclosure in an amount determined based on the percentage of FRa-positive cells determined in the subject.

[0437] In one aspect, a method of producing a unit dose of an ADC for the treatment of cancer in a human subject, comprises formulating an ADC of the disclosure in an amount determined based on the body weight of the subject to be treated.

[0438] In one aspect, a method of producing a unit dose of an ADC for the treatment of cancer in a human subject, comprises formulating an ADC of the disclosure in an amount determined based on (i) the percentage of FRa-positive cells determined in the subject, and (ii) the body weight of the subject.

[0439] In some embodiments of any of the methods of producing a unit dose as disclosed herein, the amount of the ADC in said unit dose is from about 0.8 mg / kg to about 5.0 mg / kg, based on the body weight of the human subject.

[0440] In some embodiments, the amount of the ADC in said unit dose is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg about 4.9 mg / kg or about 5.0 mg / kg, based on the body weight of the human subject.

[0441] In some embodiments, the amount of the ADC in said unit dose is about 0.8 mg / kg to about 4.8 mg / kg, about 0.8 mg / kg to about 4.6 mg / kg, about 0.8 mg / kg to about 4.4 mg / kg, about 0.8 mg / kg to about 4.2 mg / kg, about 0.8 mg / kg to about 4.0 mg / kg, about 0.8 mg / kg to about 3.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.4 mg / kg, about 0.8 mg / kg to about 3.2 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.0 mg / kg to about 2.6 mg / kg, about 1.0 mg / kg to about 2.4 mg / kg, about 1.0 mg / kg to about 2.2 mg / kg, about 1.0 mg / kg to about 2.0 mg / kg, about 1.0 mg / kg to about 1.8 mg / kg, about 1.0 mg / kg to about 1.6 mg / kg, about 1.0 mg / kg to about 1.4 mg / kg, about 1.0 mg / kg to about 1.2 mg / kg, about 1.2 mg / kg to about 3.0 mg / kg, about 1.2 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.2 mg / kg to about 2.4 mg / kg, about 1.2 mg / kg to about 2.2 mg / kg, about 1.2 mg / kg to about 2.0 mg / kg, about 1.2 mg / kg to about 1.8 mg / kg, about 1.2 mg / kg to about 1.6 mg / kg, about 1.2 mg / kg to about 1.4 mg / kg, about 1.4 mg / kg to about 3.0 mg / kg, about 1.4 mg / kg to about 2.8 mg / kg, about 1.4 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg, about 1.4 mg / kg to about 2.2 mg / kg, about 1.4 mg / kg to about 2.0 mg / kg, about 1.4 mg / kg to about 1.8 mg / kg, about 1.4 mg / kg to about 1.6 mg / kg, about 1.6 mg / kg to about 3.0 mg / kg, about 1.6 mg / kg to about 2.8 mg / kg, about 1.6 mg / kg to about 2.6 mg / kg, about 1.6 mg / kg to about 2.4 mg / kg, about 1.6 mg / kg to about 2.2 mg / kg, about 1.6 mg / kg to about 2.0 mg / kg, about 1.6 mg / kg to about 1.8 mg / kg, about 1.8 mg / kg to about 3.0 mg / kg, about 1.8 mg / kg to about 2.8 mg / kg, about 1.8 mg / kg to about 2.6 mg / kg, about 1.8 mg / kg to about 2.4 mg / kg, about 1.8 mg / kg to about 2.2 mg / kg, about 1.8 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 3.0 mg / kg, about 2.0 mg / kg to about 2.8 mg / kg, about 2.0 mg / kg to about 2.6 mg / kg, about 2.0 mg / kg to about 2.4 mg / kg, about 2.0 mg / kg to about 2.2 mg / kg, about 2.2 mg / kg to about 3.0 mg / kg, about 2.2 mg / kg to about 2.8 mg / kg, about 2.2 mg / kg to about 2.6 mg / kg, about 2.2 mg / kg to about 2.4 mg / kg, about 2.4 mg / kg to about 3.0 mg / kg, about 2.4 mg / kg to about 2.8 mg / kg, about 2.4 mg / kg to about 2.6 mg / kg, about 2.6 mg / kg to about 3.0 mg / kg, about 2.6 mg / kg to about 2.8 mg / kg or about 2.8 mg / kg to about 3.0 mg / kg, based on the body weight of the human subject.

[0442] In one embodiment, the amount of the ADC in said unit dose is about 0.8 mg / kg to about 4.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg or about 1.6 mg / kg to about 2.4 mg / kg, based on the body weight of the human subject. In particular embodiments, the amount of the ADC in said unit dose is about 1.6 mg / kg to about 2.4 mg / kg.

[0443] In one embodiment, the amount of the ADC in said unit dose is about 0.8 mg / kg, about 1.2 mg / kg, about 1.6 mg / kg, about 2.0 mg / kg, about 2.4 mg / kg, about 2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg or about 4.8 mg / kg, based on the body weight of the human subject.

[0444] In one embodiment, the amount of the ADC in said unit dose is about 1.6 mg / kg, based on the body weight of the human subject.

[0445] In one embodiment, the amount of the ADC in said unit dose is about 2.0 mg / kg, based on the body weight of the human subject.

[0446] In one embodiment, the amount of the ADC in said unit dose is about 2.4 mg / kg, based on the body weight of the human subject. In some embodiments, the percentage of FRa-positive cells is assayed using immunohistochemistry (IHC) or the Quantitative Continuous Scoring (QCS) assay. In some embodiments, the IHC or QCS assay is performed with an antibody reagent.

[0447] In some embodiments of any aspects of the disclosure (e.g. the kit, the article of manufacture, the method for manufacturing a medicament, the method of producing a unit dose of an ADC), the ADC can have any antibody or antigen binding fragment thereof as described herein. In some embodiments of any aspects of the disclosure, the ADC can have any linker as described herein. In a particular embodiment according to all aspects of the disclosure, the ADC is AZD5335.

[0448] In some embodiments of any aspects of the disclosure (e.g. the kit, the article of manufacture, the pharmaceutical composition such as a unit dose, the method for manufacturing a medicament, method of producing a unit dose of an ADC):

[0449] (i) the anti-FRa antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8, optionally wherein the anti-FRa antibody has a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10;

[0450] (ii) the anti-FRa antibody or antigen-binding fragment is conjugated to SG3932

[0451] (iii) the DAR of the ADC is about 8. Therapy

[0452] The present disclosure encompasses therapies which involve administering an anti-FRa antibody-drug conjugate (ADC) of the disclosure, medicaments as described herein, pharmaceutical compositions as described herein, or unit doses as described herein to a subject, for preventing, treating, or ameliorating symptoms associated with cancer.

[0453] To “treat” refers to therapeutic measures that cure, slow down, alleviate symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder. In some embodiments, a subject is successfully “treated” for a disease or disorder (particularly, cancer), according to the methods provided herein if the patient shows, e.g. total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder (particularly, cancer).

[0454] To “prevent” refers to prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of prevention include those prone to have or susceptible to the disorder. In some embodiments, a disease or disorder (particularly, cancer) is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g. fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who has not been subject to the methods of the disclosure.

[0455] The terms “subject”, “individual” and “patient” are used interchangeably herein to refer to a mammalian subject. In some embodiments the “subject” is a human, domestic animals, farm animals, sports animals, and zoo animals, e.g. humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, etc. In some embodiments, the subject is a cynomolgus monkey (Macaca fascicularis). In a particular embodiment, the subject is a human. In methods of the disclosure, the subject may not have been previously diagnosed as having cancer. Alternatively, the subject may have been previously diagnosed as having cancer. The subject may also be one who exhibits disease risk factors, or one who is asymptomatic for cancer. The subject may also be one who is suffering from or is at risk of developing cancer. In some embodiments, the subject has been previously administered a cancer therapy.

[0456] In one aspect, there is provided a method of treating a disease or disorder (e.g. cancer) comprising administering to a subject a therapeutically effective amount of an anti-FRa ADC of the disclosure (e.g. AZD5335); a pharmaceutical composition of the disclosure; or a unit dose of the disclosure, wherein the method comprises administering to the human subject the ADC in an amount from about 0.8 mg / kg to about 5.0 mg / kg. In one aspect, there is provided an anti-FRa ADC of the disclosure (e.g. AZD5335) of the disclosure; a pharmaceutical composition of the disclosure; or a unit dose of the disclosure, for use in therapy, for example for treating a disease or disorder (e.g. cancer), wherein the treatment comprises administering to the human subject the ADC in an amount from about 0.8 mg / kg to about 5.0 mg / kg.

[0457] In one aspect, there is provided a method for preventing the onset of a disease or disorder (e.g. cancer) comprising administering to a subject a therapeutically effective amount of an anti-FRa ADC of the disclosure; a pharmaceutical composition of the disclosure; or a unit dose of the disclosure, wherein the method comprises administering to the human subject the ADC in an amount from about 0.8 mg / kg to about 5.0 mg / kg.

[0458] In one aspect, there is provided an anti-FRa ADC of the disclosure (e.g. AZD5335); a pharmaceutical composition of the disclosure; or a unit dose of the disclosure, for use in a method for preventing the onset of a disease or disorder (e.g. cancer), wherein the method comprises administering to the human subject the ADC in an amount from about 0.8 mg / kg to about 5.0 mg / kg.

[0459] In one aspect, there is provided a method for treating a cancer, the method comprising administering an anti-FRa ADC of the disclosure (e.g. AZD5335); a pharmaceutical composition of the disclosure; or a unit dose of the disclosure to a subject, wherein the method comprises administering to the human subject the ADC in an amount from about 0.8 mg / kg to about 5.0 mg / kg.

[0460] In one aspect, there is provided an anti-FRa ADC of the disclosure (e.g. AZD5335); a pharmaceutical composition of the disclosure; or a unit dose of the disclosure, for use in any of the methods described herein. In a related aspect, there is provided the use of an anti-FRa ADC of the disclosure (e.g. AZD5335); a pharmaceutical composition of the disclosure; or a unit dose of the disclosure, in the manufacture of a medicament for use in any of the methods described herein.

[0461] In one aspect, there is provided an anti-FRa ADC of the disclosure (e.g. AZD5335); a pharmaceutical composition of the disclosure; or a unit dose of the disclosure, for use in treating a cancer, wherein the treatment comprises administering to the human subject the ADC in an amount from about 0.8 mg / kg to about 5.0 mg / kg.

[0462] In some embodiments, the subject is a human subject in need thereof.

[0463] In some embodiments, the cancer is a solid tumour. In some embodiments the cancer is an epithelial cancer. In some embodiments the cancer is a carcinoma. In some embodiments the epithelial cancer is an adenoma or papilloma. In particular embodiments, the cancer is a solid tumour and / or an epithelial tumour. In particular embodiments, the cancer is selected from ovarian cancer, lung cancer (e.g. lung adenocarcinoma), endometrial cancer, breast cancer (e.g. TNBC), cervical cancer, pancreatic cancer, gastric cancer, renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinomas (HNSCC), malignant pleural mesothelioma, peritoneal cancer and fallopian tube cancer. In particular embodiments, the cancer is ovarian cancer or lung cancer. In some embodiments, the ovarian cancer is platinum- resistant ovarian cancer. In particular embodiments, the platinum-resistant ovarian cancer is platinum-resistant refractory ovarian cancer. In other embodiments, the ovarian cancer is platinum-sensitive ovarian cancer. In some embodiments, the lung cancer is one or more nonsmall-cell lung carcinoma (NSCLC), which in particular embodiments may be selected from squamous NSCLC, adenocarcinoma NSCLC, or a combination thereof.

[0464] Further examples of cancer include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. ovarian carcinoma, lung cancer, nonsmall cell lung cancer (squamous cell carcinoma or adenocarcinoma), endometrial cancer, pancreatic cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinomas, malignant pleural mesothelioma, breast carcinoma (e.g. TNBC), and kidney cancer. Any type of cell may be treated, including but not limited to, lung, gastrointestinal, breast (mammary), ovarian, kidney (renal) and pancreas.

[0465] In some embodiments, the cancer is homologous recombination deficient (HRD) cancer. In some embodiments, the cancer comprises one or more cells having a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L. Mutation in an HRD gene may also be termed a mutation in a gene involved with homologous recombination repair (HRRm+). In some embodiments, the mutated HRD gene is selected from BRCA1, BRCA2, nd ATM. In some embodiments, the mutated HRD gene isBRCAl. In some embodiments, the mutated HRD gene is BRCA2. In some embodiments, the mutated HRD gene is ATM.

[0466] In one aspect, there is provided a method for depleting a population of FRa-positive cells in a subject, the method comprising administering a therapeutically effective amount of an anti-FRa ADC of the disclosure (e.g. AZD5335); a pharmaceutical composition of the disclosure; or a unit dose of the disclosure to a subject, wherein the method comprises administering to the human subject the ADC in an amount from about 0.8 mg / kg to about 5.0 mg / kg. In one aspect, there is provided an anti-FRa ADC of the disclosure (e.g. AZD5335); a pharmaceutical composition of the disclosure; or a unit dose of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject, wherein the method comprises administering to the human subject the ADC in an amount from about 0.8 mg / kg to about 5.0 mg / kg.

[0467] In some embodiments of any aspects of the disclosure (e.g. the method of treating a disease or disorder, the anti-FRa ADC of the disclosure for use in therapy, the pharmaceutical composition of the disclosure for use in therapy, the unit dose of the disclosure for use in therapy, the anti-FRa ADC of the disclosure for use in a method for preventing the onset of a disease or disorder, the pharmaceutical composition of the disclosure for use in a method for preventing the onset of a disease or disorder, the unit dose of the disclosure for use in a method for preventing the onset of a disease or disorder, the method of treating a cancer, the anti-FRa ADC of the disclosure for use in treating a cancer, the pharmaceutical composition of the disclosure for use in treating a cancer, the unit dose of the disclosure for use in treating a cancer, the method of depleting a population of FRa-positive cells in a subject, the anti-FRa ADC of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject, the pharmaceutical composition of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject, the unit dose of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject),

[0468] (i) the anti-FRa antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8, optionally wherein the anti-FRa antibody has a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10;

[0469] (ii) the anti-FRa antibody or antigen-binding fragment thereof is conjugated to SG3932

[0470]

[0471] (iii) the DAR of the ADC is about 8.

[0472] Dosage regimen

[0473] The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.

[0474] In some embodiments of any aspects of the disclosure (e.g. the method of treating a disease or disorder, the anti-FRa ADC of the disclosure for use in therapy, the pharmaceutical composition of the disclosure for use in therapy, the unit dose of the disclosure for use in therapy, the anti-FRa ADC of the disclosure for use in a method for preventing the onset of a disease or disorder, the pharmaceutical composition of the disclosure for use in a method for preventing the onset of a disease or disorder, the unit dose of the disclosure for use in a method for preventing the onset of a disease or disorder, the method of treating a cancer, the anti-FRa ADC of the disclosure for use in treating a cancer, the pharmaceutical composition of the disclosure for use in treating a cancer, the unit dose of the disclosure for use in treating a cancer, the method of depleting a population of FRa-positive cells in a subject, the anti-FRa ADC of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject, the pharmaceutical composition of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject, the unit dose of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject), the amount of the ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. For example, in some embodiments, the amount of the ADC administered is about 0.8 mg / kg, about 1.2 mg / kg, about 1.6 mg / kg, about 2.0 mg / kg, about 2.4 mg / kg, about 2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg or about 4.8 mg / kg.

[0475] In some embodiments of any aspects of the disclosure, the amount of the ADC administered is about 0.8 mg / kg to about 4.8 mg / kg, about 0.8 mg / kg to about 4.6 mg / kg, about 0.8 mg / kg to about 4.4 mg / kg, about 0.8 mg / kg to about 4.2 mg / kg, about 0.8 mg / kg to about 4.0 mg / kg, about 0.8 mg / kg to about 3.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.4 mg / kg, about 0.8 mg / kg to about 3.2 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.0 mg / kg to about 2.6 mg / kg, about 1.0 mg / kg to about 2.4 mg / kg, about 1.0 mg / kg to about 2.2 mg / kg, about 1.0 mg / kg to about 2.0 mg / kg, about 1.0 mg / kg to about 1.8 mg / kg, about 1.0 mg / kg to about 1.6 mg / kg, about 1.0 mg / kg to about 1.4 mg / kg, about 1.0 mg / kg to about 1.2 mg / kg, about 1.2 mg / kg to about 3.0 mg / kg, about 1.2 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.2 mg / kg to about 2.4 mg / kg, about 1.2 mg / kg to about 2.2 mg / kg, about 1.2 mg / kg to about 2.0 mg / kg, about 1.2 mg / kg to about 1.8 mg / kg, about 1.2 mg / kg to about 1.6 mg / kg, about 1.2 mg / kg to about 1.4 mg / kg, about 1.4 mg / kg to about 3.0 mg / kg, about 1.4 mg / kg to about 2.8 mg / kg, about 1.4 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg, about 1.4 mg / kg to about 2.2 mg / kg, about 1.4 mg / kg to about 2.0 mg / kg, about 1.4 mg / kg to about 1.8 mg / kg, about 1.4 mg / kg to about 1.6 mg / kg, about 1.6 mg / kg to about 3.0 mg / kg, about 1.6 mg / kg to about 2.8 mg / kg, about 1.6 mg / kg to about 2.6 mg / kg, about 1.6 mg / kg to about 2.4 mg / kg, about 1.6 mg / kg to about 2.2 mg / kg, about 1.6 mg / kg to about 2.0 mg / kg, about 1.6 mg / kg to about 1.8 mg / kg, about 1.8 mg / kg to about 3.0 mg / kg, about 1.8 mg / kg to about 2.8 mg / kg, about 1.8 mg / kg to about 2.6 mg / kg, about 1.8 mg / kg to about 2.4 mg / kg, about 1.8 mg / kg to about 2.2 mg / kg, about 1.8 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 3.0 mg / kg, about 2.0 mg / kg to about 2.8 mg / kg, about 2.0 mg / kg to about 2.6 mg / kg, about 2.0 mg / kg to about 2.4 mg / kg, about 2.0 mg / kg to about 2.2 mg / kg, about 2.2 mg / kg to about 3.0 mg / kg, about 2.2 mg / kg to about 2.8 mg / kg, about 2.2 mg / kg to about 2.6 mg / kg, about 2.2 mg / kg to about 2.4 mg / kg, about 2.4 mg / kg to about 3.0 mg / kg, about 2.4 mg / kg to about 2.8 mg / kg, about 2.4 mg / kg to about 2.6 mg / kg, about 2.6 mg / kg to about 3.0 mg / kg, about 2.6 mg / kg to about 2.8 mg / kg or about 2.8 mg / kg to about 3.0 mg / kg.

[0476] In one embodiment of any aspects of the disclosure, the amount of ADC administered is about 0.8 mg / kg to about 4.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg or about 1.6 mg / kg to about 2.4 mg / kg.

[0477] In one embodiment of any aspects of the disclosure, the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, the amount of the ADC administered is about 1.6 mg / kg to about 2.4 mg / kg.

[0478] In one embodiment of any aspects of the disclosure, the amount of the ADC administered is about 1.6 mg / kg.

[0479] In one embodiment of any aspects of the disclosure, the amount of the ADC administered is about 2.0 mg / kg.

[0480] In one embodiment of any aspects of the disclosure, the amount of the ADC administered is about 2.4 mg / kg.

[0481] In some embodiments of any aspects of the disclosure, the ADC is administered multiple times to the same subject as part of the same course of treatment. In some embodiments, the amount of ADC administered as described herein is the amount of ADC administered in a single administration. In some embodiments, the amount of ADC administered as described herein is the amount of ADC administered in each of more than one administrations to a subject (e.g. as part of a single course of treatment). In some embodiments, the amount of ADC administered as described herein is the amount of ADC administered in each administration (e.g. as part of a single course of treatment). For example, in some embodiments in which the ADC is administered multiple times to the same subject (e.g. as part of a single course of treatment), each administration is an administration of a dose or unit dose as described herein.

[0482] In some embodiments of any aspects of the disclosure, the ADC is administered to the subject once every week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W) or once every eight weeks (Q8W).

[0483] In a specific embodiment of any aspects of the disclosure, the ADC is administered to the subject once every three weeks (Q3W).

[0484] In a specific embodiment of any aspects of the disclosure, a unit dose as described herein of the ADC is administered to the subject once every three weeks (Q3W). In a specific embodiment of any aspects of the disclosure, a unit dose comprising about 2.0 mg / kg of the ADC is administered to the subject once every three weeks (Q3W). In a specific embodiment of any aspects of the disclosure, about 2.0 mg / kg of the ADC is administered to the subject once every three weeks (Q3W).

[0485] In some embodiments of the medicaments of the disclosure, the kits of the disclosure, and the pharmaceutical compositions such as unit doses of the disclosure, the medicament, kit, pharmaceutical composition or unit dose, is provided in a form that is suitable for, or intended for, administration to the subject once every week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W) or once every eight weeks (Q8W), such as suitable for, or intended for, administration to the subject once every three weeks (Q3W).

[0486] In a specific embodiment of any aspects of the disclosure, the kits of the disclosure, and the pharmaceutical compositions such as unit doses of the disclosure, the medicament, kit, pharmaceutical composition or unit dose, is provided in a form that is suitable for, or intended for, administration to the subject once every three weeks (Q3W), and comprises an amount of 2.0 mg / kg of the ADC.

[0487] In some embodiments of any aspects of the disclosure, the ADC, pharmaceutical composition or unit dose as described herein can be administered to a patient by any appropriate systemic or local route of administration.

[0488] In a specific embodiment, the ADC is administered intravenously.

[0489] In some embodiments of the medicaments of the disclosure, the kits of the disclosure, and the pharmaceutical compositions such as unit doses of the disclosure, the medicament, kit, pharmaceutical composition, or unit dose, is provided in a form that is suitable for, or intended for, administration to the subject via any suitable route of administration, such as suitable for, or intended for, intravenous administration to the subject.

[0490] Therapy based on expression of FRa

[0491] In some embodiments of any aspects of the disclosure, the cancer is associated with FRa expression. In other words, a cancer referred to herein may comprise a cancerous cell that expresses FRa. Said cancerous cell may be comprised within a tumour. In some embodiments, the cancer is a tumour or other mass of malignant cells comprising a cancer cell which expresses FRa. In one aspect, the FRa molecule is expressed in the cancer cell at a level higher than the level of expression in a non-cancer cell. In some embodiments, the cancer further comprises a cancer cell that does not express FRa. In some embodiments of any aspects of the disclosure, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 95% of the cells in said cancer (or a sample obtained therefrom) are FRa-positive cells.

[0492] In some embodiments of any aspects of the disclosure, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 95% of the cancer cells in the subject are FRa-positive cells.

[0493] In some embodiments of any aspects of the disclosure, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 95% of the cancer cells in said cancer (or a sample obtained therefrom) are FRa-positive cells.

[0494] In a specific embodiment, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 95% of the cells in said cancer (or a sample obtained therefrom) have FRa staining at >2+ intensity by immunohistochemistry.

[0495] In a specific embodiment, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 95% of the cancer cells in the subject have FRa staining at >2+ intensity by immunohistochemistry.

[0496] In a specific embodiment, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 95% of the cancer cells in said cancer (or a sample obtained therefrom) have FRa staining at >2+ intensity by immunohistochemistry.

[0497] In some embodiments of any aspects of the disclosure, at least about 75% of the cells in said cancer (or a sample obtained therefrom) are FRa-positive cells. In a specific embodiment, at least about 75% of the cells in said cancer (or a sample obtained therefrom) have FRa staining at >2+ intensity by immunohistochemistry.

[0498] In some embodiments of any aspects of the disclosure, at least about 75% of the cancer cells in the subject are FRa-positive cells. In a specific embodiment, at least about 75% of the cancer cells in the subject have FRa staining at >2+ intensity by immunohistochemistry.

[0499] In some embodiments of any aspects of the disclosure, at least about 75% of the cancer cells in said cancer (or a sample obtained therefrom) are FRa-positive cells. In a specific embodiment, at least about 75% of the cancer cells in said cancer (or a sample obtained therefrom) have FRa staining at >2+ intensity by immunohistochemistry.

[0500] In some embodiments of any aspects of the disclosure, at least about 25% of the cells in said cancer (or a sample obtained therefrom) are FRa-positive cells. In a specific embodiment, at least about 25% of the cells in said cancer (or a sample obtained therefrom) have FRa staining at >1+ intensity by immunohistochemistry. In a specific embodiment, the cells in said cancer (or a sample obtained therefrom) have FRa staining in the range: (i) at least about 25% of the cells having FRa staining >1+ intensity by immunohistochemistry; and (ii) less than about 75% of the cells having FRa staining >2+ intensity by immunohistochemistry.

[0501] In some embodiments of any aspects of the disclosure, at least about 25% of the cancer cells in the subject are FRa-positive cells. In a specific embodiment, at least about 25% of the cancer cells in the subject have FRa staining at >1+ intensity by immunohistochemistry. In a specific embodiment, the cancer cells in the subject have FRa staining in the range: (i) at least about 25% of the cancer cells having FRa staining >1+ intensity by immunohistochemistry; and (ii) less than about 75% of the cancer cells having FRa staining >2+ intensity by immunohi stochemi stry .

[0502] In some embodiments of any aspects of the disclosure, at least about 25% of cancer cells in said cancer (or a sample obtained therefrom) are FRa-positive cells. In a specific embodiment, at least about 25% of the cancer cells in said cancer (or a sample obtained therefrom) have FRa staining at >1+ intensity per immunohistochemistry. In a specific embodiment, the cancer cells in said cancer (or a sample obtained therefrom) have FRa staining in the range: (i) at least about 25% of the cancer cells having FRa staining >1+ intensity by immunohistochemistry; and (ii) less than about 75% of the cancer cells having FRa staining >2+ intensity by immunohistochemistry.

[0503] In some embodiments, less than 25% of the cells in said cancer (or a sample obtained therefrom) are FRa-positive cells. In a specific embodiment, less than 25% of the cells in said cancer (or a sample obtained therefrom) have FRa staining at >1+ intensity by immunohi stochemi stry .

[0504] In some embodiments, less than 25% of the cancer cells in the subject are FRa-positive cells. In a specific embodiment, less than 25% of the cancer cells in the subject have FRa staining at >1+ intensity by immunohistochemistry.

[0505] In some embodiments, less than 25% of the cancer cells in said cancer (or a sample obtained therefrom) are FRa-positive cells. In a specific embodiment, less than 25% of the cancer cells in said cancer (or a sample obtained therefrom) have FRa staining at >1+ intensity by immunohistochemistry.

[0506] In some embodiments of any aspects of the disclosure, at least about 1% of the cells in said cancer are FRa-positive cells. In a specific embodiment, at least about 1% of the cells in said cancer have FRa staining at >1+ intensity by immunohistochemistry. In a specific embodiment, the cells in said cancer (or a sample obtained therefrom) have FRa staining in the range: (i) at least about 1% of the cells having FRa staining >1+ intensity by immunohistochemistry; and (ii) less than about 25% of the cells having FRa staining >1+ intensity by immunohistochemistry.

[0507] In some embodiments of any aspects of the disclosure, at least about 1% of the cancer cells in the subject are FRa-positive cells. In a specific embodiment, at least about 1% of the cancer cells in the subject have FRa staining at >1+ intensity by immunohistochemistry. In a specific embodiment, the cancer cells in the subject have FRa staining in the range: (i) at least about 1% of the cancer cells having FRa staining >1+ intensity by immunohistochemistry; and (ii) less than about 25% of the cancer cells having FRa staining >1+ intensity by immunohi stochemi stry .

[0508] In some embodiments of any aspects of the disclosure, at least about 1% of the cancer cells in said cancer are FRa-positive cells. In a specific embodiment, at least about 1% of the cancer cells in said cancer have FRa staining at >1+ intensity by immunohistochemistry. In a specific embodiment, the cancer cells in said cancer (or a sample obtained therefrom) have FRa staining in the range: (i) at least about 1% of the cancer cells having FRa staining >1+ intensity by immunohistochemistry; and (ii) less than about 25% of the cancer cells having FRa staining >1+ intensity by immunohistochemistry. Thus, in particular embodiments, the cells in said cancer (or a sample obtained therefrom) have FRa staining in the range:

[0509] (a) at least about 75% of the cells in said cancer (or a sample obtained therefrom) have FRa staining at >2+ intensity by immunohistochemistry;

[0510] (b) (i) at least about 25% of the cells having FRa staining >1+ intensity by immunohistochemistry; and

[0511] (ii) less than about 75% of the cells having FRa staining >2+ intensity by immunohi stochemi stry ; or

[0512] (c) (i) at least about 1% of the cells having FRa staining >1+ intensity by immunohistochemistry; and (ii) less than about 25% of the cells having FRa staining >1+ intensity by immunohistochemistry.

[0513] In particular embodiments, the cancer cells in the subject have FRa staining in the range:

[0514] (a) at least about 75% of the cancer cells in the subject have FRa staining at >2+ intensity by immunohistochemistry;

[0515] (b) (i) at least about 25% of the cancer cells in the subject having FRa staining >1+ intensity by immunohistochemistry; and

[0516] (ii) less than about 75% of the cancer cells in the subject having FRa staining >2+ intensity by immunohistochemistry; or

[0517] (c) (i) at least about 1% of the cancer cells in the subject having FRa staining >1+ intensity by immunohistochemistry; and (ii) less than about 25% of the cancer cells in the subject having FRa staining >1+ intensity by immunohistochemistry.

[0518] In particular embodiments, the cancer cells in said cancer (or a sample obtained therefrom) have FRa staining in the range:

[0519] (a) at least about 75% of the cancer cells in said cancer (or a sample obtained therefrom) have FRa staining at >2+ intensity by immunohistochemistry;

[0520] (b) (i) at least about 25% of the cancer cells having FRa staining >1+ intensity by immunohistochemistry; and

[0521] (ii) less than about 75% of the cancer cells having FRa staining >2+ intensity by immunohi stochemi stry ; or (c) (i) at least about 1% of the cancer cells having FRa staining >1+ intensity by immunohistochemistry; and (ii) less than about 25% of the cancer cells having FRa staining >1+ intensity by immunohistochemistry.

[0522] In some embodiments of any aspects of the disclosure (e.g. the method of treating a disease or disorder, the anti-FRa ADC of the disclosure for use in therapy, the pharmaceutical composition of the disclosure for use in therapy, the unit dose of the disclosure for use in therapy, the anti-FRa ADC of the disclosure for use in a method for preventing the onset of a disease or disorder, the pharmaceutical composition of the disclosure for use in a method for preventing the onset of a disease or disorder, the unit dose of the disclosure for use in a method for preventing the onset of a disease or disorder, the method of treating a cancer, the anti-FRa ADC of the disclosure for use in treating a cancer, the pharmaceutical composition of the disclosure for use in treating a cancer, the unit dose of the disclosure for use in treating a cancer, the method of depleting a population of FRa-positive cells in a subject, the anti-FRa ADC of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject, the pharmaceutical composition of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject, the unit dose of the disclosure for use in a method of depleting a population of FRa-positive cells in a subject), the method or treatment further comprises a step of determining the percentage of FRa-positive cancer cells in the human subject.

[0523] In some embodiments of any aspects of the disclosure, the method or treatment further comprises a step of determining the percentage of FRa-positive cells in the cancer before administering the ADC to the human subject.

[0524] In some embodiments of any aspects of the disclosure, the method or treatment further comprises a step of determining the percentage of FRa-positive cells in a sample from the cancer before administering the ADC to the human subject.

[0525] In some embodiments of any aspects of the disclosure, the method or treatment further comprises a step of determining the percentage of FRa-positive cancer cells in the cancer before administering the ADC to the human subject.

[0526] In some embodiments of any aspects of the disclosure, the method or treatment further comprises a step of determining the percentage of FRa-positive cancer cells in a sample from the cancer before administering the ADC to the human subject.

[0527] In some embodiments of any aspects of the disclosure, the method or treatment further comprises a step of determining the percentage of FRa-positive cancer cells in the subject, such as in the cancer, such as in a sample obtained from the cancer, before administering the ADC to the human subject, and selecting the human subject as suitable for said treatment with the ADC based on a determination that the subject, cancer, or sample, comprises a percentage of FRa-positive cancer cells, as described above, such as a percentage of cancer cells having FRa staining at >1+ or >2+ intensity by immunohistochemistry as described above.

[0528] In some embodiments: (a) at least about 75% of the cancer cells in said subject, such as said cancer, such as said sample, are FRa-positive cells; (b) at least about 25% of the cancer cells in said subject, such as said cancer, such as said sample are FRa-positive cells; (c) at least about 1% of the cancer cells in said subject, such as said cancer, such as said sample, are FRa-positive cells; (d) at least about 25% to about 75% of the cancer cells in said subject, such as said cancer, such as said sample are FRa-positive cells; (e) at least about 1% to about 25% of the cancer cells in said subject, such as said cancer, such as said sample are FRa-positive cells; or (f) less than about 25% of the cancer cells in said subject, such as said cancer, such as said sample are FRa-positive cells, such as cancer cells having FRa staining at >1+ or >2+ intensity by immunohistochemistry as described above.

[0529] In some embodiments, at least about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about

[0530] 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, at least about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, at least about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 1.6 mg / kg to about

[0531] 2.4 mg / kg.

[0532] In a specific embodiment, at least about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 2.0 mg / kg. In a specific embodiment, at least about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0533] In some embodiments, at least about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about

[0534] 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, at least about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, at least about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 1.6 mg / kg to about

[0535] 2.4 mg / kg.

[0536] In a specific embodiment, at least about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 2.0 mg / kg. In a specific embodiment, at least about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0537] In some embodiments, at least about 1% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, at least about 1% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, at least about 1% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 1.6 mg / kg to about 2.4 mg / kg.

[0538] In a specific embodiment, at least about 1% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 2.0 mg / kg. In a specific embodiment, at least about 1% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0539] In some embodiments, about 25% to about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, about 25% to about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, about 25% to about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 1.6 mg / kg to about 2.4 mg / kg.

[0540] In a specific embodiment, about 25% to about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 2.0 mg / kg. In a specific embodiment, about 25% to about 75% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa- positive cancer cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks. In some embodiments, less than about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, less than about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, less than about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 1.6 mg / kg to about 2.4 mg / kg.

[0541] In a specific embodiment, less than about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and the amount of the ADC administered is about 2.0 mg / kg. In a specific embodiment, less than about 25% of the cancer cells in said subject, such as in said cancer, such as in said sample, are FRa-positive cancer cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0542] In some embodiments of any aspects of the disclosure, the method or treatment further comprises a step of determining the percentage of FRa-positive cells in the cancer, such as in a sample obtained from the cancer, before administering the ADC to the human subject, and selecting the human subject as suitable for said treatment with the ADC based on a determination that the cancer, or sample, comprises a percentage of FRa-positive cells, as described above, such as a percentage of cells having FRa staining at >1+ or >2+ intensity by immunohistochemistry as described above.

[0543] In some embodiments: (a) at least about 75% of the cells in said cancer or sample are FRa-positive cells; (b) at least about 25% of the cells in said cancer or sample are FRa-positive cells; (c) at least about 1% of the cells in said cancer or sample are FRa-positive cells; (d) at least about 25% to about 75% of the cells in said cancer or sample are FRa-positive cells; (e) at least about 1% to about 25% of the cells in said cancer or sample are FRa-positive cells; or (f) less than about 25% of the cells in said cancer or sample are FRa-positive cells, such as a percentage of cells having FRa staining at >1+ or >2+ intensity per immunohistochemistry as described above.

[0544] In some embodiments, at least about 75% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, at least about 75% of the cells in said cancer, such as in said sample, are FRa- positive cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, at least about 75% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of the ADC administered is about 1.6 mg / kg to about

[0545] 2.4 mg / kg.

[0546] In a specific embodiment, at least about 75% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 2.0 mg / kg. In a specific embodiment, at least about 75% of the cells in said cancer, such as in said sample, are FRa-positive cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0547] In some embodiments, at least about 25% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about

[0548] 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, at least about 25% of the cells in said cancer, such as in said sample, are FRa- positive cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, at least about 25% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of the ADC administered is about 1.6 mg / kg to about

[0549] 2.4 mg / kg.

[0550] In a specific embodiment, at least about 25% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 2.0 mg / kg. In a specific embodiment, at least about 25% of the cells in said cancer, such as in said sample, are FRa-positive cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0551] In some embodiments, at least about 1% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about

[0552] 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, at least about 1% of the cells in said cancer, such as in said sample, are FRa- positive cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, at least about 1% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of the ADC administered is about 1.6 mg / kg to about

[0553] 2.4 mg / kg.

[0554] In a specific embodiment, at least about 1% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 2.0 mg / kg. In a specific embodiment, at least about 1% of the cells in said cancer, such as in said sample, are FRa-positive cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0555] In some embodiments, about 25% to about 75% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about

[0556] 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, about 25% to about 75% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, about 25% to about 75% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of the ADC administered is about 1.6 mg / kg to about 2.4 mg / kg.

[0557] In a specific embodiment, about 25% to about 75% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 2.0 mg / kg. In a specific embodiment, about 25% to about 75% of the cells in said cancer, such as in said sample, are FRa-positive cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0558] In some embodiments, less than about 25% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg or about 5.0 mg / kg. In some embodiments, less than about 25% of the cells in said cancer, such as in said sample, are FRa- positive cells, and the amount of the ADC administered is about 0.8 mg / kg to about 3.0 mg / kg. In a specific embodiment, less than about 25% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of the ADC administered is about 1.6 mg / kg to about 2.4 mg / kg.

[0559] In a specific embodiment, less than about 25% of the cells in said cancer, such as in said sample, are FRa-positive cells, and the amount of ADC administered is about 2.0 mg / kg. In a specific embodiment, less than about 25% of the cells in said cancer, such as in said sample, are FRa-positive cells, and about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

[0560] Assays for assessing the expression of FRot

[0561] In some embodiments, the percentage of FRa-positive cells is assayed using immunohistochemistry (IHC) or the Quantitative Continuous Scoring (QCS) assay. In one embodiment, the IHC or QCS assay is performed with an antibody reagent.

[0562] An exemplary QCS assay is described in WO 2023 / 170216, which is incorporated by reference.

[0563] In some embodiments, the assay comprises: (a) staining a tissue sample immunohistochemically using a dye linked to a diagnostic antibody, wherein the diagnostic antibody binds to the protein (e.g. FRa) on the cells (e.g. the cancer cells) in the tissue sample; (b) acquiring a digital image of the tissue sample; (c) detecting cancer cells in the digital image; (d) determining for each cancer cell a mean optical density of staining by the dye in a membrane of the cancer cell; (e) determining a median optical density of all cancer cells in the digital image.

[0564] In certain embodiments, the assay further comprises determining the recommended dosage based on whether the median optical density falls below a lower optical density threshold, between the lower optical density threshold and an upper optical density threshold, or above the upper optical density threshold, wherein the recommended dosage is zero if the median optical density falls below the lower optical density threshold, a higher dosage if the median optical density falls between the lower optical density threshold and the upper optical density threshold, and a lower dosage if the median optical density falls above the upper optical density threshold, and wherein the lower optical density threshold and the upper optical density threshold are correlated to responses of a cohort of training patients treated with the ADC; and optionally recommending to the cancer patient a therapy involving the ADC and the recommended dosage.

[0565] In certain embodiments, the assay further comprises generating a predicted efficacy score for the tissue sample based on the median optical density, wherein the predicted efficacy score is positive if the median optical density is equal to or greater than an optical density threshold and negative if the median optical density is less than the optical density threshold, and wherein the optical density threshold is correlated to responses of a cohort of training patients treated with the ADC; and optionally recommending a therapy involving the ADC to the cancer patient if the predicted efficacy score is positive. In some embodiments, the assay comprises: (a) staining a tissue sample immunohistochemically using a dye linked to a diagnostic antibody, wherein the diagnostic antibody binds to the protein (e.g. FRa) on the cancer cells in the tissue sample; (b) acquiring a digital image of the tissue sample; (c) detecting cancer cells in the digital image; (d) determining for each cancer cell an optical density of staining of the dye in the membrane of the cancer cell; (e) identifying each cancer cell as either optical-density positive if the optical density of the cancer cell is equal to or greater than an optical density threshold or optical- density negative if the mean optical density of the cancer cell is less than the optical density threshold; (f) generating a predicted efficacy score for the tissue sample based on a percentage of cancer cells in the digital image that are optical-density positive, wherein the predicted efficacy score is positive if the percentage of cancer cells that are optical-density positive is equal to or greater than a percentage threshold and negative if the percentage of cancer cells that are optical-density positive is less than the percentage threshold, and wherein the optical density threshold and the percentage threshold are correlated to responses of a cohort of training patients treated with the ADC; and optionally (g) recommending a therapy involving the ADC to the cancer patient if the predicted efficacy score is positive.

[0566] In some embodiments, the assay comprises: (a) staining a tissue sample from the cancer patient immunohistochemically using a dye linked to a diagnostic antibody, wherein the diagnostic antibody binds to the protein (e.g. FRa) on the cancer cells in the tissue sample; (b) acquiring a digital image of the tissue sample; detecting cancer cells in the digital image; determining for each cancer cell a mean optical density of staining by the dye in a membrane of the cancer cell; and (c) identifying each cancer cell as either optical-density positive if the mean optical density of the cancer cell is equal to or greater than an optical density threshold or optical-density negative if the mean optical density of the cancer cell is less than the optical density threshold; generating a proximity score for the tissue sample equaling a percentage of cancer cells in the digital image that are either optical-density positive or optical-density negative but within a predefined distance of an optical-density positive cancer cell, and optionally (d) identifying the cancer patient as one who will likely benefit from administration of the ADC if the proximity score exceeds a predetermined percentage threshold.

[0567] In some embodiments, the assay comprises: (a) staining a tissue sample immunohistochemically using a dye linked to a diagnostic antibody, wherein the diagnostic antibody binds to the protein (e.g. FRa) on the cancer cells in the tissue sample; (b) acquiring a digital image of the tissue sample; (c) detecting cancer cells in the digital image; (d) determining for each cancer cell a mean optical density of staining by the dye in a membrane of the cancer cell; (e) determining a median optical density of all cancer cells in the digital image; (f) determining a median absolute deviation of the optical densities of the cancer cells from the median optical density of all cancer cells in the digital image; (g) generating a predicted efficacy score for the tissue sample based on the median absolute deviation, wherein the predicted efficacy score is positive if the median absolute deviation is equal to or greater than a deviation threshold and negative if the median absolute deviation is less than the deviation threshold, and wherein the deviation threshold is correlated to responses of a cohort of training patients treated with the ADC; and optionally (h) recommending a therapy involving the ADC to the cancer patient if the predicted efficacy score is positive.

[0568] In some embodiments, the assay comprises: (a) staining a tissue sample immunohistochemically using a dye linked to a diagnostic antibody, wherein the diagnostic antibody binds to the protein (e.g. FRa) on the cancer cells in the tissue sample; (b) acquiring a digital image of the tissue sample; detecting cancer cells in the digital image; (c) determining for each cancer cell a mean optical density of staining by the dye in the membrane of the cancer cell; (d) determining for each cancer cell a mean optical density of staining by the dye in the cytoplasm of the cancer cell; (e) determining a difference between the mean optical density of staining of the membrane and the mean optical density of staining of the cytoplasm for each cancer cell in the digital image; (f) identifying from among all cancer cells in the digital image an 85% quantile of the difference between the mean optical density of staining of the membrane and the mean optical density of staining of the cytoplasm; and optionally (g) recommending a therapy involving the ADC to the cancer patient if the 85% quantile of the difference exceeds a predetermined difference threshold.

[0569] The diagnostic antibody used in the assays binds to FRa on the cells (e.g. cancer cells) in the tissue sample of the cancer patient. In particular embodiments, the diagnostic antibody specifically binds to human FRa. The diagnostic antibody is labelled to aid detection of cell binding. In some embodiments, the label may be a fluorophore or a dye. In some embodiments, the label could be used for immunohistochemistry. In a specific embodiment, the diagnostic antibody is linked to 3,3 ’-Diaminobenzidine (DAB). In some embodiments, the diagnostic antibody is an anti-FRa antibody obtained from Ventana (VMSI, #742-5065). In some embodiments, the diagnostic antibody is an anti-rabbit FRa antibody. In a particular embodiment, the diagnostic antibody is an anti-rabbit FRa antibody obtained from Abeam (#ab221543). In some embodiments, the diagnostic antibody is closely associated with the ADC antibody or antigen-binding fragment as described herein. In some embodiments, the diagnostic antibody is closely associated with the ADC antibody of the ADC AB1370049-SG3932. In some embodiments, the diagnostic antibody binds to cancer cells that express FRa, such as cancer cells that express FRa at their surface. In some embodiments, the diagnostic antibody binds to cancer cells from ovarian cancer, lung cancer, endometrial cancer, colon cancer, breast cancer (e.g. TNBC), cervical cancer, pancreatic cancer, gastric cancer, renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinomas (HNSCC) and malignant pleural mesothelioma. In particular embodiments, the diagnostic antibody binds to cancer cells from ovarian cancer, lung cancer, endometrial cancer, breast cancer, peritoneal cancer or fallopian tube cancer, and may also be included or be made available to a patient or a medical service provider. In some embodiments, the diagnostic antibody binds to solid tumour cells and / or an epithelial tumour cells. In one embodiment, the diagnostic antibody binds to platinum-resistant ovarian cancer cells, which may be platinum-resistant refractory ovarian cancer cells. In another embodiment, the diagnostic antibody binds to platinum-sensitive ovarian cancer cells. In some embodiments, the diagnostic antibody binds to cancer cells from non-small-cell lung carcinoma (NSCLC). In particular embodiments, the NSCLC is selected from squamous NSCLC, adenocarcinoma NSCLC, and a combination thereof.

[0570] In some embodiments, the assay for IHC is VENTANA FOLR1 Assay (VENTANA®). In epithelial ovarian cancer (EOC) tissue, neoplastic cells labelled with the VENTANA FOLR1 Assay are evaluated for percent tumour cell staining of the diaminobenzidine (DAB) signal. VENTANA FOLR1 Assay staining in EOC tissue follows a cytoplasmic and membranous pattern. The signal is classified as strong, moderate, weak, or negative based on membrane localisation only. The VENTANA FOLR1 Stain Intensity Reference Slide (Cat. No. 09382780001) is used as reference for determination of moderate signal intensity.

[0571] Negative (0) signal intensity is characterised by an absence of any detectable signal. Negative cases may still exhibit pale grey cytoplasmic and / or membranous discolouration. Weak (1+) signal intensity is characterised by a faint gold / light brown hue that may be partial or circumferential. Moderate (2+) or Strong (3+) signal intensity is characterised by a chocolate brown to thickened dark brown, black hue that may be partial or circumferential. The signal may be distributed heterogeneously having more than one intensity level. The relative percentages of neoplastic cells staining at each of the following signal intensities: strong (3+), moderate (2+), weak (1+), and negative (0), are visually estimated and used to generate a diagnostic score.

[0572] In certain embodiments, FRa-positive cells as described herein have a cell staining at >1+ or >2+ intensity by immunohistochemistry. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure.

[0573] The above embodiments are to be understood as illustrative examples. Further embodiments are envisaged. It is to be understood that any feature described in relation to any some embodiments may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments or aspects, or any combination of any other of the embodiments or aspects. Furthermore, equivalents and modifications not described above may also be employed without departing from the scope of the disclosure, which is defined in the accompanying claims.

[0574] In the context of the present disclosure other examples and variations of the antibodies and methods described herein will be apparent to a person of skill in the art. Other examples and variations are within the scope of the disclosure, as set out in the appended claims.

[0575] All documents cited herein are each entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited documents.

[0576] EXAMPLES

[0577] EXAMPLE 1 - IV Administration of AZD5335 in cynomolgus monkey (2 dose, 3 weeks apart)

[0578] Aim

[0579] To determine the potential toxicity and tolerability profile of AZD5335 in male cynomolgus monkeys following 2 single (each 3 weeks apart) IV dose administrations.

[0580] To investigate the pharmacokinetics (PK) of the unconjugated antibody, free payload, and the ADC.

[0581] Materials and methods

[0582] Four groups, each consisting of 3 male monkeys, received 2 doses of either unconjugated antibody (FRa-mAb AB1370049) at 15 mg / kg or AZD5335 at 15, 20, and 25 mg / kg by IV bolus injection 3 weeks apart (dosing on Days 1 and 22). Injection volume was 1.4 to 2.5 ml / kg. Scheduled necropsies were conducted 21 days after the final dose (Day 43) for monkeys administered AZD5335 and monkeys administered FRa-mAb were released from study on Day 44. The following parameters and endpoints were evaluated in this study: clinical evaluations (including morbidity / mortality and cage side observations), body weights, qualitative food consumption, injection site observations, ophthalmic observations, respiratory rate and pulse oximetry, clinical pathology parameters (haematology, clinical chemistry, coagulation, urinalysis, and urine clinical chemistry), PK, anti-drug antibodies, cytokine levels, and anatomic pathology (macroscopic, microscopic, and organ weights).

[0583] Results

[0584] Systemic exposures (total antibody, total ADC, and TOPOi payload Co, Cmax, and AUC(o-t)) of AZD5335 were dose proportional over the dose range from 15 to 25 mg / kg, but did not appear to increase from 15 to 20 mg / kg on either dosing occasion. Following administration of AZD5335, mean Co / Cmax and AUC(o-2id) for total antibody, total ADC, and TOPOi payload were similar or within 2-fold on Day 22 relative to Day 1. The exposure of AZD5335 for the 15 mg / kg dose group was slightly lower than the exposure of the unconjugated anti-FRa antibody at 15 mg / kg.

[0585] Following the second dose administration of AZD5335 at 20 mg / kg, 1 of 3 animals was euthanised on Day 33 due to declining clinical condition, including dehydration, liquid faeces, and failure to respond to treatment. Body weight reductions compared with Day 1 were noted following each dose administration: 13% on Day 14, 4% on Day 21 (due to a 0.2 kg gain prior to the Day 22 dose), 9% on Day 28, and 13% on Day 33 prior to necropsy. Changes in clinical pathology (decreased white blood cells, lymphocytes, monocytes, and RBC mass [with no concurrent increase in reticulocytes], albumin, cholesterol, phosphorus, and sodium; and increased red cell distribution width, platelet count, and urea nitrogen) were observed. There were no AZD5335-related effects on cytokine levels, macroscopic evaluation, or organ weights. AZD5335-related microscopic changes were observed in the bone marrow (decreased erythropoiesis) and small and large intestine (minimal to mild glandular degeneration / atrophy, minimal glandular regeneration, and / or minimal glandular dilatation with cellular debris). Other microscopic findings were observed that were consistent with stress and / or inanition. The unscheduled euthanasia of this animal was considered of uncertain relationship to AZD5335 administration due to lack of similar occurrence of findings in the surviving animals at 20 mg / kg that had similar exposure and all animals dosed at 25 mg / kg, low severity of the clinical pathology and direct AZD5335-related microscopic findings, and undetermined cause for moribundity based on macroscopic and microscopic assessments. In animals administered AZD5335 at 15, 20, and 25 mg / kg that survived to scheduled euthanasia, AZD5335-related findings were limited to haematological and GI tract changes. AZD5335-related changes in haematology parameters consisted of minimal to mild decreases in lymphocytes and monocytes throughout the dosing period in animals at > 15 mg / kg, though most notably on Days 4 through 15, 29, and / or 36. The decreases in lymphocytes and monocytes generally approximated or trended toward baseline by Day 43 at all dose levels, except for lymphocytes at 25 mg / kg. Additionally, animals at > 15 mg / kg generally had minimal to mild test item-related decreases in RBC mass throughout the dosing period, but most notably on Days 4, 8, 29, and / or 36. Two animals at 20 mg / kg had moderately decreased RBC mass on Days 22 or 36, respectively. Reticulocytes were mildly to moderately decreased in animals at

[0586] > 20 mg / kg within 1 week of each dose, and at 15 mg / kg on Day 29 only (1 week after second dose), with subsequent minimal to mild increases in reticulocytes observed on Days 15, 22, 36, and / or 46 (by 2 weeks after each dose). By Day 43, the decreases in RBC mass were no longer present at 15 mg / kg and trended towards or approximated baseline at > 20 mg / kg. Animals at

[0587] > 20 mg / kg had minimal increases in platelets on Days 15, 29, and / or 36, which were no longer present by Day 43. Though test item effects on RBC mass and platelets were considered likely based on the mechanism of action (MOA) of the payload, a procedure-effect could not be entirely excluded given the general minimal magnitude of the changes and lack of dose dependency.

[0588] Salient microscopic changes associated with the administration of AZD5335 were noted in mucosal glands in the duodenum, jejunum, ileum, cecum, colon, and rectum of individual animals dosed at > 20 mg / kg and were characterised by one or more of the following: minimal degeneration / atrophy, regeneration, dilatation, and cellular debris. However, due to the presence of 4+ entamoebic cyst load detected by faecal swab in 1 animal given 20 mg / kg, and recognition that intestinal parasite infestation can be associated with similar low-grade microscopic glandular changes, a direct correlation between these findings and the test item was equivocal in this animal / group.

[0589] Conclusion

[0590] In conclusion, administration of FRa-mAb by IV (slow bolus) injection on Days 1 and 22 was well-tolerated at 15 mg / kg (total antibody Co 456 pg / ml and AUC(o-2id) 3180 day-pg / ml) on Day 22. There was 1 unscheduled euthanasia at 20 mg / kg of AZD5335 (mean total ADC Co 416 pg / ml and AUC(o-2id) 1720 day-pg / ml) due to declining condition. This was considered of uncertain relationship to AZD5335 administration since 2 of 3 animals survived to scheduled termination and there were no apparent intra-group exposure differences. There was no dose dependency and the cause of moribundity was undetermined following macroscopic and microscopic assessment.

[0591] Administration of AZD5335 by IV (slow bolus) injection on Days 1 and 22 was well- tolerated at 25 mg / kg (total ADC Co 773 pg / ml and AUC(o-2id) 3990 day-pg / ml on Day 22).

[0592] EXAMPLE 2 - A Repeat-dose 6-week Study of AZD5335 by Intravenous Injection in Cynomolgus Monkeys with a 6-week Recovery Period

[0593] Aim

[0594] To determine the potential toxicity profile and local tolerability of AZD5335 when given by repeat-dose IV bolus injection (2 administrations, 3 weeks apart) to cynomolgus monkeys, and to evaluate the potential reversibility of any findings following a 6-week recovery period.

[0595] To investigate the toxicokinetics (TK) of AZD5335 and the TOPOi payload.

[0596] Materials and methods

[0597] Groups of male and female monkeys (n = 3 / sex for Groups 1 to 4) were administered 15, 20 or 25 mg / kg / dose of AZD5335 by IV bolus injection or buffer solution (acting as a negative control) at 3-week intervals (dosing on Days 1 and 22). Injection volume was 2.5 ml / kg. Scheduled necropsies were conducted 21 days after the final dose (main study necropsy on Day 43). Three additional groups of male and female monkeys (n = 2 / sex for Groups 1, 3 and 4) were administered control buffer solution, 20 or 25 mg / kg / dose as above, but were retained for an additional 6-week treatment-free period with scheduled necropsy 62 days after the last dose (recovery necropsy on Day 84). Due to early terminations, the 25 mg / kg group only received the first dose.

[0598] The following parameters and endpoints were evaluated in this study: mortality, clinical observations, injection site observations / dermal scoring, body weights, qualitative food consumption, neurologic examinations, qualitative electrocardiology, blood pressure, heart rate, ophthalmology, respiratory rate, pulse oximetry, clinical pathology parameters (haematology, coagulation, clinical chemistry, cytokines, and urinalysis), bioanalysis and TK parameters, gross necropsy findings, organ weights, and histopathologic examinations. In addition, anti-drug antibody samples were collected. Results

[0599] Pharmacologically relevant species are required to assess potential toxicity of the ADC that could be driven by both target engagement and subsequent supra-pharmacology responses, and / or off-target toxicity due to exposure of non-target tissue to the cytotoxic payload. The cynomolgus monkey met the criteria as a pharmacologically relevant species, based on high amino acid sequence homology of the FRa antigen, expression levels, and comparable affinity of the antibody component of the ADC to the FRa antigen in the monkey, when these criteria were assessed against human and other animal species.

[0600] In the pivotal 6-week repeat-dose (Q3W x 2) IV study with a 6-week recovery period in the cynomolgus monkey, in which dose levels of 15, 20, and 25 mg / kg were evaluated, the highest nonseverely toxic dose (HNSTD) was identified as 15 mg / kg. A tabular summary of the principal treatment-related findings and comparisons of systemic exposure to AZD5335 is presented in Table 9.

[0601] Table 9: Summary of Key Findings and Exposure Data in Pivotal Toxicology Study with

[0602] AZD5335

[0603] All major organs and functions (respiratory, cardiovascular, renal, and nervous system) were assessed as part of this study, and at the HNSTD, no AZD5335-related effects were noted on survival, local (skin) tolerance, body weight, ECG blood pressure, respiration rate, ophthalmoscopy, neurological endpoints, clinical chemistry, urinalysis, systemic cytokine release, or coagulation parameters. At the HNSTD, treatment-related changes were minimal, recoverable, and associated with anticipated pharmacological activity of AZD5335 on the haematopoietic system and included mild to moderate and transient effects on some red and white cell parameters. At dose levels above the HNSTD; although, no effects on ECG blood pressure, respiration rate, ophthalmoscopy, neurological endpoints, or systemic cytokine release were observed, clinical observations that led to moribund state included hunched posture and liquid / mucoid / nonformed faeces. Mild to moderate, reversible effects were also observed on the haematopoietic system and the histopathological findings confirmed that the target organs were the GI system, bone marrow, and ovary, consistent with the mechanism of action for the TOPOi payload, in animals dosed with > 20 mg / kg / dose. In animals administered 20 mg / kg that survived to scheduled recovery termination, toxicity to target organs either fully resolved or showed signs of recovery. This toxicity profile was similar to that determined in the non-GLP dose range finding toxicity study (Example 1) in male monkeys with key organs identified as bone marrow and GI; although, in the present Example (GLP study) the severity of the AZD5335-related findings was more prominent as the dose range finding study identified the HNSTD of 25 mg / kg with minimal to mild effects on the haematopoietic system (minimal to mild decreased white blood cell and minimal to moderate reticulocyte counts) with no histopathological correlate 21 days post-dose. Findings in the GI tract were noted in mucosal glands in the duodenum, jejunum, ileum, cecum, colon, and rectum of individual animals at > 20 mg / kg and were characterised by one or more of the following: minimal degeneration / atrophy, regeneration, dilatation, and cellular debris.

[0604] Conclusion

[0605] In conclusion, at 15 mg / kg / dose, AZD5335-related changes were limited to clinical observations (i.e. liquid / nonformed faeces) and haematology parameters (i.e. transient decreased white blood cell count, neutrophils, and reticulocytes) which trended towards baseline or reversed by the end of the dosing period. No microscopic findings were evident in animals administered 15 mg / kg. The highest nonseverely toxic dose (HNSTD) was determined to be 15 mg / kg / dose (IV Q3W x 2 doses).

[0606] EXAMPLE 3 - Dose escalation study of AZD5335 monotherapy in human

[0607] AZD5335 was administered (0.8, 1.6, 2.0, 2.4, or 3.0 mg / kg intravenously) every 3 weeks (Q3W) in human patients with platinum-resistant refractory OC (PRROC). Figure 3 summarises the study design. Outcomes analysed included (A) safety and tolerability, (B) pharmacokinetics, and (C) efficacy, as set out below.

[0608] Blood samples were collected to enable the characterisation of AZD5335 PK and, where appropriate, the investigation of additional metabolites.

[0609] Starting Dose Justification

[0610] AZD5335 monotherapy was investigated at dose levels ranging from 0.8 mg / kg to 3.0 mg / kg Q3W intravenously. A starting dose for single agent AZD5335 of 0.8 mg / kg was selected based on the results of the good laboratory practice (GLP) toxicology study in cynomolgus monkeys, scaling of pharmacokinetics (PK) to humans from cynomolgus monkeys, and FDA regulatory guidelines. Dose increments and clinically efficacious dose range (0.8 to 3.0 mg / kg) were determined, and modelled exposure were estimated.

[0611] The highest nonseverely toxic dose (HNSTD) of AZD5335 in the cynomolgus monkey was identified as 15 mg / kg Q3W for a total of 2 doses in Example 2, and the human equivalent dose (HED) for this HNSTD was calculated to be 4.8 mg / kg (Farres et al. 2015, FDA 2005). The selection of the clinical starting dose of 0.8 mg / kg Q3W for a first-time-in-human (FTiH) study was based on l / 6th the HED of 0.8 mg / kg. At a dose of 0.8 mg / kg AZD5335 Q3W, administered over 1 hour IV infusion, the steady state plasma exposures of AZD5335 are predicted to be a Cmax at steady state (Cmax,ss) of 20 pg / ml and an AUC from time 0 to Day 21 (AUCo-2id) of 94 pg.d / ml.

[0612] Definitions

[0613] The following terms are used in the present Example.

[0614] Dose-limiting Toxicity (DLT)

[0615] The DLTs were evaluated during dose escalation. The DLT-evaluation period was 21 days from the first dose of AZD5335 on Cycle 1 Day 1 or up to and including the planned end of Cycle 1 if an alternative dosing schedule was explored. A DLT is defined as any > Grade 3 treatment-emergent AE that occurs during the DLT evaluation period, not attributable to primary disease or disease-related processes or intercurrent illnesses, with modifications or exceptions.

[0616] Efficacy Assessments

[0617] Tumour response is assessed by RECIST vl .1 (Eisenhauer et al, 2009) according to the schedule. Tumour Evaluation

[0618] Tumour assessments use images from computerised tomography (CT, preferred) or magnetic resonance imaging (MRI), with IV contrast, of the chest, abdomen, and pelvis, and additionally investigate areas that may be involved based on signs and symptoms of individual subjects, collected during screening / baseline and at regular (follow-up) intervals during study intervention. Brain scans (MRI preferred) are mandatory at screening / baseline for all subjects in this study. For ovarian cancer participants without known brain metastases, a CT scan is sufficient. Post baseline MRI of the brain only has to be performed in subjects with brain metastases at baseline, while subjects without brain metastases do not need additional brain scans for subsequent tumour assessments unless clinically indicated. The imaging modality used for baseline tumour assessment is kept the same consistently at each subsequent followup assessment throughout the study if possible.

[0619] Screening / baseline imaging is performed no more than 28 days before start of study intervention and ideally is performed as close as possible to and prior to the start of study intervention. Scans obtained as part of standard clinical practice, prior to informed consent, but within the 28-day period are acceptable. Tumour assessments are performed per the schedule in the respective sub-studies until objective disease progression as defined by RECIST vl .1 and assessed by the Investigator, or withdrawal of consent. Response (complete response (CR) or partial response (PR)) is confirmed by a repeat, consecutive scan at least 4 weeks after the first documentation of response.

[0620] Tumour markers are not used for tumour response assessments as per RECIST vl.l. Tumour markers are collected for separate analyses. However, the results will not contribute to tumour response based on RECIST vl. l assessment.

[0621] Eastern Cooperative Oncology Group (ECOG) Performance Status (PS)

[0622] The subject's performance status is assessed as detailed in the schedule of the respective sub-study using the ECOG PS scale.

[0623] Adverse Events

[0624] An adverse event (AE) is the development of any untoward medical occurrence in a subject or clinical study subject administered a medicinal product and which does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavourable and unintended sign (e.g. an abnormal laboratory finding), symptom (for example nausea, chest pain), or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. The term AE is used to include both serious and non-serious AEs and can include a deterioration of a pre-existing medical occurrence. An AE may occur at any time, including run-in or washout periods, even if no study intervention has been administered.

[0625] Serious Adverse Events

[0626] A serious adverse event (SAE) is an AE occurring during any study phase (i.e. run-in, treatment, washout, follow-up), that fulfils one or more of the following criteria: (1) results in death; (2) is immediately life-threatening; (3) requires subject hospitalisation or prolongation of existing hospitalisation; (4) results in persistent or significant disability or incapacity; (5) is a congenital anomaly or birth defect; (6) is an important medical event that may jeopardise the subject or may require medical treatment to prevent one of the outcomes listed above.

[0627] Disease Progression

[0628] Disease progression can be considered as a worsening of a subject's condition attributable to the disease for which the investigational product is being studied. It may be an increase in the severity of the disease under study and / or increases in the symptoms of the disease. The development of new or progression of existing metastasis to the primary cancer under study should be considered as disease progression and not an AE. Events that are unequivocally due to disease progression should not be reported as AEs during the study.

[0629] AZD5335

[0630] AZD5335 is supplied as either a lyophilised or liquid product. Lyophilised AZD5335 is supplied in 20R amber vials as a sterile lyophilised solid containing 100 mg AZD5335 (nominal) per vial. The AZD5335 investigational product (IP) must be stored at 2°C to 8°C (refrigerated) prior to use and protected from light exposure. AZD5335 vials must not be frozen.

[0631] Liquid AZD5335 is supplied as an intravenous bag protectant (IVBP) solution to ensure compatibility of AZD5335 to the IV infusion components and diluent solution. The IVBP is stored at 2°C to 8°C (refrigerated). The lyophilised AZD5335 must not be reconstituted with the IVBP solution. Sterile water for injection is used for reconstitution.

[0632] All IPs should be kept in a secure and dry place. Vials should be stored protected from light at 2°C to 8°C (refrigerated) and must not be frozen.

[0633] Weight-based doses are calculated using the following formula: Study subjects

[0634] Inclusion criteria:

[0635] • Age >18 years.

[0636] • Eastern Cooperative Oncology Group performance status 0 or 1.

[0637] • Histologically confirmed diagnosis of relapsed, high-grade epithelial, platinum-resistant recurrent OC, primary peritoneal cancer, or fallopian tube cancer.

[0638] • Measurable disease per Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST vl .l).

[0639] • Received adequate prior therapy in accordance with local practice or a clinical trial is deemed the best option for further treatment based on response and / or tolerability to prior therapy.

[0640] • Prior chemotherapy regimens must have contained bevacizumab, unless ineligible.

[0641] • No restriction on the number of prior lines of therapy received; prior FRa-targeted therapies, including ADCs, were permitted.

[0642] • All patients provided an archival or baseline tumour sample, but patients who received prior FRa-targeted therapy provided a fresh baseline biopsy.

[0643] • No limits on the FRa expression levels, which were determined retrospectively.

[0644] • Adequate organ and marrow function as defined in Table 10.

[0645] • Life expectancy >12 weeks.

[0646] Table 10: Criteria for Adequate Organ and Marrow Function aPTT = partial prothrombin time; CrCl = creatinine clearance; D1C1 = Day 1 Cycle 1; INR = international normalised ratio; ULN = upper limit of normal. Exclusion criteria:

[0647] • Spinal cord compression or a history of leptomeningeal carcinomatosis.

[0648] • Brain metastases, unless asymptomatic and stable.

[0649] • Unresolved grade >2 toxicities from prior therapy (exclusions apply, e.g. vitiligo and alopecia). • Interstitial lung disease (ILD) / pneumonitis or a history of (non-infectious)

[0650] ILD / pneumonitis that required oral or intravenous steroids or supplemental oxygen, or where suspected ILD / pneumonitis cannot be ruled out by imaging at screening. Table 11: Baseline patient and disease characteristics

[0651] ECOG PS, Eastern Cooperative Oncology Group performance status; FRa, folate receptor a;

[0652] PARP, poly-ADP ribose polymerase; TOPOi, topoisomerase 1 inhibitor

[0653] Collection of tumour samples

[0654] Provision of a tumour tissue sample (block or unstained slides) at baseline was mandatory for all participants. Expression of FRa was evaluated from a baseline tumour sample as determined prospectively by an analytically validated IHC or QCS assay. If feasible, at each fresh tumour sample timepoint, subjects were asked to undergo image-guided core needle or excisional tumour biopsies. Per institutional practice, image- guided fresh core needle tumour biopsies (preferably 18 gauge or larger needle) should be preferentially obtained from tumour tissues that are safely accessible, as determined by the Investigator. The tumour biopsy procedure was performed by core needle, under radiological guidance, or surgically if the site of disease was superficial and palpable or visible. It was mandated that the core biopsy be removed directly from the tumour in situ and not cored from a surgically removed tumour. This was to ensure the best possible quality of the biopsy, so the blood / nutrient supply to the tumour was not disrupted prior to biopsy collection.

[0655] (A) - Safety and tolerability

[0656] Aim

[0657] To assess the safety and tolerability of AZD5335 monotherapy (0.8, 1.6, 2.0, 2.4, or 3.0 mg / kg intravenously; every 3 weeks (Q3W)) in patients with platinum-resistant refractory OC (PRROC) by evaluating adverse events (AEs), serious AEs (SAEs), and dose-limiting toxicities (DLTs).

[0658] Materials and methods

[0659] Eligible subjects as set out above received AZD5335 Q3W administered via intravenous (IV) infusion at the selected dose starting on Cycle 1 Day 1 for up to or over 20 cycles. Subjects were treated with study intervention until disease progression, unacceptable toxicity, investigator's decision, completion of the maximum of treatment cycles, or withdrawal of consent. All subjects were followed for survival until the end of the study.

[0660] After the intervention period, the subjects were further evaluated for disease progression, end of treatment condition, and follow-up condition.

[0661] Results

[0662] The safety results are summarised in Table 12. Of the 39 patients tested, 37 (94.9%) patients had at least 1 AE considered by the investigator to be possibly related to AZD5335. The majority of AEs were CTCAE Grade 1.

[0663] SAEs considered by the investigator to be possibly related to AZD5335 were reported in 6 (15.4%) patients. No DLTs or deaths due to treatment were reported. The maximum tolerated dose has not been reached.

[0664] AZD5335 dose reduction due to AEs considered by the investigator as possibly related to AZD5335 were observed in 4 patients. AZD5335 treatment discontinuation due to AEs considered by the investigator as possibly related to AZD5335 was observed in 3 patients.

[0665] The most common treatment-related AEs (TRAEs) were nausea (61.5%), neutropenia (38.5%), and fatigue (30.8%; Table 13). The most common grade >3 TRAEs were neutropenia (17.9%) and anaemia (15.4%). Table 12: Safety summary of AZD5335 monotherapy by the investigator. The table includes AEs with an onset date on or after the date of first dose of AZD5335 up to and including 30 days following the date of last dose but prior to subsequent cancer therapy. Patients with multiple occurrences in the same category are counted once per category regardless of the number of occurrences.

[0666] AE, adverse event; SAE, serious adverse event; TRAE, treatment-related adverse event

[0667] Table 13: TRAEs of any grade occurring in >15% of patients

[0668] *Combined preferred terms: neutropenia, neutrophil count decreased + neutropenia; anaemia, haemoglobin decreased + anaemia; fatigue, asthenia + fatigue. Treatment-related events were defined as reasonable possibility that the AE was caused by AZD5335, as assessed by the investigator. The table includes AEs with an onset date or that worsen on or after the date of first dose and up to and including 30 days following the date of last dose of treatment. Patients with multiple occurrences are counted once per preferred term regardless of the number of occurrences.

[0669] AE, adverse event; TRAE, treatment-related adverse event

[0670] Conclusion

[0671] AZD5335 demonstrated a manageable safety profile consistent with other TOPOi-based ADCs, with manageable levels of dose modifications.

[0672] (B) - Pharmacokinetics

[0673] Aim

[0674] To characterise the pharmacokinetics (PK) of AZD5335.

[0675] Materials and methods

[0676] Plasma samples were collected from the patients treated with AZD5335 as described above, for measurement of plasma concentrations of AZD5335 (ADC assay), total anti-FRa antibody AB1370049 (total antibody assay), and total unconjugated payload.

[0677] Concentrations of unconjugated payload, total antibody and total ADC from AZD5335 in human plasma samples were measured via HPLC with MS / MS detection using validated methods.

[0678] An immunoaffinity approach using SMART Digest IA magnetic beads coated with anti- ID antibody (AB1670104 mouse IgGl) was used to enrich AZD5335 from human plasma. Because AZD5335 is too large for practical direct quantitative analysis using LC / MS / MS technology, the bound proteins were subjected to “on-bead” and in-solution proteolysis with trypsin. As a result of the trypsin digestion, characteristic peptide fragments originating from the antibody were produced. In short, for each test sample a 20.0-pL aliquot was mixed with SMART Digest IA streptavidin magnetic beads coated with the anti-ID antibody. After washing, the beads were then incubated at 70 °C for 2 hours to complete the trypsin digestion. In addition, an aliquot of the trypsin digest was further hydrolysed using papain to release free payload. The characteristic peptides were quantified as surrogates for the Total Antibody concentrations and the released payload was quantified as a surrogate for the ADC concentration. Calibration curves and quality controls are prepared by spiking AZD5335 in blank human plasma at concentrations ranging from 0.300 - 45.0 pg / mL. To quantitate unconjugated payload, a nominal payload concentration range of 0.0500 to 50.0 ng / mL was used in control samples.

[0679] To quantitate total antibody and total ADC from AZD5335, a 20.0-pL sample aliquot is isolated using SMART Digest IA streptavidin magnetic beads coated with anti-ID antibody. After washing, the beads are then incubated at 70 °C for 2 hours to complete the trypsin digestion. A 50-pL aliquot of the digest is removed and further digested with papain to release payload for Total ADC analysis. The remaining of the original extract is further digested with regular trypsin at 37 °C overnight for the Total Antibody assay. The final extract is analysed via HPLC and MS / MS detection using positive ion electrospray.

[0680] Results

[0681] Available PK data for total ADC (AZD5335), total antibody (AB 1370049), and total unconjugated payload were obtained from a total of 37 patients following IV infusion of 0.8 to 3.0 mg / kg AZD5335 Q3W. The PK parameters of the plasma concentration-nominal time data for total ADC (AZD5335), total antibody (AB1370049), and total unconjugated payload were derived using non-compartmental analysis methods in Phoenix® WinNonlin® Version 8.3 or higher (Certara).

[0682] Mean PK profiles of total ADC (AZD5335), total antibody, and total unconjugated payload following a single AZD5335 dose are presented in Figure 4. ADC and total antibody concentrations were similar, indicating that conjugation of AZD5335 was stable in patients (Figure 4). Minimal accumulation of ADC was observed within the dose range tested (half-life of 5.25-6 days). AZD5335 and total unconjugated warhead exhibited approximately linear PK, with exposure increasing in proportion to dose over the dose range (Figures 4 and 5). Figure 5 depicts the summary of dose-normalised PK parameters of ADC (AZD5335) and total unconjugated payload: dose-normalised maximal concentration (Cmax / Dose) and dose- normalised area under the concentration curve (AUC / Dose). Conclusion

[0683] In summary, the PK of AZD5335 and its payload were linear from 0.8 to 3.0 mg / kg. AZD5335 was stable in circulation with minimal accumulation prior to the second dose. The linearity of the PK profile and the demonstrated stability of AZD5335 in circulation supports Q3W dosing.

[0684] (C) - Efficacy

[0685] Aim

[0686] To determine the preliminary antitumour activity of AZD5335 monotherapy in terms of the objective response rate (ORR), disease control rate, and duration of response.

[0687] To determine the relationship between AZD5335 efficacy and FRa expression on cancer cells.

[0688] Results

[0689] The objective response rate (ORR) (confirmed and unconfirmed) following treatment with AZD5335 as described above was 34.2% (13 / 38) across all patients, with responses observed at all doses. Of these, 26.3% (10 / 38) were confirmed objective responses. There were no complete responses. Confirmed responses for patients in the 3.0 mg / kg group were not available due to the short duration of follow-up.

[0690] Best percentage change in target lesion size is shown in Figure 6 and best objective response and treatment status is shown in Figure 7.

[0691] 46.2% (6 / 13) patients with high FRa expression had a confirmed objective response (Table 14). High FRa expression was defined as >75% of tumour cells staining at >2+ intensity by immunohistochemistry. Of those with high FRa expression and receiving AZD5335 at doses >1.6 mg / kg (n=9), 5 (55.6%) had a confirmed objective response.

[0692]

[0693] *One patient was excluded from the 3 mg / kg cohort due to not having the opportunity to complete any on-treatment scan at the time of the data cutoff. fDenominator is the number of patients in the FRa subgroup.

[0694] CI, confidence interval; FRa, folate receptor a; ORR, objective response rate including both confirmed and unconfirmed responses; cORR, confirmed objective response rate; PR, partial response

[0695] Advantageously, 35.7% (5 / 14) patients with low FRa expression had an objective response (Table 14), with 21.4% (3 / 14) patients having a confirmed objective response. Low FRa expression was defined as >25% tumour cells staining at >1+ intensity by immunohistochemistry but less than >75% of tumour cells staining at >2+ intensity by immunohistochemistry. Of those with low FRa expression and receiving AZD5335 at doses >1.6 mg / kg (n=12), 41.7% (5 / 12) had an objective response, with 25% (3 / 12) having a confirmed objective response.

[0696] Conclusion

[0697] Promising efficacy data have been observed in a heavily pretreated population of patients with PRROC. Radiological responses were observed at all explored doses of AZD5335. 46.2% (6 / 13) patients with high FRa expression had a confirmed objective response. Advantageously, 35.7% (5 / 14) patients with low FRa expression had an objective response, with 21.4% (3 / 14) patients having a confirmed objective response. Efficacy data improved in patients receiving doses >1.6 mg / kg, but objective responses were observed at all doses tested.

[0698] EXAMPLE 4 - AZD5335 monotherapy in human

[0699] This Example presents a combined dataset including results from the dose escalation study of Example 3 including some additional participants not shown in Example 3 (together indicated as “MIA”), and results from an additional randomised study (“M1B1”). In the “Ml Bl” study, AZD5335 was administered (1.6 mg / kg, 2.0 mg / kg or 2.4 mg / kg intravenously) every 3 weeks (Q3W) in human patients with platinum-resistant ovarian cancer (PROC).

[0700] Outcomes analysed included (A) efficacy, (B) clinical safety, and (C) clinical pharmacology, as set out below. For efficacy and clinical safety, the dose levels of 1.6 mg / kg, 2.0 mg / kg and 2.4 mg / kg from the combined data set (“MIA” and “M1B1”) were analysed. For clinical pharmacology, the dose levels of 1.6 mg / kg, 2.0 mg / kg and 2.4 mg / kg from the combined data set (“MIA” and “M1B1”) were analysed, as well as data for dose levels of 0.8 mg / kg and 3.0 mg / kg from the “MIA” dataset, included for completeness. Across the MIA and M1B1 studies, a total of 166 patients were treated at the three selected dose levels (1.6 mg / kg [N = 50], 2.0 mg / kg [N = 56], and 2.4 mg / kg [N = 60]), including 124 participants from the M1B1 study (1.6 mg / kg [N = 41], 2.0 mg / kg [N = 41], and 2.4 mg / kg [N = 42]). Dose level cohorts were broadly balanced with respect to baseline characteristics. Table 15 presents data on baseline characteristics for participants in the combined dataset (MIA + M1B1) who were treated with the three selected AZD5335 dose levels (1.6, 2.0 and 2.4 mg / kg).

[0701] Table 15: Baseline demographic and disease characteristics of the combined monotherapy dataset

[0702] N: Number of subjects per treatment group. Percentages are calculated based on the number of patients in the full analysis set under each treatment arm / total. PARPi, poly-ADP ribose polymerase inhibitor; TOPOi, topoisomerase 1 inhibitor; FRa, folate receptor a. (A) - Efficacy

[0703] Objective response rate (ORR) and duration of response (DoR)

[0704] The objective response rate (ORR) showed efficacy across the three selected AZD5335 dose levels, with no clear dose response relationship (see Table 16), and large overlaps of the corresponding confidence intervals.

[0705] Duration of response (DoR) analysis suggested that AZD5335 dose levels >2.0 mg / kg were associated with more durable responses. DoR5 was higher in the 2.0 mg / kg and 2.4 mg / kg doses than for the 1.6 mg / kg dose (see Table 16).

[0706] Table 16: Objective response analysis, combined for MIA and M1B1

[0707] ORR - objective response rate (confirmed responses only); Time to Response - time from first IMP dose until first reported objective response per RECIST 1.1; DoR5 - probability that patients in response per RECIST 1.1 will remain alive and progression-free at least 5 months after responding.

[0708] Durability of response was analysed by assessing individual patient tumour size changes over time (data not shown). Patients treated with 1.6 mg / kg showed an absence of complete response (CR) (disappearance of target lesions (TLs) or % change from baseline in TL of - 100%). Reduction in target lesion size appeared more sustained (% change from baseline in TL < -30% for beyond -220 days from dosing) in the 2.0 mg / kg and 2.4 mg / kg dose cohorts versus 1.6 mg / kg.

[0709] Progression-free survival

[0710] More than 50% of patients in all three groups across the combined MIA and M1B1 dataset showed progression free survival during the time frame assessed. The proportion of participants who remained progression-free was higher in the 2.0 mg / kg dose group (64.0%) and also the 2.4 mg / kg dose group (56.7%) as compared to the 1.6 mg / kg dose group (53.8%) (Table 17)

[0711] This finding was more pronounced in the M1B1 cohort: 1.6 mg / kg dose group 48.5%, 2.0 mg / kg dose group 66.7%, 2.4 mg / kg dose group 68.8%.

[0712] It is noted that many patients had not had the chance to reach 6 months of follow-up at the time of assessment, but the median duration of follow-up was comparable across the dose levels (1.6 mg / kg, 2.0 mg / kg, and 2.4 mg / kg dose groups; median of 4.9-5.4 months as indicated in Table 17)

[0713] In the combined dataset (MIA + M1B1), the fraction of participants with PFS events was higher (38.0%) in the 1.6 mg / kg cohort than either of the 2.0 mg / kg or 2.4 mg / kg cohorts (26.8% and 28.3% respectively). In the M1B1 cohort, where the dose level cohorts were opened broadly simultaneously, a similar pattern was observed (with PFS events at 29.3%, 17.1% and 19.0% in the 1.6 mg / kg, 2.0 mg / kg and 2.4 mg / kg cohorts respectively).

[0714] Table 17: Progression-free survival analysis, combined for MIA and M1B1

[0715] PFS event: Percentage of patients with PFS events (# of patients with events / # in full analysis set); PFS6 probability that patients are progression free and alive for at least 6 months, or for the full period of follow up available at the time of assessment where 6 months had not yet passed, following first dose; The calculation is based on the Kaplan-Meier technique. CI Confidence interval; Patients with at least one post-baseline scan are included in this table.

[0716] Pharmacodynamic Biomarker Data

[0717] Pharmacodynamic biomarker data were generated by longitudinal assessment of circulating tumour DNA (ctDNA) at C2D1 (week 3), C3D1 (week 6) and C4D1 (week 9). Mean variant allele frequency (mVAF) was measured using the Guardant Health G360 assay, with buffy coat to correct for clonal haematopoiesis of indeterminate potential (CHIP). In parallel, cancer antigen 125 (CA-125) was assessed at the same timepoints per standard local protocols. A dose-dependent reduction in ctDNA mVAF and CA-125 was observed across all timepoints assessed (see Figure 8). Additionally, a trend to greater reduction from baseline at doses > 2.0 mg / kg was observed in both ctDNA mVAF and CA-125 at C2D1 (week 3), C3D1 (week 6) and C4D1 (week 9), with results consistent between study M1B1 (data not shown) and the combined M1A+M1B1 datasets.

[0718] Conclusion

[0719] The ORR data demonstrated AZD5335 efficacy across the three tested dose ranges without a clear discrimination between doses; however, dose levels > 2.0 mg / kg were associated with deeper tumour shrinkage and more durable responses.

[0720] The PFS data suggested incremental efficacy benefit with higher dose.

[0721] The pharmacodynamic biomarker data showed a dose-dependent reduction in ctDNA mVAF and CA-125 across all timepoints assessed, with greater reduction from baseline at doses > 2.0 mg / kg in both ctDNA and CA-125 at early timepoints (3, 6 and 9 weeks).

[0722] (B) - Clinical Safety

[0723] Treatment emerging adverse events (AEs) were consistent with known mechanisms of action (haematological, Gl-related toxicities). Overall, the available safety data across studies MIA and M1B1 suggests a dose-dependent increase in the frequency of Grade >3 AEs, serious adverse events (SAEs) and AEs requiring dose delays, reductions and discontinuations, with a substantial increase observed at 2.4 mg / kg dose (see Table 18). SAEs were reported approximately twice as frequently (43.3% vs 23.2%), and Grade 3 / 4 events were markedly higher (75.0% vs. 51.8%) in the 2.4 mg / kg cohort in comparison to the 2.0 mg / kg cohort. One SAE with an outcome of death was reported in 1 participant (1.6 mg / kg, cause of death small intestinal obstruction), which was assessed as not related to AZD5335.

[0724] In the 2.4 mg / kg cohort, the frequencies of dose discontinuations were approximately three times higher than those observed for the 2.0 or 1.6 mg / kg cohorts (11.7%, 3.6%, and 4.0% respectively), and dose reductions were markedly higher at 2.0 or 1.6 mg / kg (33.3%, 19.6% and 8.0% respectively). For the 2.4 mg / kg cohort, these discontinuations typically occurred early, within the first 2 to 3 cycles of treatment and this may impact the benefit. Table 18: Safety summary

[0725] [a] Possibly related is defined as reasonable possibility that the AE was caused by investigational product, [b] Grade 3 : severe, Grade 4: life-threatening, Grade 5 : fatal. The table includes adverse events with an onset date or that worsen on or after the date of first dose and up to and including 30 days (90 days for pneumonitis) following the date of last dose of treatment. Subjects with multiple occurrences in the same category are counted once per category regardless of the number of occurrences, n: Number of participants per category; N Number of participants per treatment group. AE: adverse event; SAE: serious adverse event

[0726] Conclusion

[0727] Safety data demonstrated a clear dose dependence across the range of dose levels tested, with incremental increases in AE incidence and severity with higher doses. (C) - Clinical Pharmacology

[0728] Pharmacokinetics (PK) and population PK (PopPK) analysis

[0729] The pharmacokinetic properties of AZD5335 were evaluated in participants with PROC from the studies “MIA” and “M1B1”, following IV infusion of AZD5335 administered Q3W as monotherapy. In addition to patients as described above who were administered 1.6 mg / kg, 2.0 mg / kg, or 2.4 mg / kg, pharmacokinetic analysis was also performed on patients administered 0.8 mg / kg or 3.0 mg / kg AZD5335 Q3W as monotherapy. Non-compartmental analyses were performed using plasma concentration data of ADC (AZD5335), total antibody, and total unconjugated payload.

[0730] Table 19 presents a summary of the PK parameters for ADC, total antibody, and unconjugated payload. Following administration of the first dose, AZD5335, total antibody, and unconjugated payload exhibited linear PK, with exposure increasing proportionately for doses ranging from 0.8 mg / kg and 3.0 mg / kg. The terminal half-life for AZD5335 was between 5.5 and 7.5 days, while the half-life for the payload was between 5.3 and 6.4 days. The AUC ratio between ADC and total antibody was approximately 80% and elimination tl / 2 were relatively similar, indicating that conjugated AZD5335 was stable in plasma. No significant accumulation of ADC and unconjugated payload at steady state was observed within the tested dose range. Maximal concentrations of the total unconjugated payload were approximately 100- fold lower than ADC in molar concentration. The PK exposures were comparable regardless of FRa expression status (see Figure 9).

[0731] Table 19: Summary of non-compartmental PK parameters of ADC (AZD5335), total antibody, and payload by cohort and dose following the first dose

[0732] PK parameters are estimated and presented as mean (SD).a’b’c’d’e’fdenotes 1, 3, 4, 7, 8, or 13 of total participants whose PK parameters could not be estimated.

[0733] An interim population PK (PopPK) analysis was performed which included a total of 153 participants across 5 different dose levels: (9 participants at 0.8 mg / kg, 41 participants at 1.6 mg / kg, 47 participants at 2.0 mg / kg, 50 participants at 2.4 mg / kg, and 6 participants at 3.0 mg / kg). The PopPK model effectively described the observed PK data of the ADC and total unconjugated payload and suggested a linear proportional PK profile across the range of dose levels.

[0734] Pharmacokinetic-Pharmacodynamic (PK / PD) analysis

[0735] The effect of AZD5335 on the magnitude of tumour reduction from baseline and longitudinal tumour growth inhibition was evaluated. The analysis was based on PK exposure quantiles to assess which dose level would provide the optimal PK exposure range for optimal anti-tumour activities. It was determined that ADC AUC (Cycle 1) was the most relevant PK metric to evaluate changes in tumour dynamics. There were 139 participants with available PK exposure data and tumour growth inhibition data (Interim Response Evaluable Set), which were divided into four equal PK quantiles (QI, Q2, Q3, Q4, with 35, 35, 34 and 35 participants in each quantile, respectively) (see Table 20). There were participants that fell in all four PK exposure quantiles for 1.6 mg / kg, 2.0 mg / kg, and 2.4 mg / kg. However, in QI, there were 1.6- fold more participants for 1.6 mg / kg as compared to that of 2.0 mg / kg (37.8% vs 23.3%, respectively). In contrast, there were higher percentages of participants in Q2+Q3+Q4 for the > 2.0 mg / kg dose group than those in < 1.6 mg / kg. Table 20: The percentage of participants in each PK (ADC AUC1) exposure quantile from all tested dose levels

[0736] Figure 10 shows the effect of AZD5335 on the best overall change of tumour reduction from baseline. The data show that a higher tumour reduction was observed with higher PK exposure, particularly in Q2, Q3, and Q4 and regardless of participants with FRa high or low expression.

[0737] Figure 11 shows the effect of AZD5335 on the longitudinal tumour growth inhibition as a function of time (cycles). The available PK / PD data allowed for assessment of tumour dynamic changes by AZD5335 up to 8 cycles. The data show that longer extent of tumour suppression was observed with higher PK exposure, particularly in Q2, Q3, and Q4 and regardless of participants with FRa high or low expression.

[0738] Exposure-response analysis for both efficacy and safety (ER-ES)

[0739] Based on the PopPK predicted exposure metrics, exposure-response analyses for objective response rate (ORR) were performed. The exposure-response analyses included all participants treated at 5 different dose levels and with evaluable PK samples following the first Cycle of treatment (9 participants at 0.8 mg / kg, 37 participants at 1.6 mg / kg, 39 participants at 2.0 mg / kg, 40 participants at 2.4 mg / kg and 5 participants at 3.0 mg / kg).

[0740] The average ADC concentration until time of event (Cav until the time of event), used as a PK exposure metric, was identified to be the most significant in correlation to clinical response. This PK metric estimates the average ADC concentration in an individual participant, including any dose modifications that happened prior to the time of event [defined as (a) the time to the first clinical response scan if the response occurred, or (b) the time until the last recorded dose or the last tumour scan assessment, whichever was earlier, if the response did not occur]. The model indicated that higher ADC exposure was associated with higher probability of response. At dose of 2.0 mg / kg, the mean predicted probability of ORR at 50th [5th, 95th exposure percentile] was 0.54 [0.39, 0.69], At 1.6 mg / kg, the predicted probability was 0.47 [0.38, 0.59], and for 2.4 mg / kg, the mean probability was 0.57 [0.44, 0.70], The data showed that there was a larger difference in mean probability between 1.6 and 2.0 mg / kg than that between 2.0 mg / kg and 2.4 mg / kg.

[0741] Exposure-safety analyses were further performed, which included all participants in the safety analysis set treated at 5 different dose levels and with evaluable PK samples following the first Cycle of treatment (9 participants at 0.8 mg / kg, 41 participants at 1.6 mg / kg, 47 participants at 2.0 mg / kg, 50 participants at 2.4 mg / kg and 6 participants at 3.0 mg / kg).

[0742] Safety endpoints included:

[0743] • Any Grade 3+ AE

[0744] • Any SAE

[0745] • Any AE leading to treatment discontinuation

[0746] • Any AE leading to dose reduction

[0747] • Any AE leading to dose delay

[0748] • Any AE leading to dose reduction, interruption or discontinuation

[0749] • Any grade 3+ haematological toxicity

[0750] • Any grade 1+ haematological toxicity

[0751] • Neutropenia (Grade 3+)

[0752] • Anaemia (Grade 3+)

[0753] • Thrombocytopenia (Grade 3+)

[0754] • Nausea or vomiting (Grade 3+)

[0755] • Diarrhea (Grade 3+)

[0756] • Any eye disorder (Gr 1+)

[0757] • Pneumonitis (Grl+)

[0758] Following the exposure-safety analyses, the best fit relationship across all safety endpoints was selected and the differences in risk between dose levels were compared, to evaluate any potential increase in risk associated with an increased dose. All safety endpoints analysed were based on a statistically significant model fit (p < 0.05). The most common exposure metric was payload AUC1. Using the exposure-safety relationships for all the safety endpoints based on payload AUC1, the mean, as well as the 5th and 9th percentiles were estimated, for the predicted safety risk at each dose level. Safety endpoints with significant exposure-response relationship are highlighted in Table 21. Table 21: Differentiation between dose levels and predicted efficacy and key safety events based on ER-ES analyses

[0759] Pr(p50): Predicted probability of AE at the 50th percentile of related exposure; Pr(p5): Predicted probability of AE at the 5th percentile of related exposure; Pr(p95): Predicted probability of AE at the 95th percentile of related exposure; PL: payload.

[0760] This analysis indicated that with an increase in PK exposure of the payload, there was an increase in the probability of an AE. The probability of any haematologic Gr3+ AEs is the most significant relationship with increasing payload exposure, which may lead to dose modifications. The differences in median probability of dose reduction between 1.6 mg / kg and 2.0 mg / kg was relatively minor (0.04 probability difference). The median probability of predicted ORR for 0.8 mg / kg was 0.35 [0.31, 0.45], which was lower than the predicted median ORR for 1.6 and 2.0 mg / kg (0.47 and 0.54, respectively). There was no participant treated with 2.0 mg / kg that had two dose level reduction to 0.8 mg / kg. In addition, the probability of dose discontinuation was low and comparable between 1.6 mg / kg and 2.0 mg / kg (only 0.01 probability difference).

[0761] Conclusion

[0762] Clinical data showed higher tumour reduction from baseline and longer extent of tumour growth inhibition with higher clinical PK exposure (e.g. > 2.0 mg / kg). The PK exposure range of 2.0 mg / kg (Q2+Q3+Q4) had a higher proportion of patient with optimal anti-tumour activities than that of 1.6 mg / kg.

[0763] A positive exposure-ORR relationship indicated a higher PK exposure is warranted for improving probability of ORR. While positive exposure-safety relationships were noted, the difference in probability of dose reduction and discontinuation due to an AE is minimal and comparable between 1.6 mg / kg and 2.0 mg / kg. However, a higher probability of dose modifications due to an AE was estimated to occur for 2.4 mg / kg compared to doses < 2.0 mg / kg.

Claims

CLAIMS1. A method of treating cancer in a human subject in need thereof, comprising administering to the human subject an antibody-drug conjugate (ADC) in an amount from about 0.8 mg / kg to about 5.0 mg / kg, wherein the ADC comprises an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor.

2. The method according to claim 1, wherein the amount of ADC administered is about 0.8 mg / kg, about 1.2 mg / kg, about 1.6 mg / kg, about 2.0 mg / kg, about 2.4 mg / kg, about2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg, about4.8 mg / kg or about 5.0 mg / kg.

3. The method according to claim 1, wherein the amount of ADC administered is about 0.8 mg / kg to about 4.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg or about 1.6 mg / kg to about 2.4 mg / kg.

4. The method according to any one of claims 1-3, wherein the amount of ADC administered is about 1.6 mg / kg.

5. The method according to any one of claims 1-3, wherein the amount of ADC administered is about 2.0 mg / kg.

6. The method according to any one of claims 1-3, wherein the amount of ADC administered is about 2.4 mg / kg.

7. The method according to any one of claims 1-6, wherein the ADC is administered to the subject once every week, once every two weeks, once every three weeks or once every four weeks.

8. The method according to claim 7, wherein the ADC is administered to the subject once every three weeks.

9. The method according to claim 1, wherein about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

10. The method according to any one of claims 1-9, wherein the ADC is administered intravenously.

11. The method according to any one of claims 1-10, wherein at least about 75% of the cells in said cancer are FRa-positive cells.

12. The method according to any one of claims 1-10, wherein about 25% to about 75% of the cells in said cancer are FRa-positive cells.

13. The method according to any one of claims 1-10, wherein about 1% to about 25% of the cells in said cancer are FRa-positive cells.

14. The method according to any one of claims 1-13, further comprising a step of determining the percentage of FRa-positive cells in a sample obtained from the cancer before administering the ADC to the human subject.

15. The method according to any one of claims 1-10, further comprising a step of determining the percentage of FRa-positive cells in a sample obtained from the cancer before administering the ADC to the human subject, and selecting the human subject as suitable for said treatment with the ADC based on:(a) at least about 75% of the cells in said sample are FRa-positive cells;(b) at least about 25% of the cells in said sample are FRa-positive cells; or(c) at least about 1% of the cells in said sample are FRa-positive cells.

16. A method of producing a unit dose of an ADC for the treatment of cancer in a human subject, wherein the ADC comprises an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor, the method comprising:(a) determining the percentage of FRa-positive cells in a sample from the cancer; and(b) producing a unit dose of the ADC by formulating the ADC in an amount determined based on the percentage of FRa-positive cells determined in the subject.

17. The method according to claim 16, wherein the amount of the ADC in said unit dose is:(a) about 0.8 mg / kg, about 1.2 mg / kg, about 1.6 mg / kg, about 2.0 mg / kg, about 2.4 mg / kg, about 2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg, about 4.8 mg / kg or about 5.0 mg / kg, based on the body weight of the human subject;(b) about 0.8 mg / kg to about 4.8 mg / kg, about 0.8 mg / kg to about 3.6 mg / kg, about 0.8 mg / kg to about 3.0 mg / kg, about 1.0 mg / kg to about 2.8 mg / kg, about 1.2 mg / kg to about 2.6 mg / kg, about 1.4 mg / kg to about 2.4 mg / kg or about 1.6 mg / kg to about 2.4 mg / kg, based on the body weight of the human subject;(c) about 1.6 mg / kg, based on the body weight of the human subject;(d) about 2.0 mg / kg, based on the body weight of the human subject; or(e) about 2.4 mg / kg, based on the body weight of the human subject.

18. The method according to any one of claims 11-17, wherein the percentage of FRa- positive cells is assayed using immunohistochemistry (IHC) or the Quantitative Continuous Scoring (QCS) assay.

19. The method according to claim 18, wherein the IHC is performed with an antibody reagent.

20. The method according to any one of claims 1-19, wherein the cancer is a solid tumour and / or an epithelial tumour.

21. The method according to any one of claims 1-20, wherein the cancer is ovarian cancer, lung cancer (e.g. lung adenocarcinoma), endometrial cancer, pancreatic cancer, gastric cancer, renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinomas (HNSCC), breast cancer (e.g. TNBC), cervical cancer, malignant pleural mesothelioma, peritoneal cancer or fallopian tube cancer.

22. The method according to any one of claims 1-21, wherein the cancer is ovarian cancer, optionally wherein the ovarian cancer is platinum-resistant ovarian cancer or platinumsensitive ovarian cancer.

23. The method according to any one of claims 1-21, wherein the cancer is lung cancer.

24. The method according to claim 23, wherein the lung cancer is a non-small-cell lung cancer (NSCLC), optionally wherein the NSCLC is selected from squamous NSCLC, adenocarcinoma NSCLC, or a combination thereof.

25. The method according to any one of claims 1-24, wherein the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT).

26. The method according to any one of claims 1-25, wherein the anti-FRa antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8.

27. The method according to claim 26, wherein the anti-FRa antibody or antigen-binding fragment thereof comprises:(a) L at the N-terminus (e.g. position 1) of the VH;(b) E at the N-terminus (e.g. position 1) of the VH; or(c) Q at the N-terminus (e.g. position 1) of the VH.

28. The method according to any one of claims 1-27, wherein the anti-FRa antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8.

29. The method according to any one of claims 1-28, wherein the anti-FRa antibody comprises a constant heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19 or 21 and a constant light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

30. The method according to any one of claims 1-29, wherein the anti-FRa antibody comprises a constant heavy chain amino acid sequence of SEQ ID NO: 19 or 21 and a constant light chain amino acid sequence of SEQ ID NO: 20.

31. The method according to any one of claims 1-28, wherein the antigen-binding fragment comprises a constant heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19 and a constant light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

32. The method according to any one of claims 1-28 or 31, wherein the antigen-binding fragment comprises a constant heavy chain amino acid sequence of SEQ ID NO: 19 and a constant light chain amino acid sequence of SEQ ID NO: 20.

33. The method according to any one of claims 1-30, wherein the anti-FRa antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9 and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10.

34. The method according to any one of claims 1-30 or 33, wherein the anti-FRa antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10.

35. The method according to any one of claims 1-28, 31 or 32, wherein the antigen-binding fragment is a Fab fragment, a Fab’ fragment, or a F(ab’)2 fragment.

36. The method according to any one of claims 1-35, wherein the anti-FRa antibody or antigen-binding fragment thereof is fully human.

37. The method according to any one of claims 1-36, wherein the anti-FRa antibody or antigen-binding fragment thereof is monoclonal, polyclonal, recombinant, or multispecific.

38. The method according to any one of claims 1-37, wherein the anti-FRa antibody or antigen-binding fragment thereof is of the IgGl, IgG2, IgG3 or IgG4 type.

39. The method according to claim 38, wherein the anti-FRa antibody or antigen-binding fragment thereof is of the IgGl type.

40. The method according to any one of claims 1-39, wherein the topoisomerase I inhibitor is represented by formula A* :and salts and solvates thereof.

41. The method according to any one of claims 1-40, wherein the topoisomerase I inhibitor is linked to the anti-FRa antibody or antigen-binding fragment thereof via a linker, wherein the linker + topoisomerase I inhibitor is:

42. The method according to claim 41, wherein the linker + topoisomerase I inhibitor is:

43. The method according to any one of claims 1-42, wherein the drug to antibody ratio (DAR) of the ADC is in the range of about 1 to 20, optionally wherein the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10.

44. The method according to claim 43, wherein the DAR is about 8 or about 4.

45. The method according to claim 44, wherein the DAR is about 8.

46. The method according to any one of claims 1-45, wherein:(i) the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8, optionally wherein the anti-FRa antibody has a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10;(ii) the anti-FRa antibody or antigen-binding fragment thereof is conjugated to SG3932(iii) the DAR of the ADC is about 8.

47. A method of treating cancer in a human subject in need thereof, comprising administering to the human subject an antibody-drug conjugate (ADC) in an amount from about 0.8 mg / kg to about 5.0 mg / kg, wherein the ADC comprises an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin, wherein the cytotoxin is a topoisomerase I inhibitor, wherein:(i) the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigenbinding fragment thereof has a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8, optionally wherein the anti-FRa antibody has a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10;(ii) the anti-FRa antibody or antigen-binding fragment thereof is conjugated toSG3932(iii) the DAR of the ADC is about 8.

48. The method according to claim 47, wherein the amount of ADC administered is about 1.6 mg / kg.

49. The method according to claim 47, wherein the amount of ADC administered is about 2.0 mg / kg.

50. The method according to claim 47, wherein the amount of ADC administered is about 2.4 mg / kg.

51. The method according to claim 47, wherein about 2.0 mg / kg of the ADC is administered to the subject once every three weeks.

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