WO2025257350A1

WO2025257350A1 - Pai-1 antigen-binding molecules

Info

Publication number: WO2025257350A1
Application number: PCT/EP2025/066468
Authority: WO
Inventors: Chin-Ann Johnny ONG; Ying Liu; Josephine HENDRIKSON; Qiu Xuan Suzanne TAN; Wen Xuan Gillian NG; Wee Shan Joey TAN; Yieh Lin Clara CHONG; Wanyu GUO
Original assignee: Singapore Health Services Pte Ltd
Current assignee: Singapore Health Services Pte Ltd
Priority date: 2024-06-13
Filing date: 2025-06-12
Publication date: 2025-12-18
Anticipated expiration: 2026-12-13

Abstract

The present disclosure provides antigen-binding molecules capable of binding to PAI-1. Also provided are nucleic acids encoding such antigen-binding molecules, vectors comprising such nucleic acids, cells comprising such antigen-binding molecules, and compositions comprising such antigen-binding molecules, nucleic acids, vectors, and/or cells. Further provided are methods for treating/preventing diseases/conditions using such antigen-binding molecules, nucleic acids, vectors, cells and/or compositions.

Description

PAI-1 Antigen-Binding Molecules

This application claims priority from SG 10202401700P filed 13 June 2024, the contents and elements of which are herein incorporated by reference for all purposes.

Technical Field

The present disclosure relates to the fields of molecular biology, more specifically antibody technology. The present disclosure also relates to methods of medical treatment and prophylaxis.

Background

Plasminogen Activator Inhibitor 1 (PAI-1 ; or serpin E1 ) belongs to serine protease inhibitor (serpin) superfamily. PAI-1 plays a critical role in the regulation of fibrinolysis and tissue remodelling. Elevated PAI-1 has been reported to be implicated in various pathologies including cancer,¹ obesity, inflammation, metabolic syndrome,² and senescence.³

PAI-1 is the principal inhibitor of both the tissue-type (tPA) and the urokinase-type (uPA) plasminogen activators, enzymes responsible to activate plasminogen by cleaving a specific Arg-Val peptide bond located within the protease domain.² Different from tPA which is mainly involved in intravascular fibrinolysis, uPA exerts proteolytic effects as well as intracellular signalling functions by binding to its high- affinity receptor on the cell surface.⁴

Peritoneal metastases (PM) refers to the shedding, dissemination, and implantation of tumour deposits to the peritoneal serosa or intra-abdominal organs.⁶ Malignancies from any intra-abdominal organ (e.g. stomach, colon, pancreas, ovary) can develop PM, though the incidence varies in different histological subtypes. Up to 70% of patients with locally advanced colorectal cancer develop PM whereas 40-50% of gastric and ovarian cancer patients present with PM during the course of the disease.⁷⁸ The estimated incidence per year of the three most common PM subtypes (colorectal, gastric and ovarian) is more than 2 million in the world, exemplifying the magnitude of disease burden posed by PM.⁷'^9-11

Regardless of the histological origin, the development of PM portends a dismal prognosis for patients.¹² Left untreated, the median survival ranges from 6 to 12 months.¹²'¹³ In highly selected PM patients (approximately 5% of all PM patients), cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) can potentially provide a cure. CRS refers to a series of visceral resections and peritonectomy procedures to remove all macroscopic tumours. This procedure is highly morbid with each operation taking 8-12 hours. Remaining viable microscopic lesions are eradicated with the instillation of HIPEC at the time of surgery.¹⁴ However, current HIPEC regimens rely on cytotoxic effect of conventional chemotherapeutic agents without exploiting the molecular vulnerabilities of PM for therapy. A larger majority of PM patients is not suitable for CRS and HIPEC, and receive systemic chemotherapy or intraperitoneal instillation of chemotherapy as a palliation. Prognosis of these patients remains uniformly poor.^15-17 There are currently no targeted therapies for the treatment of PM, particularly, in the form of direct intraperitoneal instillation. Hence, there is a pressing unmet need to develop such therapeutics in order to improve patient outcome. Summary

In a first aspect, the present disclosure provides an antigen-binding molecule, optionally isolated, that binds to PAI-1 .

In some embodiments, the antigen-binding molecule comprises:

(a)

(I) a variable heavy domain of heavy chain (VHH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:195 HC-CDR2 having the amino acid sequence of SEQ ID NO:196 HC-CDR3 having the amino acid sequence of SEQ ID NO:197; or

(b)

(I) a variable heavy domain of heavy chain (VHH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:188 HC-CDR2 having the amino acid sequence of SEQ ID NO:189 HC-CDR3 having the amino acid sequence of SEQ ID NQ:190; or

(c)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:31 HC-CDR2 having the amino acid sequence of SEQ ID NO:32 HC-CDR3 having the amino acid sequence of SEQ ID NO:33; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:38 LC-CDR2 having the amino acid sequence of SEQ ID NO:39 LC-CDR3 having the amino acid sequence of SEQ ID NQ:40; or

(d)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:45 HC-CDR2 having the amino acid sequence of SEQ ID NO:46 HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:51 LC-CDR2 having the amino acid sequence of SEQ ID NO:52 LC-CDR3 having the amino acid sequence of SEQ ID NO:53; or

(e)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:57 HC-CDR2 having the amino acid sequence of SEQ ID NO:58 HC-CDR3 having the amino acid sequence of SEQ ID NO:59; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:63 LC-CDR2 having the amino acid sequence of SEQ ID NO:64 LC-CDR3 having the amino acid sequence of SEQ ID NO:65; or

(f)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:31 HC-CDR2 having the amino acid sequence of SEQ ID NO:32 HC-CDR3 having the amino acid sequence of SEQ ID NQ:70; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:72 LC-CDR2 having the amino acid sequence of SEQ ID NO:73 LC-CDR3 having the amino acid sequence of SEQ ID NO:74; or

(g)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:79 HC-CDR2 having the amino acid sequence of SEQ ID NQ:80 HC-CDR3 having the amino acid sequence of SEQ ID NO:81 ; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:84 LC-CDR2 having the amino acid sequence of SEQ ID NO:85 LC-CDR3 having the amino acid sequence of SEQ ID NO:86; or

(h)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:31 HC-CDR2 having the amino acid sequence of SEQ ID NO:32 HC-CDR3 having the amino acid sequence of SEQ ID NO:91 ; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:93 LC-CDR2 having the amino acid sequence of SEQ ID NO:94 LC-CDR3 having the amino acid sequence of SEQ ID NO:95; or

(i)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:99 HC-CDR2 having the amino acid sequence of SEQ ID NQ:100 HC-CDR3 having the amino acid sequence of SEQ ID NQ:101 ; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NQ:105 LC-CDR2 having the amino acid sequence of SEQ ID NQ:106 LC-CDR3 having the amino acid sequence of SEQ ID NQ:107; or

0)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:110 HC-CDR2 having the amino acid sequence of SEQ ID NO:111 HC-CDR3 having the amino acid sequence of SEQ ID NO:112; and (ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:116 LC-CDR2 having the amino acid sequence of SEQ ID NO:117 LC-CDR3 having the amino acid sequence of SEQ ID NO:118; or

(k)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:122 HC-CDR2 having the amino acid sequence of SEQ ID NO:123 HC-CDR3 having the amino acid sequence of SEQ ID NO:124; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NQ:105 LC-CDR2 having the amino acid sequence of SEQ ID NO:129 LC-CDR3 having the amino acid sequence of SEQ ID NQ:130; or

(l)

(I) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:79 HC-CDR2 having the amino acid sequence of SEQ ID NQ:80 HC-CDR3 having the amino acid sequence of SEQ ID NO:134; and (ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:135 LC-CDR2 having the amino acid sequence of SEQ ID NO:39 LC-CDR3 having the amino acid sequence of SEQ ID NO:136; or

(m)

(I) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:31 HC-CDR2 having the amino acid sequence of SEQ ID NO:32 HC-CDR3 having the amino acid sequence of SEQ ID NO:139; and (ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:141 LC-CDR2 having the amino acid sequence of SEQ ID NQ:106 LC-CDR3 having the amino acid sequence of SEQ ID NO:142; or

(n)

(I) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:147 HC-CDR2 having the amino acid sequence of SEQ ID NO:148 HC-CDR3 having the amino acid sequence of SEQ ID NO:149; and (ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:152 LC-CDR2 having the amino acid sequence of SEQ ID NO:153

LC-CDR3 having the amino acid sequence of SEQ ID NO:154; or

(o)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:158 HC-CDR2 having the amino acid sequence of SEQ ID NO:159 HC-CDR3 having the amino acid sequence of SEQ ID NQ:160; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:165 LC-CDR2 having the amino acid sequence of SEQ ID NO:166 LC-CDR3 having the amino acid sequence of SEQ ID NO:167; or

(P)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:172 HC-CDR2 having the amino acid sequence of SEQ ID NO:173 HC-CDR3 having the amino acid sequence of SEQ ID NO:174; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NQ:105 LC-CDR2 having the amino acid sequence of SEQ ID NO:178 LC-CDR3 having the amino acid sequence of SEQ ID NO:179; or

(q)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:31 HC-CDR2 having the amino acid sequence of SEQ ID NO:32 HC-CDR3 having the amino acid sequence of SEQ ID NO:181 ; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:182 LC-CDR2 having the amino acid sequence of SEQ ID NO:129 LC-CDR3 having the amino acid sequence of SEQ ID NO:183.

In some embodiments, the antigen-binding molecule comprises:

(a) a VHH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:187 or 186; or

(b) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15; and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30. In some embodiments, the antigen-binding molecule binds to active PAI-1 and binds to latent PAI-1.

In some embodiments, the antigen-binding molecule binds to, or in proximity to, the s4A groove of PAI-1 .

In some embodiments, the antigen-binding molecule exhibits competitive binding to PAI-1 with an agent known to bind to the s4A groove of PAI-1 , optionally wherein the agent is TM5441 and/or Tiplasinin.

In some embodiments, the antigen-binding molecule binds to PAI-1 via contact with one or more amino acid residues of the region shown in SEQ ID NO:265.

In some embodiments, the antigen-binding molecule is a multispecific antigen-binding molecule, wherein the antigen-binding molecule further comprises an antigen-binding domain which binds to an antigen other than PAI-1 .

In some embodiments, the antigen-binding molecule is conjugated to a drug moiety or a detectable moiety.

The present disclosure also provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to the present disclosure.

The present disclosure also provides a nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule or CAR according to the present disclosure.

The present disclosure also provides an expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to the present disclosure.

The present disclosure also provides a cell comprising an antigen-binding, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors according to the present disclosure.

The present disclosure also provides a method comprising culturing a cell according to the present disclosure under conditions suitable for expression of an antigen-binding molecule or CAR by the cell.

The present disclosure also provides a composition comprising an antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, or cell according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

The present disclosure also provides an antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell, or composition according to the present disclosure, for use in a method of medical treatment or prophylaxis. The present disclosure also provides an antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell, or composition according to the present disclosure, for use in a disease or condition in which PAI-1 is pathologically-implicated.

The present disclosure also provides for use of an antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell, or composition according to the present disclosure, in the manufacture of a medicament for treating or preventing a disease or condition in which PAI-1 is pathologically-implicated.

The present disclosure also provides a method of treating or preventing a disease or condition in which PAI-1 is pathologically-implicated, comprising administering to a subject a therapeutically- or prophylactically-effective amount of an antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell, or composition according to the present disclosure.

In some embodiments, the disease or condition is cancer.

In some embodiments, the cancer is associated with ascites characterised by the presence of PAI-1 .

In some embodiments, the cancer is selected from: ovarian cancer, endometrial cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, pancreatic cancer, hepatobiliary cancer and peritoneal metastasis.

In some embodiments, the peritoneal metastasis is selected from: colorectal peritoneal metastasis, small bowel peritoneal metastasis, mesothelioma, endometrial peritoneal metastasis, gastric peritoneal metastasis, ovarian peritoneal metastasis, appendiceal peritoneal metastasis, pancreatic peritoneal metastasis, urothelial peritoneal metastasis, Pseudomyxoma peritonei (PMP), breast peritoneal metastasis, esophageal peritoneal metastasis, lung peritoneal metastasis, hepatobilliary peritoneal metastasis, peritoneal metastasis of unknown origin, and primary peritoneal carcinoma.

In some embodiments, the cancer is characterised by:

(i) high STAT3 activation;

(ii) high PAI-1 ;

(iii) high STAT3 activation and high PAI-1 ; or

(iv) high STAT3 activation and low PAI-1 .

In some embodiments, the disease or condition is characterised by coagulation.

In some embodiments, the disease or condition is selected from: thrombosis, e.g. deep vein thrombosis (DVT), portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, cerebral venous sinus thrombosis, thrombotic stroke; myocardial infarction; antiphospholipid syndrome (APS); disseminated intravascular coagulation (DIC); activated protein C resistance, e.g. Factor V Leiden; and cancer

The present disclosure also provides, an in vitro complex, optionally isolated, comprising an antigenbinding molecule according to the present disclosure bound to PAI-1 .

The present disclosure also provides a method for detecting PAI-1 in a sample, comprising contacting a sample containing, or suspected to contain, PAI-1 with an antigen-binding molecule according to the present disclosure, and detecting the formation of a complex of the antigen-binding molecule with PAI-1 .

The present disclosure also provides a method of selecting or stratifying a subject for treatment with a PAI-1 -targeted agent, the method comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to the present disclosure, and detecting the formation of a complex of the antigen-binding molecule with PAI-1 .

The present disclosure also provides for use of an antigen-binding molecule according to the present disclosure as an in vitro or in vivo diagnostic or prognostic agent.

Description

PAI-1 is secreted in large amounts into the ascitic fluid of patients with peritoneal metastases (PM), a common end point of many epithelial cancers including colorectal, ovarian, gastric, and appendiceal tumours. Paracrine inhibition of this target is highly efficacious in reducing tumour burden in vitro and in in vivo mouse models of PM. The present invention relates to novel antigen-binding molecules that are able to perturb the functions of cancer cells via targeting and neutralising PAI-1 . Surprisingly, this effect cannot be achieved with existing commercial anti-PAI-1 antibodies. The novel antigen-binding molecules of the invention are useful in the treatment of diseases that are dependent on PAI-1 for their pathogenesis, including the treatment of peritoneal metastases in the presence of ascites.

PAI-1

The present disclosure relates to PAI-1 -specific antigen-binding molecules.

Human plasminogen activator inhibitor 1 (PAI-1 ) (also known as Serpin E1 , endothelial plasminogen activator inhibitor) is the protein identified by UniProt P05121 . The structure and function of PAI-1 is described e.g. in Sillen and Declerck, Front Cardiovasc Med. (2020) 7:622473 and Aertgeerts et al., Nat Struct Biol. (1995) Oct;2(10):891-7, which are hereby incorporated by reference in their entirety. PAI-1 is encoded by the SERPINE1 gene in humans.

The canonical isoform of human PAI-1 (isoform 1 ) has the amino acid sequence shown in SEQ ID NO:250. Alternative splicing of mRNA encoded by the human SERPINE1 gene yields two main PAI-1 isoforms: isoform 1 (SEQ ID NO:250), and isoform 2 (SEQ ID NO:251 ). Isoform 2 differs from isoform 1 in that positions 33 to 27 of SEQ ID NQ:250 are absent. The canonical isoform of human PAI-1 comprises an N-terminal signal peptide (SEQ ID NO:252). The mature form of human PAI-1 isoform 1 is shown in SEQ ID NO:253.

PAI-1 comprises three p-sheets (referred to as A-C) and nine a-helices (referred to as hA-hl). The - strands which form the p-sheets are referred to as s(#)A, s(#)B and s(#)C. For example, ‘s3A’ refers to the third p-strand in p-sheet A. Plasminogen activator recognises PAI-1 as a (pseudo) substrate. PAI-1 comprises a flexible surface-exposed reactive center loop (RCL) of 26 amino acids designated P16-P10’ (SEQ ID NO:254) that presents a substrate-mimicking peptide sequence (Arg346-Met347, designated P1-P1’).

PAI-1 has three interconvertible conformations: active, latent and substrate forms.

PAI-1 at active conformation possesses the exposed reactive centre loop (RCL) (Figure 2B). In the active conformation, PAI-1 p-sheet A comprises p-strands s1 A, s2A, s3A, s5A and s6A (SEQ ID NO: 255, 256, 257, 259 and 260, respectively). Insertion of a portion of the RCL is observed in both active-to-latent conversion and complex formation with plasminogen activator (see “PA” in Figure 1 ). RCL insertion forms a strand between strands 3 and 5 of the antiparallel sheet A, referred to as s4A (SEQ ID NO:258). The s4A structure is shown as a dark grey sheet in Figure 2.

In complex formation, interaction between PAI-1 and plasminogen activator gives rise to a noncovalent Michaelis complex formation in which the P1-P1 ' bond in the RCL docks into the active site of plasminogen activator. Then, cleavage of P1-P1 ' bond causes covalent linkage of P1 residue to the active site serine of plasminogen activator by an ester bond. After that, the N-terminal residues of the RCL becomes inserted into p-sheet A. As a result plasminogen activator is relocated to the opposite pole of PAI-1 . Plasminogen activator is inhibited as a consequence of distortion of its active site during the conformational change. If insertion of the RCL is prevented, release of PAI-1 from plasminogen activator is more likely and hence PAI-1 acts only as a substrate of the protease and not as an inhibitor.

The structure and mechanism of action of PAI-1 is reviewed in Sillen and Declerck, Front Cardiovasc Med. (2020) 7:622473 and Aertgeerts et al., Nat Struct Biol. (1995) Qct;2(10):891-7, which are hereby incorporated by reference in their entirety.

In this specification ‘PAI-1 ’ refers to PAI-1 from any species, and includes isoforms, fragments, variants or homologues from any species. In some embodiments PAI-1 is PAI-1 from a mammal ((e.g. a therian, placental, epitherian, preptotheria, archontan, primate (rhesus, cynomolgous, non-human primate or human)). In some embodiments, the PAI-1 is PAI-1 from a human or a mouse.

As used herein, isoforms, fragments, variants or homologues of a given reference protein (e.g. PAI-1 ) may be characterised as having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of the reference protein.

A ‘fragment’ generally refers to a fraction of the reference protein. A ‘variant’ generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein. An ‘isoform’ generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein. A ‘homologue’ generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein. Homologues include orthologues. Homologues of human PAI-1 include e.g. mouse PAI-1 (UniProt P22777).

Isoforms, fragments, variants or homologues of a given reference protein (e.g. PAI-1 ) may optionally be characterised as having at least 70%, preferably one of >80%, >85%, >90%, >91 %, >92%, >93%, £94%, £95%, £96%, £97%, £98%, £99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature (i.e. after processing to remove signal peptide) form of a specified isoform of the relevant protein from a given species, e.g. human.

In some embodiments, the PAI-1 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of £80%, £85%, £90%, £91 %, £92%, £93%, £94%, £95%, £96%, £97%, £98%, £99% or 100% amino acid sequence identity to SEQ ID NO: 250 or 251 .

In some embodiments, the PAI-1 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of £80%, £85%, £90%, £91 %, £92%, £93%, £94%, £95%, £96%, £97%, £98%, £99% or 100% amino acid sequence identity to SEQ ID NO: 253.

A ‘fragment’ of a reference protein may be of any length (by number of amino acids), although may optionally be at least 25% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

A fragment of PAI-1 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 300 or 400 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 300 or 400 amino acids.

In some embodiments, a fragment of PAI-1 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of £80%, £85%, £90%, £91 %, £92%, £93%, £94%, £95%, £96%, £97%, £98%, £99% or 100% amino acid sequence identity to SEQ ID NO:253.

In some embodiments, a fragment of PAI-1 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of £80%, £85%, £90%, £91 %, £92%, £93%, £94%, £95%, £96%, £97%, £98%, >99% or 100% amino acid sequence identity to SEQ ID NO:254, 255, 256, 257, 258, 259, 260, 264, 265 or 266.

PAI-1 ’s main function is the inhibition of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Urokinase-type plasminogen activator (uPA) is the protein identified by UniProt P00749. The canonical isoform of human uPA (isoform 1 ) has the amino acid sequence shown in SEQ ID NO:267. Tissue-type plasminogen activator (tPA) is the protein identified by UniProt P00750. The canonical isoform of human tPA (isoform 1 ) has the amino acid sequence shown in SEQ ID NO:268.

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are enzymes responsible for the cleavage of plasminogen to form plasmin. Plasmin mediates the degradation of the extracellular matrix (ECM), either by itself or in conjunction with matrix metalloproteinases. In this scenario, PAI-1 inhibits uPA via active site binding, preventing the formation of plasmin. Additional inhibition is mediated by PAI-1 binding to the uPA:uPA receptor (uPAR) complex, resulting in the latter’s degradation. PAI-1 can be said to inhibit the serine proteases tPA and uPA and hence is an inhibitor of fibrinolysis, the physiological process that degrades blood clots. In addition, PAI-1 inhibits the activity of the matrix metalloproteinases, which play a crucial role in invasion of malignant cells through the basal lamina. In humans, PAI-1 is mainly produced by the endothelium (cells lining blood vessels), but is also secreted by other tissue types such as adipose tissue and stromal tissue. In pathological conditions, such as cancer, PAI-1 may be expressed in fibroblasts and mesothelial cells.

Antigen-binding molecules

The present disclosure provides antigen-binding molecules capable of binding to PAI-1 . Such antigenbinding molecules may also be described as an antigen-binding molecules that bind to PAI-1 .

An ‘antigen-binding molecule’ refers to a molecule that binds to a given target antigen. Antigen-binding molecules include antibodies ( .e. immunoglobulins (Igs)) and antigen-binding fragments thereof. As used herein, ‘antibodies’ include monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antibody-derived antigen-binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc. Antigen-binding fragments of antibodies include e.g. Fv, Fab, F(ab’)2 and F(ab’) fragments. In some embodiments, an antigen-binding molecule may be an antibody or an antigen-binding fragment thereof.

Antigen-binding molecules according to the present disclosure also include antibody-derived molecules, e.g. molecules comprising an antigen-binding region/domain derived from an antibody. Antibody-derived antigen-binding molecules may comprise an antigen-binding region/domain that comprises, or consists of, the antigen-binding region of an antibody (e.g. an antigen-binding fragment of an antibody). In some embodiments, the antigen-binding region/domain of an antibody-derived antigen-binding molecule may be or comprise the Fv (e.g. provided as an scFv) or the Fab region of an antibody, or the whole antibody. For example, antigen-binding molecules according to the present disclosure include antibody-drug conjugates (ADCs) comprising a (cytotoxic) drug moiety (e.g. as described hereinbelow). Antigen-binding molecules according to the present disclosure also include multispecific antigen-binding molecules such as immune cell engager molecules comprising a domain for recruiting (effector) immune cells (reviewed e.g. in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17: 418-434 and Ellerman, Methods (2019) 154:102-117, both of which are hereby incorporated by reference in their entirety), including BiTEs, BiKEs and TriKEs. Antigen-binding molecules according to the present disclosure also include chimeric antigen receptors (CARs), which are recombinant receptors providing both antigen-binding and T cell activating functions (CAR structure, function and engineering is reviewed e.g. in Dotti et al., Immunol Rev (2014) 257(1 ) and Jayaraman et al., EBioMedicine (2020) 58:102931 , both of which are hereby incorporated by reference in their entirety).

The antigen-binding molecule of the present disclosure comprises a moiety or moieties capable of binding to a target antigen(s). In some embodiments, the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen. In some embodiments, the moiety capable of binding to a target antigen comprises a nanobody (j.e. a single-domain antibody (sdAb). In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an aptamer capable of binding to the target antigen, e.g. a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3): 181 -202). In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e. a single-domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011 ) 286:41273-85 and Emanuel et al., Mabs (2011 ) 3:38-48).

As used herein, a ‘peptide’ refers to a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A ‘polypeptide’ is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.

In some aspects of the present disclosure, the antigen-binding molecules generally comprise an antigenbinding domain comprising a VH and a VL of an antibody capable of specific binding to the target antigen. The antigen-binding domain formed by a VH and a VL may also be referred to herein as an Fv region.

An antigen-binding molecule may be, or may comprise, an antigen-binding polypeptide, or an antigenbinding polypeptide complex. An antigen-binding molecule may comprise more than one polypeptide which together form an antigen-binding domain. The polypeptides may associate covalently or non- covalently. In some embodiments, the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL). An antigen-binding molecule may refer to a non-covalent or covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, or 8 polypeptides), e.g. an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.

The antigen-binding molecules of the present disclosure may be designed and prepared using the sequences of monoclonal antibodies (mAbs). Antigen-binding regions of antibodies, such as single chain variable fragment (scFv), Fab and F(ab’)2 fragments may also be used/provided. An ‘antigen-binding region’ is any fragment of an antibody that binds to the target for which the given antibody is specific.

Antibodies generally comprise six complementarity-determining regions CDRs; three in the heavy chain variable (VH) region: HC-CDR1 , HC-CDR2 and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1 , LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody that binds to the target antigen.

The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH regions comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term.

In some aspects of the present disclosure, the antigen-binding molecules are single domain antibodies (sdAbs). Single domain antibodies (sdAbs) - also referred to variously in the art as ‘single variable domain on a heavy chain antibodies’, ‘VHHs’, ‘nanobodies’ and ‘heavy chain only antibodies (HcAbs)’ are described e.g. in Henry and MacKenzie, Front Immunol. (2018) 9:41 and Bever et al., Anal Bioanal Chem. (2016) 408(22): 5985-6002, both of which are hereby incorporated by reference in their entirety.

Single-domain antibodies are formed of a single, monomeric antibody variable domain. The first singledomain antibodies were engineered from heavy-chain antibodies found in camelids, and cartilaginous fishes also have heavy-chain antibodies.

Single-domain antibodies generally comprise three complementarity-determining regions CDRs: CDR1 , CDR2 and CDR3. The three CDRs together define the paratope of the molecule, which is the part through which it binds to its target antigen.

Single domain antibodies further comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, single-domain antibodies comprise the following structure: N term-[FR1]-[CDR1]-[FR2]-[CDR2]-[FR3]-[CDR3]-[FR4]-C term.

There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991 ), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1 ): D671-D674. The CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In preferred embodiments, the CDRs and FRs of antigenbinding molecules referred to herein are defined according to the IMGT information system.

In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule that binds to PAI-1. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule that binds to PAI-1 . In some embodiments, the antigen-binding molecule comprises the CDRs and the FRs of an antigen-binding molecule that binds to PAI-1. That is, in some embodiments, the antigen-binding molecule comprises the VH region and the VL region of an antigenbinding molecule that binds to PAI-1. In some embodiments, the antigen-binding molecule comprises the VHH region of an antigen-binding molecule that binds to PAI-1 .

In some embodiments, the antigen-binding molecule comprises the CDRs, FRs and/or the VH and/or VL regions of a PAI-1 -binding antibody/nanobody clone described herein, or CDRs, FRs and/or VH and/or VL regions which are derived from those of a PAI-1 -binding antibody clone described herein. In some embodiments, the antigen-binding molecule comprises the CDRs, FRs and/or the VHH regions of a PAI- 1 -binding nanobody clone described herein, or CDRs, FRs and/or VHH regions which are derived from those of a PAI-1 -binding nanobody clone described herein.

In some embodiments, a PAI-1 -binding antibody/nanobody clone is selected from: A5, D4, E8, A7, A9, A10, B1 , B6, C11 , D6, D7, E2, F4, F5, F8, F10, and G11 . In some embodiments, a PAI-1-binding antibody/nanobody clone is selected from: A5, D4, E8, A10, B6, C11 , D6, D7, E2, F4, F8, F10, and G11 . In some embodiments, a PAI-1 -binding antibody/nanobody clone is selected from: A5, D4, E8, C11 and D6. In some embodiments, a PAI-1 -binding antibody/nanobody clone is selected from: A5, D4, and E8. In some embodiments, a PAI-1 -binding antibody/nanobody clone is E8.

In some embodiments, the antigen-binding molecule comprises the CDRs, FRs and/or the VH and/or VL regions of an antibody described herein (e.g. an antibody of Table C(i) herein), or CDRs, FRs and/or VH and/or VL regions which are derived from those of antibody described herein (e.g. an antibody of Table C(i) herein). In some embodiments, the antigen-binding molecule comprises the CDRs, FRs and/or the VHH region of an antibody described herein (e.g. an antibody of Table C(ii) herein), or CDRs, FRs and/or VHH region which are derived from those of antibody described herein (e.g. an antibody of Table C(i I) herein).

In some embodiments, the antigen-binding molecule comprises: a VH or VHH region comprising HC-CDR1 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid), HC-CDR2 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid) and HC-CDR3 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid) as indicated in Column A of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table A(i), or in Column A of row 1 or 2 of Table A(i I), wherein the HC-CDR1 , HC-CDR2 and HC-CDR3 sequences of Column A are selected from the same row of Table A(i) or from the same row of Table A(ii).

By way of illustration, in some embodiments the antigen-binding molecule comprises a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO:31 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:31 are substituted with another amino acid), HC-CDR2 having the amino acid sequence of SEQ ID NO:32 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:32 are substituted with another amino acid) and HC-CDR3 having the amino acid sequence of SEQ ID NO:33 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:33 are substituted with another amino acid). It will be appreciated that the HC-CDR1 , HC-CDR2 and HC-CDR3 sequences of the preceding sentence are selected from Column A of the same row (row 1 ) of Table A(i).

In some embodiments, the antigen-binding molecule comprises: a VH or VHH region comprising HC-FR1 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR1 are substituted with another amino acid), HC-FR2 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR2 are substituted with another amino acid), HC-FR3 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR3 are substituted with another amino acid) and HC-FR4 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid) as indicated in Column A of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table B(i) or in Column A of row 1 or 2 of Table B(ii), wherein the HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 sequences of Column A are selected from the same row of Table B(i) or from the same row of Table B (I I ) .

By way of illustration, in some embodiments, the antigen-binding molecule comprises a VH region comprising HC-FR1 having the amino acid sequence of SEQ ID NO:34 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:34 are substituted with another amino acid), HC-FR2 having the amino acid sequence of SEQ ID NO:35 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:35 are substituted with another amino acid), HC-FR3 having the amino acid sequence of SEQ ID NO:36 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:36 are substituted with another amino acid) and HC-FR4 having the amino acid sequence of SEQ ID NO:37 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:37 are substituted with another amino acid). It will be appreciated that the HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 sequences of the preceding sentence are selected from Column A of the same row (row 1 ) of Table B(i).

In some embodiments, the antigen-binding molecule comprises: a VH or VHH region comprising:

HC-CDR1 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid), HC-CDR2 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid) and HC-CDR3 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid) as indicated in Column A of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table A(i) or in Column A of row 1 or 2 of Table A(ii), wherein the HC-CDR1 , HC-CDR2 and HC-CDR3 sequences of Column A are selected from the same row of Table A(i) or from the same row of Table A(ii); and

HC-FR1 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR1 are substituted with another amino acid), HC-FR2 (or a variant thereof in which 1 or 2 or 3 amino acids in HC- FR2 are substituted with another amino acid), HC-FR3 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR3 are substituted with another amino acid) and HC-FR4 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid) as indicated in Column A of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table B (I) or in Column A of row 1 or 2 of Table B(ii), wherein the HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 sequences of Column A are selected from the same row of Table B(i) or from the same row of Table B(ii).

In some embodiments, the antigen-binding molecule comprises: a VH region comprising:

HC-CDR1 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid), HC-CDR2 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid) and HC-CDR3 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid) as indicated in Column A of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table A(i); and

HC-FR1 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR1 are substituted with another amino acid), HC-FR2 (or a variant thereof in which 1 or 2 or 3 amino acids in HC- FR2 are substituted with another amino acid), HC-FR3 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR3 are substituted with another amino acid) and HC-FR4 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid) as indicated in Column A of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table B(i); wherein the HC-CDR1 , HC-CDR2, HC-CDR3 sequences of Column A of Table A(i) or and the HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 sequences of Column B of Table B(i) are selected from rows having the same number.

In some embodiments, the antigen-binding molecule comprises: a VHH region comprising:

HC-CDR1 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 are substituted with another amino acid), HC-CDR2 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR2 are substituted with another amino acid) and HC-CDR3 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid) as indicated in Column A of row 1 or 2 Table A(ii); and

HC-FR1 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR1 are substituted with another amino acid), HC-FR2 (or a variant thereof in which 1 or 2 or 3 amino acids in HC- FR2 are substituted with another amino acid), HC-FR3 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR3 are substituted with another amino acid) and HC-FR4 (or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid) as indicated in Column A of row 1 or 2 of Table B(ii); wherein the HC-CDR1 , HC-CDR2, HC-CDR3 sequences of Column A of Table A(i I) or and the HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 sequences of Column B of Table B(i I) are selected from rows having the same number.

By way of illustration, in some embodiments, the antigen-binding molecule comprises a VH region comprising: HC-CDR1 having the amino acid sequence of SEQ ID NO:31 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:31 are substituted with another amino acid), HC-CDR2 having the amino acid sequence of SEQ ID NO:32 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:32 are substituted with another amino acid) and HC-CDR3 having the amino acid sequence of SEQ ID NO:33 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:33 are substituted with another amino acid), HC-FR1 having the amino acid sequence of SEQ ID NO:34 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:34 are substituted with another amino acid), HC-FR2 having the amino acid sequence of SEQ ID NO:35 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:35 are substituted with another amino acid), HC-FR3 having the amino acid sequence of SEQ ID NO:36 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:36 are substituted with another amino acid) and HC-FR4 having the amino acid sequence of SEQ ID NO:37 (or a variant thereof in which 1 or 2 or 3 amino acids in SEQ ID NO:37 are substituted with another amino acid). It will be appreciated that the HC-CDR1 , HC-CDR2 and HC-CDR3 sequences of the preceding sentence are selected from Column A of row 1 of Table A(i), and that the HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 sequences are selected from Column A of the row of Table B(i) having the same number (row 1 ).

In some embodiments, the antigen-binding molecule comprises a VH region comprising at least 70%, preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of a VH region sequence selected from Column A of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table C(i), or a VHH region comprising at least 70%, preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, £98%, >99% or 100% amino acid sequence identity to the amino acid sequence of a VHH region sequence selected from Column A of row 1 or 2 of Table C(i I).

In some embodiments, the antigen-binding molecule comprises: a VL region comprising LC-CDR1 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid), LC-CDR2 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid) and LC-CDR3 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid) as indicated in Column B of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table A(i), wherein the LC-CDR1 , LC-CDR2 and LC-CDR3 sequences of Column B are selected from the same row of Table A(i).

In some embodiments, the antigen-binding molecule comprises: a VL region comprising LC-FR1 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR1 are substituted with another amino acid), LC-FR2 (or a variant thereof in which 1 or 2 or 3 amino acids in LC- FR2 are substituted with another amino acid), LC-FR3 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR3 are substituted with another amino acid) and LC-FR4 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR4 are substituted with another amino acid) as indicated in Column B of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table B(i), wherein the LC-FR1 , LC-FR2, LC-FR3 and LC- FR4 sequences of Column B are selected from the same row of Table B(i).

In some embodiments, the antigen-binding molecule comprises: a VL region comprising:

LC-CDR1 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid), LC-CDR2 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid) and LC-CDR3 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid) as indicated in Column B of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table A(i), wherein the LC- CDR1 , LC-CDR2 and LC-CDR3 sequences of Column B are selected from the same row of Table A(i); and

LC-FR1 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR1 are substituted with another amino acid), LC-FR2 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR2 are substituted with another amino acid), LC-FR3 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR3 are substituted with another amino acid) and LC-FR4 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR4 are substituted with another amino acid) as indicated in Column B of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 15 of Table B(i), wherein the LC- FR1 , LC-FR2, LC-FR3 and LC-FR4 sequences of Column B are selected from the same row of Table B(i).

LC-CDR1 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 are substituted with another amino acid), LC-CDR2 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR2 are substituted with another amino acid) and LC-CDR3 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid) as indicated in Column B of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table A(i); and

LC-FR1 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR1 are substituted with another amino acid), LC-FR2 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR2 are substituted with another amino acid), LC-FR3 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR3 are substituted with another amino acid) and LC-FR4 (or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR4 are substituted with another amino acid) as indicated in Column B of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table B(i); wherein the LC-CDR1 , LC-CDR2, LC-CDR3 sequences of Column B of Table A(i) and the LC-FR1 , LC-FR2, LC-FR3 and LC-FR4 sequences of Column B of Table B(i) are selected from rows having the same number.

In some embodiments, the antigen-binding molecule comprises a VL region comprising at least 70%, preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of a VL region sequence selected from Column B of row 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of Table C(i).

In some embodiments, the antigen-binding molecule comprises a VH region according to any one embodiment as described herein, and a VL region according to any one embodiment as described herein. In some embodiments, the antigen-binding molecule comprises a VHH region according to any one embodiment as described herein.

In embodiments in accordance with the present disclosure, one or more amino acids are substituted with another amino acid. A substitution comprises substitution of an amino acid residue with a non-identical 'replacement' amino acid residue. A replacement amino acid residue of a substitution according to the present disclosure may be a naturally-occurring amino acid residue (j.e. encoded by the genetic code) which is non-identical to the amino acid residue at the relevant position of the equivalent, unsubstituted amino acid sequence, selected from: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie): leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Vai). In some embodiments, a replacement amino acid may be a non-naturally occurring amino acid residue - i.e. an amino acid residue other than those recited in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, alb, and other amino acid residue analogues such as those described in Ellman, et a/., Meth. Enzym. 202 (1991 ) 301-336.

In some embodiments, a substitution may be biochemically conservative. In some embodiments, where an amino acid to be substituted is provided in one of rows 1 to 5 of the table below, the replacement amino acid of the substitution is another, non-identical amino acid provided in the same row: By way of illustration, in some embodiments wherein substitution is of a Met residue, the replacement amino acid may be selected from Ala, Vai, Leu, lie, Trp, Tyr, Phe and Norleucine.

In some embodiments, a replacement amino acid in a substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, a replacement amino acid in a substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces:

That is, in some embodiments, a nonpolar amino acid is substituted with another, non-identical nonpolar amino acid. In some embodiments, a polar amino acid is substituted with another, non-identical polar amino acid. In some embodiments, an acidic polar amino acid is substituted with another, non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is substituted with another, non- identical basic polar amino acid. In some embodiments, a neutral amino acid is substituted with another, non-identical neutral amino acid. In some embodiments, a positive amino acid is substituted with another, non-identical positive amino acid. In some embodiments, a negative amino acid is substituted with another, non-identical negative amino acid. In some embodiments, substitution(s) may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. target binding) of the antigen-binding molecule comprising the substitution as compared to the equivalent unsubstituted molecule.

The VH and VL region of an antigen-binding region of an antibody together constitute the Fv region. In some embodiments, the antigen-binding molecule according to the present disclosure comprises, or consists of, an Fv region that binds to PAI-1 . In some embodiments, the VH and VL regions of the Fv are provided as single polypeptide joined by a linker sequence, i.e. a single chain Fv (scFv).

The VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1 ) region of an antigen-binding region of an antibody together constitute the Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising a VH, a CH1 , a VL and a CL (e.g. CK or CA). In some embodiments, the Fab region comprises a polypeptide comprising a VH and a CH1 (e.g. a VH-CH1 fusion polypeptide), and a polypeptide comprising a VL and a CL (e.g. a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and a CL (e.g. a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH (e.g. a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1 , VL and CL regions of the Fab or CrossFab are provided as single polypeptide joined by linker regions, i.e. as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).

In some embodiments, the antigen-binding molecule described herein comprises, or consists of, a whole antibody that binds to PAI-1 . As used herein, ‘whole antibody’ refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.

Immunoglobulins of type G (i.e. IgG) are -150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chains comprise a VL followed by a CL. Depending on the heavy chain, immunoglobulins may be classed as IgG (e.g.

IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM. The light chain may be kappa (K) or lambda (A).

Herein, a ‘CH1 domain’ refers to an amino acid sequence corresponding to the CH1 domain of an immunoglobulin (Ig). The CH1 domain is the region of an Ig formed by positions 118 to 215 of the immunoglobulin constant domain, according to the EU numbering system (described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1 ): 78-85). A ‘hinge domain’ refers to an amino acid sequence corresponding to the hinge domain of an Ig. The hinge domain is the region of an Ig formed by positions 216 to 230 of the immunoglobulin constant domain, according to the EU numbering system. A ‘CH2 domain’ refers to an amino acid sequence corresponding to the CH2 domain of an Ig. The CH2 domain is the region of an Ig formed by positions 231 to 340 of the immunoglobulin constant domain, according to the EU numbering system. A ‘CH3 domain’ refers to an amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig). The CH3 domain is the region of an Ig formed by positions 341 to 447 of the immunoglobulin constant domain, according to the EU numbering system. A ‘CH2-CH3 region’ refers to an amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig). The CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of the immunoglobulin constant domain, according to the EU numbering system.

In some embodiments, the antigen-binding molecule described herein comprises, or consists of, an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM that binds to PAI-1 .

In some embodiments, the antigen-binding molecule of the present disclosure comprises one or more regions (e.g. CH1 , CH2, CH3, etc.) of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. Ig A1 , lgA2), IgD, IgE or IgM, e.g. a human IgG (e.g. hlgG1 , hlgG2, hlgG3, hlgG4), hlgA (e.g. hlgA1 , hlgA2), hlgD, h Ig E or hlgM. In some embodiments, the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of a human lgG1 allotype (e.g. G1 m1 , G1 m2, G1 m3, G1 m17 or isoallotype nG1 m1 ).

In some embodiments, the antigen-binding molecule comprises (e.g. comprises one or more polypeptides comprising) a CH1 region. In some embodiments, a CH1 region comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, £94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:233.

In some embodiments, the antigen-binding molecule comprises (e.g. comprises one or more polypeptides comprising) a hinge region. In some embodiments, a hinge region comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:234.

In some embodiments, the antigen-binding molecule comprises (e.g. comprises one or more polypeptides comprising) a CH2 region. In some embodiments, a CH2 region comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, £94%, £95%, £96%, £97%, £98%, £99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:235.

In some embodiments, the antigen-binding molecule comprises (e.g. comprises one or more polypeptides comprising) a CH3 region. In some embodiments, a CH3 region comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of £70%, £75%, £80%, £85%, £86%, £87%, £88%, £89%, £90%, >91%, >92%, >93%, £94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:236 or 238.

In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region.

As used herein, an ‘Fc region’ refers to a polypeptide complex formed by interaction between two polypeptides, each polypeptide comprising the CH2-CH3 region of an immunoglobulin (Ig) heavy chain constant sequence.

In some embodiments, a CH2 region, CH3 region and/or a CH2-CH3 region according to the present disclosure corresponds to the CH2 region/CH3 region/CH2-CH3 region of an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. Ig A1 , lgA2), IgD, IgE or IgM. In some embodiments, the CH2 region, CH3 region and/or a CH2-CH3 region corresponds to the CH2 region/CH3 region/CH2-CH3 region of a human IgG (e.g. hlgG 1 , hlgG2, hlgG3, hlgG4), hlgA (e.g. hlgA1 , hlgA2), hlgD, hlgE or hlgM. In some embodiments, the CH2 region, CH3 region and/or a CH2-CH3 region corresponds to the CH2 region/CH3 region/CH2-CH3 region of a human lgG1 allotype (e.g. G1 m1 , G1 m2, G1 m3, G1 m17 or isoallotype nG1 m1 ).

Fc regions provide for interaction with Fc receptors and other molecules of the immune system to bring about functional effects. Fc-mediated effector functions are reviewed e.g. in Jefferis et al., Immunol Rev 1998 163:59-76 (hereby incorporated by reference in its entirety), and are brought about through Fc- mediated recruitment and activation of immune cells (e.g. macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells and T cells) through interaction between the Fc region and Fc receptors expressed by the immune cells, recruitment of complement pathway components through binding of the Fc region to complement protein C1q, and consequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and/or chemokine production, and antigen processing and presentation.

Modifications to antibody Fc regions that influence Fc-mediated functions are known in the art, such as those described e.g. in Wang et al., Protein Cell (2018) 9(1 ):63-73, which is hereby incorporated by reference in its entirety. Exemplary Fc region modifications known to influence antibody effector function are summarised in Table 1 of Wang et al., Protein Cell (2018) 9(1 ):63-73. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification to increase or reduce an Fc-mediated function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region. Where an Fc region comprises a modification, the modification may be present in one or both of the polypeptide chains which together form the Fc region.

Where an Fc region/CH2/CH3 is described as comprising modification(s) ‘corresponding to’ reference substitution(s), equivalent substitution(s) in the homologous Fc/CH2/CH3 are contemplated. By way of illustration, L234A/L235A substitutions in human lgG1 (numbered according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 ) correspond to L to A substitutions at positions 117 and 118 of the mouse Ig gamma-2A chain C region (UniProtKB: P01863-1 , v1 ).

Where an Fc region is described as comprising a modification, the modification may be present in one or both of the polypeptide chains which together form the Fc region.

In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification in one or more of the CH2 and/or CH3 regions.

In some embodiments, the Fc region comprises modification to increase an Fc-mediated function. In some embodiments, the Fc region comprises modification to increase ADCC. In some embodiments, the Fc region comprises modification to increase ADCP. In some embodiments, the Fc region comprises modification to increase CDC. An antigen-binding molecule comprising an Fc region comprising modification to increase an Fc-mediated function (e.g. ADCC, ADCP, CDC) induces an increased level of the relevant effector function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.

In some embodiments, the Fc region comprises modification to increase binding to an Fc receptor. In some embodiments, the Fc region comprises modification to increase binding to an Fey receptor. In some embodiments, the Fc region comprises modification to increase binding to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRllla and FcyRlllb. In some embodiments, the Fc region comprises modification to increase binding to FcyRllla. In some embodiments, the Fc region comprises modification to increase binding to FcyRlla. In some embodiments, the Fc region comprises modification to increase binding to FcyRllb. In some embodiments, the Fc region comprises modification to increase binding to FcRn. In some embodiments, the Fc region comprises modification to increase binding to a complement protein. In some embodiments, the Fc region comprises modification to increase binding to C1q. In some embodiments, the Fc region comprises modification to promote hexamerisation of the antigen-binding molecule. In some embodiments, the Fc region comprises modification to increase antigen-binding molecule half-life. In some embodiments, the Fc region comprises modification to increase coengagement.

In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions F243L/R292P/Y300L/V305I/P396L as described in Stavenhagen et al. Cancer Res. (2007) 67:8882-8890. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S239D/I332E or S239D/I332E/A330L as described in Lazar et al. , Proc Natl Acad Sci USA. (2006)103:4005-4010. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S298A/E333A/K334A as described in Shields et al., J Biol Chem. (2001 ) 276:6591-6604. In some embodiments, the Fc region comprises modification to one of heavy chain polypeptides corresponding to the combination of substitutions L234Y/L235Q/G236W/S239M/H268D/D270E/S298A, and modification to the other heavy chain polypeptide corresponding to the combination of substitutions D270E/K326D/A330M/K334E, as described in Mimoto et al., MAbs. (2013): 5:229-236. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions G236A/S239D/I332E as described in Richards et al., Mol Cancer Ther. (2008) 7:2517-2527.

In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions K326W/E333S as described in Idusogie et al. J Immunol. (2001 ) 166(4):2571-5. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S267E/H268F/S324T as described in Moore et al. MAbs. (2010) 2(2): 181 -9. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions described in Natsume et al., Cancer Res. (2008) 68(10):3863-72. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions E345R/E430G/S440Y as described in Diebolder et al. Science (2014) 343(6176):1260-3.

In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions M252Y/S254T/T256E as described in Dall’Acqua et al. J Immunol. (2002) 169:5171-5180.

In some embodiments, the Fc region comprises a CH2-CH3 region comprising an amino acid difference at one or more of the following positions, relative to the amino acid sequence of a CH2-CH3 region of a reference Fc region: 252, 254 or 256 (according to the EU numbering system). In some embodiments, the Fc region comprises a CH2-CH3 region comprising one or more of the following specified amino acid residues: Y252, T254 or E256 (according to the EU numbering system). In some embodiments, the Fc region comprises a CH2-CH3 region comprising Y252, T254 and E256. In some embodiments, the Fc region comprises a CH2-CH3 region comprising one or more of the following amino acid substitutions, relative to the amino acid sequence of a CH2-CH3 region of the reference Fc region: M252Y, S254T or T256E (according to the EU numbering system).

These so called ‘YTE’ modifications located at the CH2-CH3 interface of the Fc region have been shown to increase the binding affinity at pH 6.0 to the MHC Class I neonatal Fc receptor (FcRn), localised within the acidic endosomes of endothelial and haematopoietic cells, which increases efficient recycling of administered mAb and longer half-life in the plasma.

In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions M428L/N434S as described in Zalevsky et al. Nat Biotechnol. (2010) 28:157-159.

In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S267E/L328F as described in Chu et al., Mol Immunol. (2008) 45:3926-3933. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions N325S/L328F as described in Shang et al. Biol Chem. (2014) 289:15309-15318. In some embodiments, the Fc region comprises modification to reduce/prevent an Fc-mediated function. In some embodiments, the Fc region comprises modification to reduce/prevent ADCC. In some embodiments, the Fc region comprises modification to reduce/prevent ADCP. In some embodiments, the Fc region comprises modification to reduce/prevent CDC. An antigen-binding molecule comprising an Fc region comprising modification to reduce/prevent an Fc-mediated function (e.g. ADCC, ADCP, CDC) induces an reduced level of the relevant effector function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.

In some embodiments, the Fc region comprises modification to reduce/prevent binding to an Fc receptor. In some embodiments, the Fc region comprises modification to reduce/prevent binding to an Fey receptor. In some embodiments, the Fc region comprises modification to reduce/prevent binding to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRllla and FcyRlllb. In some embodiments, the Fc region comprises modification to reduce/prevent binding to FcyRllla. In some embodiments, the Fc region comprises modification to reduce/prevent binding to FcyRlla. In some embodiments, the Fc region comprises modification to reduce/prevent binding to FcyRllb. In some embodiments, the Fc region comprises modification to reduce/prevent binding to a complement protein. In some embodiments, the Fc region comprises modification to reduce/prevent binding to C1q. In some embodiments, the Fc region comprises modification to reduce/prevent glycosylation of the amino acid residue corresponding to N297.

In some embodiments, the Fc region is not able to induce one or more Fc-mediated functions (/.e. lacks the ability to elicit the relevant Fc-mediated function(s)). Accordingly, antigen-binding molecules comprising such Fc regions also lack the ability to induce the relevant function(s). Such antigen-binding molecules may be described as being devoid of the relevant function(s).

In some embodiments, the Fc region is not able to induce ADCC. In some embodiments, the Fc region is not able to induce ADCP. In some embodiments, the Fc region is not able to induce CDC. In some embodiments, the Fc region is not able to induce ADCC and/or is not able to induce ADCP and/or is not able to induce CDC.

In some embodiments, the Fc region is not able to bind to an Fc receptor. In some embodiments, the Fc region is not able to bind to an Fey receptor. In some embodiments, the Fc region is not able to bind to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRllla and FcyRlllb. In some embodiments, the Fc region is not able to bind to FcyRllla. In some embodiments, the Fc region is not able to bind to FcyRlla. In some embodiments, the Fc region is not able to bind to FcyRllb. In some embodiments, the Fc region is not able to bind to FcRn. In some embodiments, the Fc region is not able to bind to a complement protein. In some embodiments, the Fc region is not able to bind to C1q. In some embodiments, the Fc region is not glycosylated at the amino acid residue corresponding to N297.

In some embodiments, the Fc region comprises modification corresponding to N297A or N297Q or N297G as described in Leabman et al., MAbs. (2013) 5:896-903. In some embodiments, the Fc region comprises modification corresponding to L235E as described in Alegre et al., J Immunol. (1992) 148:3461-3468. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A/L235A or F234A/L235A as described in Xu et al., Cell Immunol. (2000) 200:16-26. In some embodiments, the Fc region comprises modification corresponding to P329A or P329G as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A/L235A/P329G as described in Lo et al. J. Biol. Chem (2017) 292(9):3900-3908. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions described in Rother et al., Nat Biotechnol. (2007) 25:1256-1264. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S228P/L235E as described in Newman et al., Clin. Immunol. (2001 ) 98:164-174. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions H268Q/V309L/A330S/P331S as described in An et al., MAbs. (2009) 1 :572-579. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions V234A/G237A/P238S/H268A/V309L/A330S/P331S as described in Vafa et al., Methods. (2014) 65:114- 126. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A/L235E/G237A/A330S/P331S as described in US 2015/0044231 A1.

The combination of substitutions ‘L234A/L235A and corresponding substitutions (such as e.g. F234A/L235A in human lgG4) are known to disrupt binding of Fc to Fey receptors and inhibit ADCC, ADCP, and also to reduce C1q binding and thus CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, hereby incorporated by reference in entirety). The substitutions ‘P329G’ and ‘P329A’ reduce C1q binding (and thereby CDC). Substitution of ‘N297’ with ‘A’, ‘G’ or ‘Q’ is known to eliminate glycosylation, and thereby reduce Fc binding to C1q and Fey receptors, and thus CDC and ADCC. Lo et al. J. Biol. Chem (2017) 292(9):3900-3908 (hereby incorporated by reference in its entirety) reports that the combination of substitutions L234A/L235A/P329G eliminated complement binding and fixation as well as Fc y receptor dependent, antibody-dependent, cell-mediated cytotoxicity in both murine lgG2a and human lgG1.

The combination of substitutions L234A/L235E/G237A/A330S/P331 S in lgG1 Fc is disclosed in US 2015/0044231 A1 to abolish induction of phagocytosis, ADCC and CDC.

In some embodiments, the Fc region comprises modification corresponding to the substitution S228P as described in Silva et al., J Biol Chem. (2015) 290(9):5462-5469. The substitution S228P in lgG4 Fc reduces Fab-arm exchange (Fab-arm exchange can be undesirable).

In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A/L235A. In some embodiments, the Fc region comprises modification corresponding to the substitution P329G. In some embodiments, the Fc region comprises modification corresponding to the substitution N297Q. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A/L235A/P329G.

In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A/L235A/P329G/N297Q.

In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A/L235E/G237A/A330S/P331 S.

In some embodiments, the Fc region comprises modification corresponding to the substitution S228P, e.g. in lgG4.

In some embodiments, the antigen-binding molecule comprises (e.g. comprises one or more polypeptides comprising) a CH2-CH3 region. In some embodiments, a CH2-CH3 region comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:239 or 240.

In some embodiments, the antigen-binding molecule comprises (e.g. comprises one or more polypeptides comprising) a CH1-hinge-CH2-CH3 region. In some embodiments, a CH1-hinge-CH2-CH3 region comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:232 or 237.

In some embodiments, the antigen-binding molecule of the present disclosure comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; CK). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CK , e.g. IGLC1 , IGLC2, IGLC3, IGLC6 or IGLC7.

In some embodiments, the antigen-binding molecule comprises (e.g. comprises one or more polypeptides comprising) a CL region. In some embodiments, a CL region comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, £94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:241 , 242, 243, 244, 245, 246. In some embodiments, the antigen-binding molecule comprises (e.g. comprises one or more polypeptides comprising) a CL region. In some embodiments, a CL region comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, £96%, £97%, £98%, £99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:241 . In some embodiments, the antigen-binding molecule is or comprises a monoclonal antibody, or an antigen-binding fragment thereof.

In some embodiments, the antigen-binding molecule is or comprises a fully human antibody/antibody fragment. A fully human antibody/antibody fragment may be encoded by human nucleic acid sequence(s). A fully human antibody/antibody fragment may be devoid of non-human amino acid sequences.

In some embodiments, the antigen-binding molecule is not an anti-PAI-1 antibody selected from: #242816, MA-33B8, MA-H4B3, MA-124K1 , MA-12V3, MA-8H9D4, #353927, LS-C91951 , #MA-33H1F7, and AF1786.

Aspects of the present disclosure relate to multispecific antigen-binding molecules. By ‘multispecific’ it is meant that the antigen-binding molecule displays specific binding to more than one target. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding domains (/.e. at least two antigen-binding domains, e.g. comprising non-identical VHs and VLs, or non-identical VHHs).

In some embodiments, the antigen-binding molecule binds to PAI-1 and another target other than PAI-1 and so is at least bispecific. The term ‘bispecific’ means that the antigen-binding molecule is able to bind specifically to at least two distinct antigenic determinants.

It will be appreciated that an antigen-binding molecule according to the present disclosure (e.g. a multispecific antigen-binding molecule) may comprise antigen-binding molecules capable of binding to the targets for which the antigen-binding molecule is specific. For example, an antigen-binding molecule that binds to PAI-1 and another target other than PAI-1 may comprise: (I) an antigen-binding molecule that binds to PAI-1 , and (ii) an antigen-binding molecule that binds to an antigen other than PAI-1 .

It will also be appreciated that an antigen-binding molecule according to the present disclosure (e.g. a multispecific antigen-binding molecule) may comprise antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to the targets for which the antigen-binding molecule is specific.

In some embodiments, a component antigen-binding molecule of a larger antigen-binding molecule (e.g. a multispecific antigen-binding molecule) may be referred to e.g. as an ‘antigen-binding domain’ or ‘antigen-binding region’ of the larger antigen-binding molecule.

Multispecific antigen-binding molecules according to the present disclosure may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety. Suitable formats include those shown in Figure 2 of Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212: antibody conjugates, e.g. lgG2, F(ab’)2 or CovX-Body; IgG or IgG-like molecules, e.g. IgG, chimeric IgG, KX-body common HC; CH1/CL fusion proteins, e.g. scFv2-CH1/CL, VHH2-CH1/CL; ‘variable domain only’ bispecific antigenbinding molecules, e.g. tandem scFv (taFV), triplebodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triple heads, tandem dAb/VHH, tertravalent dAb.VHH; Non-lg fusion proteins, e.g. scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibody, DNL-Fab2, DNL-Fab2-scFv, DNL- Fab2-lgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g. scFv-Fc(kih), scFv- Fc(CH3 charge pairs), scFv-Fc (EW-RVT), scFv-fc (HA-TF), scFv-Fc (SEEDbody), taFv-Fc(kih), scFv- Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc (SEEDbody), DART- Fc, scFv-CH3(kih), TriFabs; Fc fusions, e.g. Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-lg, scFv2-Fcab; CH3 fusions, e.g. Dia-diabody, scDb-CH3; IgE/IgM CH2 fusions, e.g. scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, e.g. Fab-scFv (bibody), Fab-scFv2 (tribody), Fab- Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-lg fusion proteins, e.g. DNL-Fabs, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g. IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab, CrossMab(kih), scFab-lgG(kih), Fab-scFab-lgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pairs + CH1/CL charge pairs, hinge/CH3 charge pairs, SEED-body, Duobody, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc*; appended and Fc-modified IgGs, e.g. lgG(kih)-Fv, IgG HA-TF-Fv, lgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-lg, DVI-lg (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, e.g. Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEEDbody, TriFab; appended IgGs - HC fusions, e.g. IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CaCP) Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab), Fab-lgG(CaCp Fab), Fab-lgG(CR3), Fab-hinge-lgG(CR3); appended IgGs - LC fusions, e.g. IgG-scFv(LC), scFv(LC)-lgG, dAb-IgG; appended IgGs - HC and LC fusions, e.g. DVD-lg, TVD-lg, CODV-lg, scFv4-lgG, Zybody; Fc fusions, e.g. Fab-scFv- Fc, scFv4-lg; F(ab’)2 fusions, e.g. F(ab’)2-scFv2; CH1/CL fusion proteins e.g. scFv2-CH1-hinge/CL; modified IgGs, e.g. DAF (two-in one-IgG), DutaMab, Mab²; and non-lg fusions, e.g. DNL-Fab4-lgG. The skilled person is readily able to design and produce multispecific antigen-binding molecules.

The present disclosure also provides Chimeric Antigen Receptors (CARs). CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. CAR structure and engineering is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1 ), hereby incorporated by reference in its entirety. CARs comprise an antigen-binding region linked to a cell membrane anchor region and a signalling region. An optional hinge region may provide separation between the antigen-binding region and cell membrane anchor region, and may act as a flexible linker.

The antigen-binding domain of a CAR according to the present disclosure comprises or consists of an antigen-binding molecule that binds to PAI-1 , as described herein. Accordingly, a CAR according to the present disclosure comprises an antigen-binding molecule as described herein.

It will be appreciated that an antigen-binding molecule according to the present disclosure forms, or is comprised in, the antigen-binding domain of the CAR. Accordingly, in some embodiments, the antigenbinding molecule of the present disclosure is comprised in a CAR. It will also be appreciated that an antigen-binding molecule according to the present disclosure may be a CAR. A CAR having an antigen-binding domain comprising or consisting of an antigen-binding molecule of the present disclosure (e.g. a PAI-1-binding Fv, or a PAI-1-binding VHH) is an antigen-binding molecule. The antigen-binding domain of the CAR of the present disclosure may be provided with any suitable format, e.g. scFv, scFab, VHH etc.

The cell membrane anchor region is provided between the antigen-binding region and the signalling region of the CAR and provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding region in the extracellular space, and signalling region inside the cell. In some embodiments, the CAR comprises a cell membrane anchor region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the transmembrane region amino acid sequence for one of CD3- , CD4, CD8 or CD28. As used herein, a region which is ‘derived from’ a reference amino acid sequence comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference sequence.

The signalling region of a CAR allows for activation of the T cell. The CAR signalling regions may comprise the amino acid sequence of the intracellular domain of CD3- , which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing T cell. Signalling regions comprising sequences of other ITAM-containing proteins such as FcyRI have also been employed in CARs (Haynes et al., 2001 J Immunol 166(1 ): 182-187). Signalling regions of CARs may also comprise co-stimulatory sequences derived from the signalling region of co-stimulatory molecules, to facilitate activation of CAR-expressing T cells upon binding to the target protein. Suitable co-stimulatory molecules include CD28, 0X40, 4-1 BB, ICOS and CD27. In some cases CARs are engineered to provide for co-stimulation of different intracellular signalling pathways. For example, signalling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, whereas the 4-1 BB-mediated signalling is through TNF receptor associated factor (TRAF) adaptor proteins. Signalling regions of CARs therefore sometimes contain co-stimulatory sequences derived from signalling regions of more than one co-stimulatory molecule. In some embodiments, the CAR of the present disclosure comprises one or more co-stimulatory sequences comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the intracellular domain of one or more of CD28, 0X40, 4-1 BB, ICOS and CD27.

An optional hinge region may provide separation between the antigen-binding domain and the transmembrane domain, and may act as a flexible linker. Hinge regions may be derived from IgG 1 or lgG4. In some embodiments, the CAR of the present disclosure comprises a hinge region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the hinge region of lgG1 or lgG4. Also provided is a cell comprising a CAR according to the present disclosure. The CAR according to the present disclosure may be used to generate CAR-expressing immune cells, e.g. CAR-T or CAR-NK cells. Engineering of CARs into immune cells may be performed during culture, in vitro.

Polypeptides and particular exemplary antigen-binding molecules

The present disclosure also provides polypeptide constituents of antigen-binding molecules. The polypeptides may be provided in isolated or substantially purified form.

The antigen-binding molecule of the present disclosure may be, or may comprise, a complex of polypeptides.

In the present specification where a polypeptide comprises more than one domain or region, it will be appreciated that the plural domains/regions are preferably present in the same polypeptide chain. That is, the polypeptide comprising more than one domain or region is a fusion polypeptide comprising the domains/regions.

In some embodiments a polypeptide according to the present disclosure comprises, or consists of, a VH as described herein. In some embodiments a polypeptide according to the present disclosure comprises, or consists of, a VL as described herein. In some embodiments a polypeptide according to the present disclosure comprises, or consists of, a VHH as described herein.

In some embodiments, the polypeptide additionally comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide additionally comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises a CH1 , CH2 region and/or a CH3 region of an immunoglobulin (Ig).

In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide comprises a CH1 region as described herein. In some embodiments, the polypeptide comprises a hinge region as described herein. In some embodiments, the polypeptide comprises a CH2 region as described herein. In some embodiments, the polypeptide comprises a CH3 region as described herein. In some embodiments, the polypeptide comprises a CH2-CH3 region as described herein. In some embodiments, the polypeptide comprises a CH1-hinge-CH2-CH3 region as described herein.

In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the polypeptide comprises a CL region as described herein.

In some embodiments, the polypeptide according to the present disclosure comprises a structure from N- to C-terminus according to one of the following:

(i) VH

(ii) VL (iii) VH-CH1

(iv) VL-CL

(v) VL-CH1

(vi) VH-CL

(vii) VH-CH1 -CH2-CH3

(viii) VL-CL-CH2-CH3

(ix) VL-CH1-CH2-CH3

(x) VH-CL-CH2-CH3

(xi) VHH

Also provided by the present disclosure are antigen-binding molecules composed of the polypeptides of the present disclosure. In some embodiments, the antigen-binding molecule of the present disclosure comprises one of the following combinations of polypeptides:

(A) VH + VL

(B) VH-CH1 + VL-CL

(C) VL-CH1 + VH-CL

(D) VH-CH1-CH2-CH3 + VL-CL

(E) VH-CL-CH2-CH3 + VL-CH1

(F) VL-CH1-CH2-CH3 + VH-CL

(G) VL-CL-CH2-CH3 + VH-CH1

(H) VH-CH1-CH2-CH3 + VL-CL-CH2-CH3

(I) VH-CL-CH2-CH3 + VL-CH1-CH2-CH3

In some embodiments, the antigen-binding molecule comprises more than one of a polypeptide of the combinations shown in (A) to (I) above. By way of example, with reference to (D) above, In some embodiments, the antigen-binding molecule comprises two polypeptides comprising the structure VH- CH1-CH2-CH3, and two polypeptides comprising the structure VL-CL.

In accordance with (I) to (xi) and (A) to (I) above, ‘VH’ refers to a VH region as described herein, ‘VL’ refers to a VL region as described herein, ‘VHH’ refers to a VHH region as described herein.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30. In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:186 or 187.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215 or 216.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231 .

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VH region comprising the heavy chain CDRs, and a VL region comprising the light chain CDRs, of an antibody selected from an antibody as shown in Table A(i) herein. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide or polypeptides comprising: (I) a VH region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 as indicated in column A of Table A(i), and (ii) a VL region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 as indicated in column B of Table A(i), wherein the sequences of Columns A and B are selected from the same row of Table A(i). In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VH region comprising the heavy chain CDRs, and a VL region comprising the light chain CDRs, of an antibody as shown in Table A(i).

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VHH region comprising the heavy chain CDRs of a nanobody selected from an antibody as shown in Table A(ii) herein. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide or polypeptides comprising a VHH region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 as indicated in column A of Table A(ii), wherein the sequences are selected from the same row of Table A(i I). In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VHH region comprising the heavy chain CDRs of a nanobody as shown in Table A(ii).

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VH region comprising the heavy chain FRs, and a VL region comprising the light chain FRs, of an antibody selected from an antibody as shown in Table B(i) herein. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide or polypeptides comprising: (I) a VH region comprising HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 as indicated in column A of Table B(i), and (ii) a VL region comprising LC-FR1 , LC-FR2, LC-FR3, and LC-FR4 as indicated in column B of Table B(i), wherein the sequences of columns A and B are selected from the same row of Table B(i). In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VH region comprising the heavy chain FRs, and a VL region comprising the light chain FRs, of an antibody as shown in Table B(i) herein.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VHH region comprising the heavy chain FRs of a nanobody selected from a nanobody as shown in Table B(i I) herein. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide or polypeptides comprising a VHH region comprising HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 as indicated in column A of Table B(ii). In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VHH region comprising the heavy chain FRs of a nanobody as shown in Table B(ii) herein.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising: (I) an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to an amino acid sequence indicated in column A of Table C(i), and (ii) an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to an amino acid sequence indicated in column B of Table C(i), wherein the sequences of columns A and B are selected from the same row of Table C(i).

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VH region and a VL region of an antibody clone selected from an antibody as shown in Table C(i) herein. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide or polypeptides comprising: (I) an amino acid sequence indicated in column A of Table C(i), and (ii) an amino acid sequence indicated in column B of Table C(i), wherein the sequences of columns A and B are selected from the same row of Table C(i). In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide or polypeptides comprising a VH region and a VL region of an antibody as shown in Table C(i) herein.

In some embodiments, the antigen-binding molecule of the present disclosure comprises: (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, £98%, >99% or 100%) amino acid sequence identity to an amino acid sequence indicated in column A of Table D, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, £95%, £96%, £97%, £98%, £99% or 100%) amino acid sequence identity to an amino acid sequence indicated in column B of Table D, wherein the sequences of columns A and B are selected from the same row of Table D.

In some embodiments, the antigen-binding molecule of the present disclosure comprises the polypeptides of an antigen-binding molecule according to Table D herein. That is, in some embodiments, the antigenbinding molecule comprises: (I) a polypeptide comprising or consisting of an amino acid sequence indicated in column A of Table D, and (II) a polypeptide comprising or consisting of an amino acid sequence indicated in column B of Table D, wherein the sequences of columns A and B are selected from the same row of Table D.

In some embodiments, the antigen-binding molecule of the present disclosure comprises:

(1 ) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:202, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:217;

(2) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:187;

(3) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 186;

(4) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:203, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:218;

(5) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:204, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:219;

(6) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:205, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:220;

(7) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:206, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:221 ;

(8) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:207, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:222;

(9) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:208, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:223;

(10) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:209, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:224; (11 ) (i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:210, and (II) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:225;

(12) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:211 , and (II) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:226;

(13) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:212, and (II) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:227;

(14) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:213, and (II) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:228;

(15) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:214, and (II) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:229;

(16) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:215, and (II) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:230; or

(17) (I) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:216, and (II) a polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:231 .

Linkers and additional sequences

The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise further amino acids or sequences of amino acids.

The antigen-binding molecules and polypeptides of the present disclosure may comprise one or more linker sequences between sequences of amino acids. For example, a linker sequence may be provided between a VH sequence and a VL sequence, providing linkage between the VH and VL (e.g. as in an scFv molecule).

Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.

In some embodiments, the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n (SEQ ID NO: 280-281 ) or (GxS)nGm (SEQ ID NO: 282-232); wherein G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1 , 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g. 1 , 2, 3, 4, 5 or 6) copies (e.g. in tandem) of the sequence motif G4S (SEQ ID NO: 279). In some embodiments, the linker sequence comprises or consists of (G4S)4 (SEQ ID NO: 277) or (G4S)e (SEQ ID NO: 278). In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.

The antigen-binding molecules and polypeptides of the present disclosure may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule/polypeptide. For example, antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a sequence of amino acids forming a detectable moiety, e.g. as described hereinbelow.

The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt and Ensembl, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

The signal peptide may be present at the N-terminus of the antigen-binding molecule/polypeptide, and may be present in the newly synthesised antigen-binding molecule/polypeptide. The signal peptide provides for efficient trafficking of the antigen-binding molecule/polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature antigen-binding molecule/polypeptide.

Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176). In some embodiments, the signal peptide comprises, or consists, of the amino acid sequence SEQ ID NO:247, 248 or 249.

Labels and conjugates

In some embodiments, the antigen-binding molecule or polypeptide of the present disclosure comprises a detectable moiety.

In some embodiments, a detectable moiety is a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label. The antigen-binding molecule or polypeptide may be covalently or non-covalently labelled with the detectable moiety.

Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5. Radiolabels include radioisotopes such as Hydrogen³, Sulfur³⁵, Carbon¹⁴, Phosphorus³², Iodine¹²³, Iodine¹²⁵, Iodine¹²⁶, Iodine¹³¹ , Iodine¹³³, Bromine⁷⁷, Technetiurn^99m, Indium¹¹¹ , lndiurn^113m, Gallium⁶⁷, Gallium⁶⁸, Ruthenium⁹⁵, Ruthenium⁹⁷, Ruthenium¹⁰³, Ruthenium¹⁰⁵, Mercury²⁰⁷, Mercury²⁰³, Rheniurn^99m, Rhenium¹⁰¹ , Rhenium¹⁰⁵, Scandium⁴⁷, Tellurium¹²¹¹⁷¹, Tellurium¹²²¹⁷¹, Tellurium¹²⁵¹¹¹, Thulium¹⁶⁵, Thuliuml¹⁶⁷, Thulium¹⁶⁸, Copper⁶⁷, Fluorine¹⁸, Yttrium⁹⁰, Palladium¹⁰⁰, Bismuth²¹⁷ and Antimony²¹¹. Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels. Immuno-detectable labels include haptens, peptides/polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers.

In some embodiments, the antigen-binding molecule/polypeptide comprises an epitope tag, e.g. a His, (e.g. 6XHis (SEQ ID NO: 269)), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g. biotin, digoxigenin, dinitrophenol), optionally at the N- or C- terminus of the antigen-binding molecule/polypeptide.

In some embodiments, the antigen-binding molecule/polypeptide comprises a moiety having a detectable activity, e.g. an enzymatic moiety. Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g. beta-galactosidase), glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.

In some embodiments, the antigen-binding molecule or polypeptide of the present disclosure comprises a chemical moiety. In some embodiments, the antigen-binding molecule/polypeptide of the present disclosure is conjugated to a chemical moiety.

The chemical moiety may be a moiety for providing a therapeutic effect, i.e. a drug moiety. A drug moiety may be a small molecule (e.g. a low molecular weight (< 1000 daltons, typically between -300-700 daltons) organic compound). Drug moieties are described e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3):14 (hereby incorporated by reference in its entirety). In some embodiments, a drug moiety may be or comprise a cytotoxic agent. In some embodiments, a drug moiety may be or comprise a chemotherapeutic agent. Drug moieties include e.g. calicheamicin, DM1 , DM4, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5 and PBD. Functional properties of the antigen-binding molecules

The antigen-binding molecules described herein may be characterised by reference to certain functional properties. In some embodiments, the antigen-binding molecule described herein may possess one or more of the following properties: binds to PAI-1 ; reduces or inhibits a function of PAI-1 ; reduces or inhibits PAI-1 -mediated inhibition of plasminogen activator (PA) (e.g. tPA and/or uPA); reduces or inhibits activation of the STAT3 signalling pathway; reduces or inhibits tumour growth; increases fibrinolysis.

It will be appreciated that a given antigen-binding molecule may display more than one of the properties recited in the preceding paragraph. A given antigen-binding molecule may be evaluated for the properties recited in the preceding paragraph using suitable assays. For example, the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays. In some embodiments, the assays may be e.g. in vivo assays, i.e. performed in non-human animals. In some embodiments, the assays may be e.g. ex vivo assays, i.e. performed using cells/tissue/an organ obtained from a subject.

Where assays are cell-based assays, they may comprise treating cells with a given antigen-binding molecule in order to determine whether the antigen-binding molecule displays one or more of the recited properties. Assays may employ species labelled with detectable entities in order to facilitate their detection. Assays may comprise evaluating the recited properties following treatment of cells separately with a range of quantities/concentrations of a given antigen-binding molecule (e.g. a dilution series). Cellbased assays may be performed in the presence of PAI-1 , e.g. the cells may be contacted with PAI-1 or a composition comprising PAI-1.

Analysis of the results of such assays may comprise determining the concentration at which 50% of the maximal level of the relevant activity is attained. The concentration of a given agent at which 50% of the maximal level of the relevant activity is attained may be referred to as the ‘half-maximal effective concentration’ of the agent in relation to the relevant activity, which may also be referred to as the ‘EC50’.

Depending on the property, the EC50 may also be referred to as the ‘half-maximal inhibitory concentration’ or ‘IC50’, this being the concentration of the agent at which 50% of the maximal level of inhibition of a given property is observed.

The antigen-binding molecules described herein bind to PAI-1.

The ability of a given antigen-binding molecule to bind specifically to a given peptide/polypeptide can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:41 1-442), Bio-Layer Interferometry (BLI; see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabelled antigen-binding assay (RIA) enzyme-linked immunosorbent assay. Through such analysis binding to a given molecule can be measured and quantified. In some embodiments, the binding may be the response detected in a given assay.

In some embodiments, an antigen-binding molecule according to the present disclosure binds to PAI-1 . In some embodiments, an antigen-binding molecule according to the present disclosure binds to a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98% or >99%) amino acid sequence identity to SEQ ID NO: 250, 251 or 253. In some embodiments, an antigen-binding molecule according to the present disclosure binds to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 250, 251 or 253. In some embodiments, an antigen-binding molecule according to the present disclosure binds to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 253.

The antigen-binding molecules described herein preferably display specific binding to PAI-1 . As used herein, ‘specific binding’ refers to binding which is selective for the antigen, and which can be discriminated from non-specific binding to non-target antigen. An antigen-binding molecule/domain that specifically binds to a target molecule preferably binds the target with greater affinity, and/or with greater duration than it binds to other, non-target molecules.

In some embodiments, the extent of binding of the antigen-binding molecule to a non-target molecule is less than about 10% of the binding of the antibody to the target molecule as measured, e.g. by ELISA, SPR, BLI or by RIA. Alternatively, binding specificity may be reflected in terms of binding affinity where the antigen-binding molecule binds with an equilibrium constant (KD) that is at least 0.1 order of magnitude (i.e. 0.1 x 10ⁿ, where n is an integer representing the order of magnitude) greater than the KD of the antigen-binding molecule towards a non-target molecule. This may optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, or 2.0.

In some embodiments, the antigen-binding molecules described herein bind to PAI-1 with an affinity in the micromolar range, i.e. KD = 9.9 x 10⁴ to 1 x 10⁶ M. In some embodiments, the antigen-binding molecules described herein bind to PAI-1 with sub-micromolar affinity, i.e. KD < 1 x 10^-6 M. In some embodiments, the antigen-binding molecules described herein bind to PAI-1 with an affinity in the nanomolar range, i.e. KD = 9.9 x 10^-7 to 1 x 10^-9 M. In some embodiments, the antigen-binding molecules described herein bind to PAI-1 with sub-nanomolar affinity, i.e. KD < 1 x 10^-9 M. In some embodiments, the antigen-binding molecules described herein bind to PAI-1 with an affinity in the picomolar range, i.e. KD = 9.9 x 10¹⁰ to 1 x IO ¹² M. In some embodiments, the antigen-binding molecules described herein bind to PAI-1 with sub- picomolar affinity, i.e. KD < 1 x 10^-12 M.

In some embodiments, the antigen-binding molecules described herein bind to PAI-1 with a KD of less than 100 nM or less, preferably one of <90 nM, <80 nM, <70 nM, <60 nM <50 nM, <40 nM, <30 nM, <20 nM, <15 nM, <12.5 nM, <10 nM, <9 nM, <8 nM, <7 nM, <6 nM, <5 nM, <4 nM <3 nM, <2 nM, <1 nM, <500 pM, <400 pM, <300 pM, <200 pM, <100 pM, <50 pM, <40 pM, <30 pM, <20 pM, <10 pM or <1 pM. In some embodiments, the antigen-binding molecules described herein bind to PAI-1 with a KD of less than 5 nM, preferably one of <2 nM, <1 nM, <500 pM, <400 pM, <300 pM, <200 pM, <100 pM, <50 pM, <40 pM, <30 pM, <20 pM, <10 pM or <1 pM.

The antigen-binding molecules of the present disclosure may bind to a particular conformation of PAI-1 . In some embodiments, the antigen-binding molecule binds to active PAI-1 and/or latent PAI-1.

As used herein, ‘active PAI-1’ refers to PAI-1 in its active conformation (j.e. PAI-1 comprising an exposed reactive centre loop (RCL)). As used herein, ‘latent PAI-1’ refers to PAI-1 in its latent conformation (i.e. PAI-1 wherein, in the absence of an interaction partner, RCL insertion forms s4A). Active-to-latent transition occurs by slowly self-inserting the N-terminal part of the RCL into the core of the protein, thereby making the P1-P1 ’ bond inaccessible for interaction partners e.g. plasminogen activator). The structural conformations of PAI-1 are reviewed in Sillen and Declerck. Front Cardiovasc Med. (2020) 7:622473, which is hereby incorporated by reference in its entirety.

The ability of a given antigen-binding molecule to bind to a specific conformation of a target peptide/polypeptide can be determined by assays comprising determining the binding of the given antigen-binding molecule to a sample comprising the target peptide/polypeptide in the given conformation. Peptide/polypeptide samples of a given conformation can be prepared by methods known in the art, e.g. methods comprising size-exclusion chromatography, ion-exchange chromatography, affinity chromatography. Target peptides/polypeptides may comprise one or more mutations to stabilise a given conformation e.g. stable active PAI-1 comprises N150H, K154T, Q319L and M354I mutations (amino acid numbering is in relation to SEQ ID NO: 253). The ability of a given antigen-binding molecule to bind to a specific conformation of PAI-1 may be assessed essentially as described in Example 1 or Example 3.

The antigen-binding molecules of the present disclosure may bind to a particular region of interest of PAI- 1 . Antigen-binding molecules according to the present disclosure may bind to a linear epitope of PAI-1 , consisting of a contiguous sequence of amino acids i.e. an amino acid primary sequence). Antigenbinding molecules according to the present disclosure may bind to a conformational epitope of PAI-1 , consisting of a discontinuous sequence of amino acids of the amino acid sequence.

In some embodiments, the antigen-binding molecule binds to p-sheet A of PAI-1 . In some embodiments the antigen-binding molecule contacts p-sheet A of PAI-1 , In some embodiments, the antigen-binding molecule binds PAI-1 via contact with one or more amino acids of p-sheet A of PAI-1 . In some embodiments, the antigen-binding molecule contacts the region of PAI-1 shown in SEQ ID NO:255, 256, 257, 259 and/or 260. In some embodiments, the antigen-binding molecule binds to PAI-1 via contact with one or more amino acids of the region shown in SEQ ID NO:255, 256, 257, 259 and/or 260. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:255, 256, 257, 259 and/or 260. In some embodiments, the antigen- binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:255, 256, 257, 259 and/or 260.

In some embodiments, the antigen-binding molecule binds to the s4A groove of PAI-1 .

As used herein, the ‘s4A groove’ refers to the groove formed by s3A and s5A (SEQ ID NO:257 and 259). The location of the s4A groove is indicated in Figure 10B.

In some embodiments, the antigen-binding molecule contacts the s4A groove of PAI-1 . In some embodiments, the antigen-binding molecule binds to PAI-1 via contact with one or more amino acids of the s4A groove of PAI-1 .

In some embodiments, the antigen-binding molecule binds in proximity to the s4A groove of PAI-1 .

Herein, the area ‘proximal to’ or ‘in proximity to’ the s4A groove, refers to the region/area of PAI-1 within 10 angstroms (A) of the s4A groove, e.g. as determined by reference to the crystal structure of PAI-1 in PDB 1 A7C. For example, the area proximal to the s4A groove may comprise amino acids of s3A and s5A. For example, an antigen-binding molecule that binds in proximity to the s4A groove of PAI-1 may exhibit competitive binding with an agent known to bind to the s4A groove of PAI-1 (e.g. TM5441 , Tiplasinin, AZ3976, CDE-096).

In some embodiments, the antigen-binding molecule contacts PAI-1 in the area proximal to the s4A groove. In some embodiments, the antigen-binding molecule binds to PAI-1 via contact with one or more amino acids in the area proximal to the s4A groove of PAI-1 . In some embodiments, the epitope of the antigen-binding molecule comprises or consists of one or more amino acids in the area proximal to the s4A groove of PAI-1 . In some embodiments, the antigen-binding molecule contacts the region of PAI-1 within 10 A of the s4A groove. In some embodiments, the antigen-binding molecule binds to PAI-1 via contact with one or more amino acids within 10 A of the s4A groove. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of one or more amino acids within 10 A of the s4A groove.

In some embodiments, the antigen-binding molecule binds to the region of PAI-1 shown in SEQ ID NO:257, 259, 264, 265 and/or 266.

In some embodiments, the antigen-binding molecule contacts the region of PAI-1 shown in SEQ ID NO:257, 259, 264, 265 and/or 266. In some embodiments, the antigen-binding molecule binds to PAI-1 via contact with one or more amino acids of the region shown in SEQ ID NO:257, 259, 264, 265 and/or 266. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:257, 259, 264, 265 and/or 266. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:257, 259, 264, 265 and/or 266. In some embodiments, the antigen-binding molecule contacts the region of PAI-1 shown in SEQ ID NO: 265. In some embodiments, the antigen-binding molecule binds to PAI-1 via contact with one or more amino acids of the region shown in SEQ ID NO:265. In some embodiments, the epitope of the antigenbinding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:265. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:265.

In some embodiments, the antigen-binding molecule is capable of binding the same region, or an overlapping region, of PAI-1 , to the region bound by an agent that is known to bind to the s4A groove of PAI-1 . In some embodiments, the antigen-binding molecule is capable of binding the same region, or an overlapping region, of PAI-1 , to the region bound by TM5441 , Tiplasinin, AZ3976, or CDE-096.

The IUPAC name of TM5441 (CAS No. 1190221-43-2) is 5-chloro-2-(2-(2-((3-(furan-3-yl)phenyl)amino)- 2-oxoethoxy)acetamido)benzoic acid. Tiplasinin (CAS No. 393105-53-8) is also known as tiplaxtinin or PAI-039. The IUPAC name of Tiplasinin is (1-benzyl-5-(4-(trifluoromethoxy)phenyl)-1 H-indol-3- yl)oxoacetic acid. AZ3976 (CAS No. 1418747-15-5; IUPAC name: tert-Butyl ester 3-(4-Oxo-3,4-dihydro- pyrido[2,3-d]pyrimidin-2-ylamino)-azetidine-1 -carboxylate; 1-Boc-3-(4-Oxo-3,4-dihydro-pyrido[2,3- d]pyrimidin-2-ylamino)-azetidine) is described in Fjellstrom et al., J Biol Chem. 2013 288(2):873-85, which is hereby incorporated by reference in its entirety. CDE-096 (CAS No. 1228357-04-7; IUPAC name: 3- (((3-(trifluoromethyl)phenoxy)carbonyl)amino)propane-1 ,2-diyl bis(3,4,5-trihydroxybenzoate)) is described in Li et al., Proc Natl Acad Sci USA. 2013 110(51 ):E4941-E4949, which is hereby incorporated by reference in its entirety.

The region of a given target molecule to which an antigen-binding molecule binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based ‘protection’ methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21 (3):145-156, which is hereby incorporated by reference in its entirety. In some embodiments, competition ELISA assays are carried out essentially as described in Example 3.

In some embodiments, the antigen-binding molecule is capable of binding the same region, or an overlapping region, of PAI-1 , to the region bound by an antibody comprising the VH and VL regions of an antibody as indicated in Table C(i) and/or the region bound by a nanobody comprising the VHH domain of a nanobody as indicated in Table C(ii). In some embodiments, the antigen-binding molecule is capable of binding the same region, or an overlapping region, of PAI-1 , to the region bound by antibody/nanobody clone A5, D4 or E8.

Whether a test antigen-binding molecule binds to the same or an overlapping region of a given target as a reference antigen-binding molecule can be evaluated, for example, by analysis of (I) interaction between the test antigen-binding molecule and the target in the absence of the reference binding molecule, and (II) interaction between the test antigen-binding molecule in the presence of the reference antigen-binding molecule, or following incubation of the target with the reference antigen-binding molecule. Determination of a reduced level of interaction between the test antigen-binding molecule and the target following analysis according to (II) as compared to (I) might support an inference that the test and reference antigen-binding molecule bind to the same or an overlapping region of the target. Suitable assays for such analysis include e.g. competition ELISA assays and epitope binning assays.

In some embodiments, the antigen-binding molecule reduces/inhibits interaction between PAI-1 and an interaction partner of PAI-1 .

Herein, an interaction partner for PAI-1 may be any molecule (e.g. protein/nucleic acid) with which PAI-1 interacts. An interaction partner for PAI-1 may be a protein capable of forming a complex with PAI-1 through protein-protein interaction. An interaction partner for PAI-1 may be a molecular complex (e.g. a multiprotein complex) in which one or more constituent components of the complex are capable of forming a complex with PAI-1 through protein-protein interaction. In some embodiments, an interaction partner for PAI-1 may be e.g. plasminogen activator (e.g. uPA, tPA), vitronectin, LRP1 , or uPA:uPAR complex

The ability of a given antigen-binding molecule to inhibit interaction between PAI-1 and an interaction partner of PAI-1 (e.g. PA) can be determined for example by analysis of interaction in the presence of, or following incubation of one or both of the interaction partners with, the antigen-binding molecule. An antigen-binding molecule which inhibits interaction between PAI-1 and an interaction partner of PAI-1 (e.g. plasminogen activator) is identified by the observation of a reduction/decrease in the level of interaction between the interaction partners in the presence of - or following incubation of the interaction partners with - the antigen-binding molecule, as compared to the level of interaction observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule known not to affect interaction between PAI-1 and the interaction partner of PAI-1 . Suitable analysis can be performed in vitro, e.g. using recombinant interaction partners, or using cells expressing the interaction partners. Cells expressing the interaction partners may do so endogenously, or may do so from nucleic acid introduced into the cell. For the purposes of such assays, one or both of the interaction partners and/or the antigen-binding molecule may be labelled or used in conjunction with a detectable entity for the purposes of detecting and/or measuring the level of interaction.

In some embodiments, an antigen-binding molecule according to the present disclosure reduces/inhibits interaction between PAI-1 and an interaction partner of PAI-1 (e.g. plasminogen activator) to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of interaction between PAI-1 and the interaction partner of PAI-1 observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule known not to affect between PAI-1 and the interaction partner of PAI-1 ).

In some embodiments, the antigen-binding molecule is an antagonist of PAI-1 . In some embodiments, the antigen-binding molecule is capable of inhibiting a function or process mediated by PAI-1 . As used herein, ‘inhibition’ refers to a refers to a reduction, decrease or lessening relative to a control condition. Suitable assays for investigating the function of PAI-1 are well known to the skilled person.

Assays for the identification of antigen-binding molecules capable of reducing/inhibiting a function of PAI- 1 may comprise treating cells/tissue/plasma in the presence of PAI-1 with a test antigen-binding molecule, and subsequently comparing the level of relevant function to the level observed in an appropriate control condition (e.g. untreated or control treated cells/tissues/plasma).

Antigen-binding molecules capable of reducing/inhibiting a function of PAI-1 , may be identified using assays comprising detecting a correlate of a function of PAI-1 . Such assays may comprise treating cells/tissue/plasma in the presence of PAI-1 with the test antigen-binding molecule, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for the functional consequences of an activity of PAI-1 to be observed) comparing the level of the correlate of a function of PAI-1 in such cells/tissue/plasma to the level of the correlate of the relevant function in an appropriate control condition (e.g. untreated or control treated cells/tissues/plasma).

In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing/inhibiting a function of PAI-1 to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of the relevant function observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in a given assay.

A correlate of a function of PAI-1 may be, e.g. plasminogen activity (e.g. tPA and/or uPA activity), STAT3 signalling, plasminogen to plasmin conversion, plasmin activity, fibrin degradation, cell detachment, and/or cell migration.

In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring or involving Fc-mediated function. In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 independently of Fc-mediated function. That is, in some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 in an Fc region-independent manner.

The ability of an antigen-binding molecule to inhibit a function of PAI-1 by a mechanism not requiring/involving Fc-mediated function can be evaluated e.g. by analyzing the ability of the antigenbinding molecule provided in a format lacking a functional Fc region to inhibit a function of PAI-1 . For example, the effect on a function of PAI-1 can be investigated using an antigen-binding molecule comprising a ‘silent’ Fc region (e.g. comprising LALA PG substitutions), or using an antigen-binding molecule provided in a format lacking an Fc region (e.g. nanobody, scFv, Fab etc.).

In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not involving ADCC. In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not involving ADCP. In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not involving CDC.

In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring binding of the antigen-binding molecule to an Fc receptor. In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring binding of the antigen-binding molecule to an Fey receptor. In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring binding of the antigen-binding molecule to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRllla and FcyRlllb. In some embodiments, the antigenbinding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring binding to FcyRllla. In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring binding to FcyRlla. In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring binding to FcyRllb. In some embodiments, the antigenbinding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring binding to a complement protein. In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring binding to C1q. In some embodiments, the antigen-binding molecule is able to inhibit a function of PAI-1 by a mechanism not requiring N297 glycosylation.

In some embodiments, the antigen-binding molecule reduces or inhibits PAI-1 -mediated inhibition of plasminogen activator (e.g. tissue plasminogen activator (tPA) and/or urokinase plasminogen activator (uPA).

In some embodiments, an antigen-binding molecule according to the present disclosure reduces/inhibits PAI-1 -mediated inhibition of plasminogen activator to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of PAI-1 -mediated inhibition of PA observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule known not to affect PAI-1 -mediated inhibition of plasminogen activator).

Assays for the identification of antigen-binding molecules capable of reducing/inhibiting PAI-1 -mediated inhibition of plasminogen activator may comprise treating a sample comprising plasminogen activator in the presence of PAI-1 with a test antigen-binding molecule, and subsequently comparing the level of plasminogen activator activity to the level observed in an appropriate control condition (e.g. untreated or control treated sample). PAI-1 -mediated inhibition of plasminogen activator can be measured using e.g. an enzymatic assay. These are well known to those skilled in the art and commercial kits are available. In some embodiments, PAI-1 -mediated inhibition of plasminogen activator can be measured essentially as described in Example 3.

In some embodiments, the antigen-binding molecule reduces or inhibits PAI-1 -mediated activation of the STAT3 signalling pathway.

In some embodiments, an antigen-binding molecule according to the present disclosure reduces/inhibits PAI-1 -mediated activation of the STAT3 signalling pathway to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of PAI-1 -mediated activation of the STAT3 signalling pathway observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule known not to affect PAI-1 -mediated activation of the STAT3 signalling pathway).

Assays for the identification of antigen-binding molecules capable of reducing/inhibiting PAI-1 -mediated activation of the STAT3 signalling pathway may comprise treating cells/tissue in the presence of PAI-1 with a test antigen-binding molecule, and subsequently comparing the level of activation of the STAT3 signalling pathway to the level observed in an appropriate control condition (e.g. untreated or control treated cells/tissues).

PAI-1 -mediated activation of the STAT3 signalling pathway can be assessed using e.g. western blot, ELISA assay e.g. to detect phosphorylated STAT3. In some embodiments, PAI-1 -mediated activation of STAT3 signalling pathway is measured essentially as described in Example 4.2.

In some embodiments, an antigen-binding molecule according to the present disclosure reduces/inhibits growth of a cancer and/or of a tumour of a cancer. In some embodiments, the antigen-binding molecule displays anticancer activity. In some embodiments, the antigen-binding molecule reduces the growth/proliferation of cancer cells. In some embodiments, the antigen-binding molecule reduces the survival of cancer cells. In some embodiments, the antigen-binding molecule reduces cell viability of cancer cells. In some embodiments, the antigen-binding molecule reduces tumour viability of a tumour. In some embodiments, the antigen-binding molecule of the present disclosure causes a reduction in the number of cancer cells e.g. in vivo. In some embodiments, the antigen-binding molecule according to the present disclosure reduces/inhibits tumour growth in the presence of ascites e.g. ascites characterised by the presence of PAI-1 .

The antigen-binding molecule of the present disclosure may be analysed for the properties described in the preceding paragraph in appropriate assays. Such assays include e.g. cell-based assays (e.g. performed essentially as described in Example 4.1 herein), in vivo models (e.g. performed essentially as described in Example 4.3 herein), and explant tumour models (e.g. performed essentially as described in Example 4.4 herein).

Assays may comprise exposing/contacting cells in cell culture to cell-free ascites. This refers to contacting cells in vitro to cell-free ascites fluid e.g. obtained from a subject. The ascites/cell-free ascites may be characterised by the presence of PAI-1 . Such assays may comprise detecting changes in biomarker levels and/or observing the overall change in the molecular or physical phenotype of cells.

As used herein, ‘cell-free ascites’ refers to the supernatant component of ascites e.g. ascites derived from a subject, e.g. a cancer patient. Methods for the preparation of cell-free ascites are well known to the skilled person. For example, cell-free ascites may be prepared by a method comprising collecting ascites from the peritoneal cavity of a subject (e.g. at the beginning of cytoreductive surgery (CRS) or during an ascitic tap (paracentesis)) and centrifuging the ascites to separate the cellular component from the fluid component. The method may further comprise sterilisation of the fluid component (e.g. filter sterilisation).

In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing/inhibiting the level of cancer cell proliferation to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of cancer cell proliferation observed in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence tumor growth), in a given assay.

In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing/inhibiting tumor growth (e.g. in an in vivo model) to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the tumor growth observed in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence tumor growth), in a given assay.

In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing the cell viability of cancer cells/tumour viability (e.g. in an explant tumour model) to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the cell viability of cancer cells/tumour viability observed in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence cancer cell/tumour viability), in a given assay. In some embodiments, an antigen-binding molecule according to the present disclosure increases the rate/level of fibrinolysis.

In some embodiments, the antigen-binding molecule of the present disclosure is capable of increasing the rate/level of fibrinolysis to more than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the rate/level of fibrinolysis observed in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence fibrinolysis), in a given assay.

Assays for the identification of antigen-binding molecules capable of increasing the rate/level of fibrinolysis may comprise treating blood/plasma in the presence of PAI-1 with a test antigen-binding molecule, and subsequently comparing the rate/level of fibrinolysis observed in an appropriate control condition (e.g. untreated or control treated blood/plasma). Assays for the identification of antigen-binding molecules capable of increasing the rate/level of fibrinolysis are known to the skilled person and are reviewed in Longstaff, Journal of Thrombosis and Haemostasis (2018) 16:652-662. Such assays may be performed, e.g. essentially as described in Example 5 herein.

Nucleic acids and vectors

The present disclosure provides a nucleic acid, or a plurality of nucleic acids, encoding an antigen-binding molecule or polypeptide according to the present disclosure. In some embodiments, the nucleic acid(s) comprise or consist of DNA and/or RNA.

An antigen-binding molecule or polypeptide according to the present disclosure may be produced within a cell by translation of RNA encoding the polypeptide(s). An antigen-binding molecule or polypeptide according to the present disclosure may be produced within a cell by transcription from nucleic acid encoding the polypeptide(s), and subsequent translation of the transcribed RNA.

In some embodiments, the nucleic acid(s) may be, or may be comprised/contained in, a vector, or a plurality of vectors. A ‘vector’ as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.

Accordingly, the present disclosure also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present disclosure. The vector may facilitate delivery of the nucleic acid(s) encoding a polypeptide according to the present disclosure to a cell. The vector may be an expression vector comprising elements required for expressing a polypeptide according to the present disclosure. The vector may comprise elements facilitating integration of the nucleic acid(s) into the genomic DNA of cell into which the vector is introduced. Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

A vector may be a vector for expression of the nucleic acid in the cell (i.e. an expression vector). Such vectors may include a promoter sequence operably linked to a nucleotide sequence encoding an antigenbinding molecule or polypeptide according to the present disclosure. A vector may also include a termination codon (i.e. 3’ in the nucleotide sequence of the vector to the nucleotide sequence encoding the polypeptide(s)) and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

The term ‘operably linked’ may include the situation where nucleic acid encoding a polypeptide according to the present disclosure and regulatory nucleic acid sequence(s) (e.g. a promoter and/or enhancers) are covalently linked in such a way as to place the expression of the nucleic acid encoding a polypeptide under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into the desired polypeptide(s).

Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g. conjugative plasmids (e.g. F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno- associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, which are both hereby incorporated by reference in their entirety. In some embodiments, a vector according to the present disclosure is a lentiviral vector.

In some embodiments, the vector may be a eukaryotic vector, i.e. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

Constituent polypeptides of an antigen-binding molecule according to the present disclosure may be encoded by different nucleic acids of the plurality of nucleic acids, or by different vectors of the plurality of vectors. Cells comprisinq/expressinq the antigen-binding molecules and polypeptides

The present disclosure also provides a cell comprising or expressing an antigen-binding molecule or polypeptide according to the present disclosure. Also provided is a cell comprising or expressing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure.

It will be appreciated that where cells are referred to herein in the singular ( .e. ‘a/the cell’), pluralities/populations of such cells are also contemplated.

The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).

In some embodiments, the cell is, or is derived from, a cell type commonly used for the expression of polypeptides for use in therapy in humans. Exemplary cells are described e.g. in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (hereby incorporated by reference in its entirety), and include e.g. CHO, HEK 293, PER.C6, NSO and BHK cells. In preferred embodiments, the cell is, or is derived from, a CHO cell.

The cell may be a prokaryotic cell, e.g. a bacterial cell. The bacterial cell may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli.

The present disclosure also provides a method for producing a cell comprising a nucleic acid(s) or vector(s) according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure into a cell. In some embodiments, introducing an isolated nucleic acid(s) or vector(s) according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction).

The present disclosure also provides a method for producing a cell expressing/comprising an antigenbinding molecule or polypeptide according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure in a cell. In some embodiments, the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the methods are performed in vitro.

The present disclosure also provides cells obtained or obtainable by the methods according to the present disclosure. Producing the antigen-binding molecules and polypeptides

Antigen-binding molecules and polypeptides according to the present disclosure may be prepared according to methods for the production of polypeptides known to the skilled person.

Antigen-binding molecules and polypeptides may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, peptides/polypeptides can be synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.

Alternatively, antigen-binding molecules and polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety. Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461 , both of which are hereby incorporated by reference in their entirety.

In some cases, the antigen-binding molecules of the present disclosure are comprised of more than one polypeptide chain. In such cases, production of the antigen-binding molecule may comprise transcription and translation of more than one polypeptide, and subsequent association of the polypeptide chains to form the antigen-binding molecule.

For recombinant production according to the present disclosure, any cell suitable for the expression of polypeptides may be used. The cell may be a prokaryote or eukaryote. In some embodiments, the cell is a prokaryotic cell, such as a cell of archaea or bacteria. In some embodiments, the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia cell. In some embodiments, the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. a cell described hereinabove.

In some cases, the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells. In addition, very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags. Specific plasmids may also be utilised which enhance secretion of the protein into the media.

In some embodiments polypeptides may be prepared by cell-free-protein synthesis (CFPS), e.g. according to a system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431 , which is hereby incorporated by reference in its entirety.

Production may involve culture or fermentation of a eukaryotic or prokaryotic cell modified to express the polypeptide(s) of interest. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air/oxygen and/or growth factors. Secreted proteins can be collected by partitioning culture media/fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted polypeptide(s). Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).

Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.

Following culturing the cells that express the polypeptide(s), the polypeptide(s) of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide, it may be necessary to separate the cells from nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated by centrifugation from the culture media that contains the secreted polypeptide(s) of interest. If the polypeptide(s) of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e.g. by sonification, rapid freeze-thaw or osmotic lysis.

It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins.

Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation or may be performed subsequently to precipitation.

Once the polypeptide(s) of interest have been isolated from culture it may be desired or necessary to concentrate the polypeptide(s). A number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.

Compositions

The present disclosure also provides compositions comprising the antigen-binding molecules, polypeptides, nucleic acids, expression vectors and/or cells described herein. The antigen-binding molecules, polypeptides, nucleic acids, expression vectors and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. Thus, the present disclosure also provides a pharmaceutical composition/medicament comprising an antigen-binding molecule, polypeptide, nucleic acid/plurality, expression vector/plurality or cell described herein.

The compositions of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents/excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).

The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

Compositions may be formulated for topical, parenteral, systemic, intracavitary, intraperitoneal, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration. In some embodiments, a pharmaceutical composition/medicament may be formulated for administration by injection or infusion, or administration by ingestion.

Suitable formulations may comprise the relevant article in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body. In some embodiments, the composition is formulated for injection or infusion, e.g. into a blood vessel, tissue/organ of interest.

The present disclosure also provides methods for the production of pharmaceutically-useful compositions and medicaments. Such methods may comprise one or more steps selected from: producing an antigenbinding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; isolating an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; and/or mixing an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent.

For example, a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease/condition (e.g. a disease/condition described herein), the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

Therapeutic and prophylactic applications

The antigen-binding molecules, polypeptides, nucleic acids, expression vectors, cells, and compositions described herein find use in therapeutic and prophylactic methods.

The present disclosure provides an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of treating or preventing a disease or condition described herein. Also provided is the use of an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

The articles, methods and uses of the present disclosure may be effective to reduce the development or progression of a disease/condition, alleviation of the symptoms of a disease/condition or reduction in the pathology of a disease/condition. The articles, methods and uses may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease/condition. In some embodiments, the articles, methods and uses may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition. In some embodiments, the articles, methods and uses may prevent development of the disease/condition a later stage (e.g. a chronic stage or metastasis).

It will be appreciated that the articles of the present disclosure may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from a reduction in the level/activity of PAI-1 .

For example, the disease/condition may be a disease/condition in which PAI-1 is pathologically- implicated, e.g. a disease/condition for which PAI-1 is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition, or for which PAI-1 is a risk factor for the onset, development or progression of the disease/condition.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by an increase in the level of expression or activity of PAI- 1 , e.g. as compared to the level of expression/activity in the absence of the disease/condition.

Treatment in accordance with the methods of the present disclosure may achieve a reduction in the activity of PAI-1 in a subject (e.g. as compared to an equivalent untreated subject, or a subject treated with an appropriate control).

Diseases in which PAI-1 is pathologically implicated include: cancer, obesity, inflammation, fibrosis, glomerulonephritis, metabolic syndrome, sarcopenia, cardiovascular disease, atherosclerosis, diabetes, stroke, Werner syndrome, major depressive disorders (MDD), and aging.

The role of PAI-1 in disease is reviewed in Placencio et al., Cancer Res. (2015) 75(15):2969-74, Cesari et al., Cardiovasc Ther. (2010) 28(5):e72-91 , and Vaughan et al., Arterioscler Thromb Vase Biol. (2017) 37(8):1446-52, each of which are hereby incorporated by reference in their entirety.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition in which PAI-1 -mediated signalling is pathologically-implicated, e.g. a disease/condition for which PAI- 1 -mediated signalling is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition, or for which PAI-1 -mediated signalling is a risk factor for the onset, development or progression of the disease/condition.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by an increased level of PAI-1 -mediated signalling, e.g. as compared to the level of PAI-1 -mediated signalling in the absence of the disease/condition. Treatment in accordance with the methods of the present disclosure may achieve a reduction in the level of PAI-1 -mediated signalling in a subject (e.g. as compared to an equivalent untreated subject, or a subject treated with an appropriate control).

PAI-1 -mediated signalling may lead to a downstream increase in STAT3 activation/signalling. In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition in which STAT3 activation/signalling (e.g. PAI-1 -mediated STAT3 activation/signalling) is pathologically-implicated, e.g. a disease/condition for which STAT3 activation/signalling is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition, or for which STAT3 activation/signalling is a risk factor for the onset, development or progression of the disease/condition.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by an increased level of STAT3 activation/signalling (e.g. PAI-1 -mediated STAT3 activation/signalling), e.g. as compared to the level of STAT3 activation/signalling in the absence of the disease/condition.

Treatment in accordance with the methods of the present disclosure may achieve a reduction in the level of STAT3 activation/signalling (e.g. PAI-1 -mediated STAT3 activation/signalling) in a subject (e.g. as compared to an equivalent untreated subject, or a subject treated with an appropriate control).

In some aspects and embodiments, the articles of the present disclosure are provided for the treatment/prevention of cancer. Cancer may refer to any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues/cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileumjejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, and/or white blood cells.

In some embodiments, the cancer is melanoma, mesothelioma, lymphoma, myeloma, leukemia, NonHodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, colorectal cancer, ovarian cancer, liver cancer, bladder cancer, renal cancer, endometrial cancer, pancreatic cancer, oral cancer (e.g. oral squamous cell carcinoma), thyroid cancer, NSCLC, hematologic cancer, sarcoma, glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma or oligodendroglioma. In some embodiments, the cancer is colorectal cancer, gastric cancer, or ovarian cancer.

In some embodiments, the cancer is associated with ascites. Ascites is the pathological accumulation of fluid within the abdominal cavity. Ascites may also be called malignant ascites and if often a sign of advanced cancer. The most common cancers associated with ascites are adenocarcinomas of the ovary, breast, colon, stomach and pancreas. Ascites may be caused e.g. by peritoneal metastases, malignant obstruction of draining lymphatics, portal vein thrombosis, elevated portal venous pressure from cirrhosis, congestive heart failure, constrictive pericarditis, nephrotic syndrome and peritoneal infections. The pathophysiology and management of ascites is reviewed in Sangisetty and Miner, World J Gastrointest Surg. (2012) 4(4):87-95, and Saif et al., Ann Saudi Med. (2009) 29(5):369-377, which are hereby incorporated by reference in their entirety.

In some embodiments, the cancer is associated with ascites characterised by the presence of PAI-1 . That is, the ascites comprise PAI-1 .

In some embodiments, the cancer associated with ascites is selected from: ovarian, endometrial, breast, esophageal, gastric, colorectal, lung, pancreatic, hepatobilliary and peritoneal (e.g. primary peritoneal) cancer. In some embodiments, the cancer associated with ascites is selected from: colorectal, gastric, and ovarian cancer.

The cancer may be a cancer of the abdomen. The cancer may be peritoneal cancer, a cancer of the peritoneum, or a cancer associated with the peritoneum. The cancer may be a cancer characterised by tumour(s) in the peritoneum. In some embodiments, the cancer is peritoneal metastasis.

Peritoneal metastases (PM) affect the peritoneum (the thin membrane surrounding the abdominal organs). Peritoneal metastasis may also be referred to as peritoneal carcinomatosis (PC) or peritoneal surface malignancy. Peritoneal metastases are reviewed in Coccolini et al., World J Gastoenterol. (2013) 19(41 ):6979-6994, which is hereby incorporated by reference in its entirety.

Peritoneal metastases may be divided into primary and secondary types.

Primary peritoneal carcinoma refers to the de novo origin of cancer in the mesothelium of the abdomen (j.e. the peritoneum).

Secondary peritoneal metastasis (or metastatic peritoneal cancer) refers to the dissemination of tumour cells in the peritoneal cavity from other sites. Secondary peritoneal metastases arise commonly from malignancies involving gastrointestinal and gynaecological structures. The metastasis occurs via transcoelomic, vascular, or lymphatic routes. Secondary peritoneal metastases are commonly caused by invading malignant cells from tumours involving the stomach, colon, pancreas, gall bladder, appendix, breast, uterus, ovary, and lungs. In some cases, the origin of the metastatic tumour can not be determined. The peritoneal involvement in appendiceal cancer is called pseudomyxoma peritonei (PMP). Metastasis from ovarian, gastric, and colorectal malignancies is associated with increased chances of recurrence and fatality, and they are also the three most common etiologies of metastatic spread in the peritoneum.

Peritoneal metastases are commonly associated with ascites. Peritoneal metastases may comprise distinct, independent deposits of tumour bathed with ascitic fluid within the abdominal cavity. Ascites are among the most common symptoms for presentation of peritoneal metastases, with non-specific abdominal symptoms and ascites occurring in 85% of patients with peritoneal metastases (Bhuyan et al., Arch Gynecol Obstet. (2010) 281 (3):561-5).

Ascites from ovarian and gastric peritoneal metastases have been found to contain elevated pro- tumorigenic factors such as IL-6, IL-10, osteoprotegerin (OPG), vascular endothelial growth factor (VEGF), progastricin (PGC) and periostin (POSTN). Ascites from peritoneal metastases have also been found to contain PAI-1 (Hendrikson et al., Cell Rep Med. (2022) 3:100526). The presence of gross ascites in a cohort of 121 patients undergoing cytoreductive surgery (CRS) having peritoneal metastases was linked to a much poorer overall survival. Data suggest that ascites contains biologically active ligands capable of supporting cellular functions of cancer cells, thus potentially explaining their link to poor outcomes. Following exposure to peritoneal metastasis ascites, cancer cells showed enrichment of signatures representing angiogenesis and epithelial-mesenchymal transition (EMT) along with upregulation of mTORCI and STAT3 signalling (Hendrikson et al., Cell Reports Medicine (2022) 3:100526).

In some embodiments, the peritoneal metastasis is primary peritoneal carcinoma. In some embodiments, the peritoneal metastasis is secondary peritoneal metastasis.

In some embodiments, the peritoneal metastasis is selected from: colorectal peritoneal metastasis, small bowel peritoneal metastasis, mesothelioma, endometrial peritoneal metastasis, gastric peritoneal metastasis, ovarian peritoneal metastasis, appendiceal peritoneal metastasis, pancreatic peritoneal metastasis, urothelial peritoneal metastasis, Pseudomyxoma peritonei (PMP), breast peritoneal metastasis, esophageal peritoneal metastasis, lung peritoneal metastasis, hepatobilliary peritoneal metastasis, peritoneal metastasis of unknown origin, and primary peritoneal carcinoma. In some embodiments, the peritoneal metastasis is selected from: colorectal peritoneal metastasis, gastric peritoneal metastasis, and ovarian peritoneal metastasis. In some embodiments, the peritoneal metastasis is colorectal peritoneal metastasis.

As used herein, reference to ‘peritoneal metastasis’ preceded by a cancer subtype (e.g. colorectal peritoneal metastasis, ovarian peritoneal metastasis, etc.) refers to secondary peritoneal metastasis caused by a cancer of that subtype which has metastasised to the peritoneum. For example, ‘colorectal peritoneal metastasis refers to peritoneal metastasis caused by a colorectal cancer which has metastasised to the peritoneum.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition described in WO 2020/197505 A1 , which is hereby incorporated by reference in its entirety.

In some embodiments, the disease/condition to be treated is a cancer associated with ascites. In some embodiments the cancer is characterised by high STAT3 activation. In some embodiments the cancer is characterised by low STAT3 activation. In some embodiments, the cancer is characterised by high PAI-1. In some embodiments, the cancer is characterised by low PAI-1 .

In some preferred embodiments, the disease/condition to be treated is a cancer associated with ascites wherein the cancer is characterised by high STAT3 activation and high PAI-1. In some preferred embodiments, the disease/condition to be treated is a cancer associated with ascites wherein the cancer is characterised by high STAT3 activation and low PAI-1 .

PAI-1 within the ascites can lead to STAT3 activation in cancer cells when these cancer cells are exposed to ascites, culminating in an epithelial-mesenchymal transition (EMT) phenotype that is responsible for the clinical manifestation of a biological aggressive tumour, leading to poor prognosis in these patients. Ascites are thought to activate STAT3 signalling in a non-canonical fashion i.e. not in the canonical fashion comprising JAK activation.

An ’increased’ or ‘high’ level of PAI-1 or level of STAT3 activation in accordance with the present disclosure refers to a level of PAI-1 /level of STAT3 activation which is greater than a reference value for the level of PAI-1 /level of STAT3 activation. In some embodiments, an ‘increased’ or ‘high’ level of PAI- 1 /level of STAT3 activation may be more than 1 times, e.g. >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the reference value. Conversely, a ‘reduced’ or ‘low’ level of PAI-1 /level of STAT3 activation in accordance with the present disclosure refers to a level of PAI-1 /level of STAT3 activation, which is less than a reference value for the level of PAI-1 /level of STAT3 activation. In some embodiments, a ‘reduced’ or ‘low’ level of PAI-1 /level of STAT3 activation may be less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the reference value.

The reference value in accordance with the preceding paragraph may be the average (e.g. the mean) value for the level of PAI-1 /level of STAT3 activation, in the context of the relevant disease/condition. In some embodiments, the reference value may be the average (e.g. the mean) value for the level of PAI- 1 /level of STAT3 activation in a tissue/organ affected by the disease/condition. In some embodiments, the reference value may be the average (e.g. the mean) value for the level of PAI-1 /level of STAT3 activation in cancers (j.e. in general), a representative subset of cancers, a given type of cancer, or a given type of tumour. In some embodiments, the reference value may be the average (e.g. the mean) value for the level of PAI-1 in ascites associated with the relevant disease/condition (e.g. ascites associated with cancer, e.g. peritoneal metastasis).

In some embodiments, a high level of PAI-1 is a concentration of > 20 ng/ml PAI-1 , e.g. as measured in a cell-free ascites sample. In some embodiments, a low level of PAI-1 is a concentration of < 20 ng/ml PAI- 1 , e.g. as measured in a cell-free ascites sample.

In some embodiments, a high level of STAT3 activation corresponds to a concentration of phosphorylated STAT3 of > 0.2 as measured at an optical density of 450 nm (OD450) in an ELISA assay. In some embodiments, a low level of STAT3 activation corresponds to a concentration of phosphorylated STAT3 of < 0.2 as measured at an optical density of 450 nm (OD450) in an ELISA assay.

The level of PAI-1 as referred to hereinabove may be determined by analysis by an appropriate method, e.g. by ELISA assay using antibodies providing for the detection of PAI-1 . The level of STAT3 activation as referred to hereinabove may be determined by analysis by an appropriate method, e.g. by ELISA assay using antibodies providing for the detection of phosphorylated STAT3.

The level of STAT3 activation as referred to hereinabove may also be determined using one or more correlates/surrogates/biomarkers of STAT3 activation. In some embodiments, STAT3 activation can be assessed by one or more of IL6, IL10, CCL2, MMP9, ANGPT1 , TGFB1 , POSTN, VSIG4, CD44 and CXCL10. In some embodiments, STAT3 activation can be assessed by one or more correlates/surrogates/biomarkers of STAT3 activation described in WO 2020/197505 A1 , which is hereby incorporated by reference in its entirety. In some embodiments, STAT3 activation can be assessed by one or more correlates/surrogates/biomarkers of STAT3 activation in ascites associated with the relevant disease/condition (e.g. ascites associated with cancer, e.g. peritoneal metastasis).

In some embodiments, a subject may be selected for treatment described herein based on the level of STAT3 activation and/or the level of PAI-1 e.g. in the organ/tissue that is affected by the disease/condition, in a tumour, or in ascites. In some embodiments, a subject may be selected for treatment described herein based on the determination that the subject has an increased/high level of STAT3 activation and/or an increased/high level of PAI-1 e.g. e.g. in the organ/tissue that is affected by the disease/condition, or in a tumour. In some embodiments, a subject may be selected for treatment described herein based on the determination that the subject has an increased/high level of STAT3 activation and an increased/high level of PAI-1 . In some embodiments, a subject may be selected for treatment described herein based on the determination that the subject has an increased/high level of STAT3 activation and/or a decreased/low level of PAI-1 . In some embodiments, the disease/condition to be treated is a cancer as stratified/identified by criteria described in WO 2020/197505 A1 , which is hereby incorporated by reference in its entirety.

It will be appreciated that the articles of the present disclosure may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from an increase in the level/activity of plasminogen activator (e.g. tissue type plasminogen activator (tPA) and/or urokinase type plasminogen activator (uPA)).

For example, the disease/condition may be a disease/condition in which reduced expression or activity of plasminogen activator (e.g. tPA and/or uPA) is pathologically-implicated, e.g. a disease/condition for which reduced expression or activity of plasminogen activator is associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition, or for which reduced expression or activity of plasminogen activator is a risk factor for the onset, development or progression of the disease/condition.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by a reduced level of expression or activity of plasminogen activator (e.g. tPA and/or uPA)), e.g. as compared to the level of expression/activity in the absence of the disease/condition.

Treatment in accordance with the methods of the present disclosure may achieve an increase in the activity of plasminogen activator (e.g. tPA and/or uPA) in a subject (e.g. as compared to an equivalent untreated subject, or a subject treated with an appropriate control).

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition in which PAI-1 -mediated inhibition of plasminogen activator (e.g. tPA and/or uPA) is pathologically-implicated, e.g. a disease/condition for which PAI-1 -mediated inhibition of plasminogen activator is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition, or for which PAI-1 - mediated inhibition of plasminogen activator is a risk factor for the onset, development or progression of the disease/condition.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by an increased level of PAI-1 -mediated inhibition of plasminogen activator (e.g. tPA and/or uPA), e.g. as compared to the level of PAI-1 -mediated inhibition of plasminogen activator in the absence of the disease/condition.

Treatment in accordance with the methods of the present disclosure may achieve a reduction in the level of PAI-1 -mediated inhibition of plasminogen activator (e.g. tPA and/or uPA) in a subject (e.g. as compared to an equivalent untreated subject, or a subject treated with an appropriate control). In some aspects and embodiments, articles of the present disclosure are provided for the treatment/prevention of coagulation, particularly pathological coagulation or hypercoagulation.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition in which coagulation is pathologically-implicated, e.g. a disease/condition for which coagulation is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition, or for which coagulation is a risk factor for the onset, development or progression of the disease/condition.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by an increased level/rate of coagulation, e.g. as compared to the level/rate of coagulation in the absence of the disease/condition. In some embodiments, the disease/condition is characterised by a decreased level/rate of fibrinolysis e.g. as compared to the level/rate of fibrinolysis in the absence of the disease/condition.

Treatment in accordance with the methods of the present disclosure may achieve a reduction in the level/rate of coagulation and/or an increase in the level/rate of fibrinolysis in a subject (e.g. as compared to an equivalent untreated subject, or a subject treated with an appropriate control).

As used herein, ‘coagulation’ refers to coagulation of the blood, i.e. the formation of blood clots. Diseases may be associated with excessive coagulation and/or aberrantly activated coagulation. As used herein, excessive and/or aberrantly activated coagulation may be referred to as ‘pathological coagulation’ or ‘hypercoagulation’. Pathological coagulation/hypercoagulation may refer to coagulation which is implicated in (i.e. which positively contributes to) the pathology of a disease.

As used herein, ‘fibrinolysis’ refers to the dissolution of blood clots.

Haemostasis is an essential physiological process that preserves the integrity of the vascular system and secures sufficient blood flow throughout the circulatory system. The balance between clot formation (coagulation) and clot dissolution (fibrinolysis) is very tightly regulated in a spatiotemporal manner and requires a dynamic interplay with other systems involved, such as the vascular system and platelets. Briefly, upon vascular injury, a sequence of cellular and molecular events is triggered that can be characterized by three distinct but overlapping phases of initiation, amplification, and propagation (coagulation). The end result of the coagulation cascade is the conversion of fibrinogen, a soluble plasma protein, into an insoluble fibrin meshwork that constitutes blood clots. To limit the coagulatory response to the site of injury and prevent vascular occlusion, the prothrombotic response is balanced by the fibrinolytic system.

Fibrinolysis revolves around the enzymatic activation of plasminogen into the key fibrinolytic enzyme plasmin through tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PAs). Tissue-type PA is produced by vascular endothelial cells and released in response to thrombin and venous occlusion. It is primarily involved in the activation of plasminogen that is required for fibrin dissolution in the circulation. In contrast, uPA is expressed by a variety of cells, including renal epithelial cells, inflammatory cells, and cancer cells. It is considered more important in pericellular proteolysis during tissue remodeling and cell migration through the activation of cell-bound plasminogen. PAI-1 is the primary inhibitor of tPA and uPA and so is a key component of the plasminogen/plasmin system. The role of PAI-1 in hemostasis and cardiovascular disease is reviewed in Sillen and Declerk, Front Cardiovasc Med. (2020) 7:622473.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by one or more of the following: reduced expression or activity of plasminogen activator (e.g. tPA and/or uPA), increased PAI-1 -mediated inhibition of plasminogen activator (e.g. tPA and/or uPA), reduced level/rate of coagulation, increased level/rate of fibrinolysis, reduced plasminogen to plasmin conversion, reduced plasmin activity, and reduced fibrin degradation.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by the presence/formation of blood clots, e.g. blood clots characterised by thrombosis/thromboembolism. Treatment in accordance with the methods of the present disclosure may promote/potentiate fibrinolysis/thrombolysis. An agents (e.g. an antigen-binding molecule) of the present disclosure may find utility as a fibrinolytic/thrombolytic agent. The present disclosure provides an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use as a fibrinolytic/thrombolytic agent.

The disease/condition to be treated/prevented in accordance with the present disclosure may be selected from: thrombosis, e.g. deep vein thrombosis (DVT), portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, cerebral venous sinus thrombosis, thrombotic stroke; myocardial infarction; antiphospholipid syndrome (APS); disseminated intravascular coagulation (DIC); activated protein C resistance, e.g. Factor V Leiden; and cancer.

In some embodiments, the articles of the present disclosure may be administered topically, parenterally, or systemically. In some embodiments, the articles of the present disclosure may be administered by a intraperitoneal, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, subcutaneous, intradermal, intrathecal, oral or transdermal route of administration. In some embodiments, the articles of the present disclosure may be administered intraperitoneally. In some embodiments, the articles of the present disclosure may be administered intravenously.

Methods of detection

The present disclosure also provides the articles of the present disclosure for use in methods for detecting, localising or imaging PAI-1. The antigen-binding molecules and compositions described herein may be used in methods that involve detecting binding of the antigen-binding molecule to PAI-1. Such methods may involve detection of the bound complex of an antigen-binding molecule and PAI-1 .

As such, a method is provided, comprising contacting a sample containing, or suspected to contain, PAI-1 with an antigen-binding molecule/combination/composition according to the present disclosure, and detecting the formation of a complex of the antigen-binding molecule and PAI-1. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell comprising PAI-1 with an antigen-binding molecule/composition according to the present disclosure, and detecting the formation of a complex of the antigen-binding molecule and PAI-1 .

Suitable method formats are well known in the art, including immunoassays such as sandwich assays, e.g. ELISA. The methods may involve labelling the antigen-binding molecule, or target(s), or both, with a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label, radiolabel, chemical, nucleic acid or enzymatic label as described herein. Detection techniques are well known to those of skill in the art and can be selected to correspond with the labelling agent.

Methods comprising detecting PAI-1 , or cells comprising PAI-1 , include methods for diagnosing/prognosing a disease/condition described herein.

Methods of this kind may be performed in vitro on a patient sample, or following processing of a patient sample. Once the sample is collected, the patient is not required to be present for the in vitro method to be performed, and therefore the method may be one which is not practised on the human or animal body. In some embodiments, the method is performed in vivo.

Such methods may involve detecting or quantifying PAI-1 and/or cells comprising PAI-1 , e.g. in a patient sample. Where the method comprises quantifying PAI-1 , the method may further comprise comparing the determined amount against a standard or reference value as part of the diagnostic or prognostic evaluation. Other diagnostic/prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis or to confirm a result obtained by using the tests described herein.

Detection in a sample may be used for the purpose of diagnosis of a disease/condition (e.g. cancer), predisposition to a disease/condition, or for providing a prognosis (prognosticating) for a disease/condition, e.g. a disease/condition described herein. The diagnosis or prognosis may relate to an existing (previously diagnosed) disease/condition.

A sample may be taken from any tissue or bodily fluid. The sample obtained from a subject may be of any kind. A biological sample may be taken from any tissue or bodily fluid, e.g. a blood sample, blood-derived sample, serum sample, lymph sample, semen sample, saliva sample, synovial fluid sample, ascites. A blood-derived sample may be a selected fraction of a patient’s blood, e.g. a selected cell-containing fraction or a plasma or serum fraction. A sample may comprise a tissue sample or biopsy; or cells isolated from a subject.

In some embodiments, the sample may consist of, or comprise, ascites obtained from a subject. In some embodiments, the sample may be an ascites-derived sample. An ascites-derived sample may be a selected fraction of a patient’s ascites, e.g. a selected cell-containing fraction or a cell-free fraction. In some embodiments, the sample is cell-free ascites.

As used herein, ‘cell-free ascites’ refers to the supernatant component of ascites derived from a subject, e.g. a patient. Methods for the preparation of cell-free ascites are well known to the skilled person. For example, cell-free ascites may be prepared by a method comprising collecting ascites from the peritoneal cavity of a subject (e.g. at the beginning of cytoreductive surgery (CRS) or during an ascitic tap (paracentesis)) and centrifuging the ascites to separate the cellular component from the fluid component. The method may further comprise sterilisation of the fluid component (e.g. filter sterilisation).

The sample may be a sample as described in WO 2020/197505 A1 , which is hereby incorporated by reference in its entirety.

A subject may be selected for diagnostic/prognostic evaluation based on the presence of symptoms indicative of a disease/condition described herein, or based on the subject being considered to be at risk of developing a disease/condition described herein.

The present disclosure also provides methods for selecting/stratifying a subject for treatment with a PAI- 1 -targeted agent. In some embodiments a subject is selected for treatment/prevention in accordance with the methods of the present disclosure, or is identified as a subject which would benefit from such treatment/prevention, based on detection/quantification of PAI-1 , or cells comprising PAI-1 , e.g. in a sample obtained from the individual.

In some embodiments, the method for selecting/stratifying a subject for treatment with a P Al -1 -targeted agent comprises analysing the level of PAI-1 and/or the level of STAT3 activation in a sample obtained from the subject, e.g. in the organ/tissue that is affected by the disease/condition, in a tumour, or in ascites.

In some embodiments, the method for selecting/stratifying a subject for treatment with a P Al -1 -targeted agent may be a method as described in WO 2020/197505 A1 , which is hereby incorporated by reference in its entirety.

Subjects

A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals (veterinary use). The subject to be administered with an article of the present disclosure (e.g. in accordance with therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient.

A subject may have (e.g. may have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease/condition, or may be at risk of developing/contracting such a disease/condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for one or more markers of such a disease/condition.

In some embodiments, a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of PAI-1 , e.g. in a sample obtained from the subject.

Kits

The present disclosure also provides kits of parts.

In some embodiments, the kit may have at least one container having a predetermined quantity of an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell, composition or combination described herein.

In some embodiments, the kit may comprise materials for producing an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell, composition or combination described herein. In some embodiments, the kit of parts may comprise materials for formulating an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell, composition or combination described herein to a pharmaceutical composition/medicament, e.g. in a composition further comprising a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.

The kit may provide the antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell, composition or combination together with instructions for administration to a patient in order to treat a specified disease/condition (e.g. a disease/condition described herein).

In some embodiments the kit may further comprise at least one container having a predetermined quantity of another therapeutic agent (e.g. as described herein). In such embodiments, the kit may also comprise a second medicament or pharmaceutical composition such that the two medicaments or pharmaceutical compositions may be administered simultaneously or separately such that they provide a combined treatment for the specific disease/condition. Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

5 Sequence identity

As used herein, ‘sequence identity’ refers to the percent of nucleotides/amino acid residues in a subject sequence that are identical to nucleotides/amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining 0 percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When 5 using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

Sequences

able A(i) able A(ii)

78

able B(i) able B(ii)

Table C(i) Table C(ii)

Table D

***

The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, an amino acid sequence or a region of a polypeptide which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60% (e.g. one of >60%, >65%, >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, £97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of the amino acid sequence/polypeptide/region. An amino acid sequence/region/position of a polypeptide/amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence/region/position of a polypeptide/amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960). It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and/or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

Methods described herein may preferably be performed in vitro. The term ‘in vitro' is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo' is intended to encompass procedures with/on intact multi-cellular organisms.

Brief Description of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

Figure 1. Schematic overview of the PAI-1 conformations as well as its structural changes in different mechanisms.

Figures 2A to 2B. The structures of latent and active PAI-1 . (A) Schematic model of latent PAI-1 and (B) a constructed model of active PAI-1 based on the structures of both latent PAI-1 and antithrombin III with some important structural domains indicated. The protein is shown from the front with the p sheet A indicated and highlighted in dark grey; strands 3A to 5A are also indicated.

Figure 3A to 3B. Identification of the s4A groove for PAI-1 inhibition. (A) Schematics showing three peptides representing distinct regions of RCL that were designed for synthesis and plotted by UCSF Chimera. Sequences of peptides were TV ASS (peptide 1 , SEQ ID NO: 261 ), TVASSSTA (peptide 2, SEQ ID NO: 262), TVASSSTAVIVSAR (peptide 3, SEQ ID NO: 263), and VTILLVTS (peptide control with random amino acid sequences, SEQ ID NO: 270). (B) Graph showing that Peptide 3 suppressed PAI-1 function in inhibiting tPA activity. PAI-1 in active conformation was incubated with DMSO or 300 pM of each peptide at 37°C for 60 min. PAI-1 activity to inhibit tPA function was quantified by a chromogenic enzymatic assay in triplicates and normalized to the DMSO-treated samples. All statistical analyses were performed on biological triplicates using unpaired two-sided t-test with p < 0.01 (**) and p > 0.05 (ns).

Figures 4A to 4H. Validation of the s4A groove for PAI-1 inhibition. (A) Binding model of Amentoflavone (indicated by arrow) was obtained by AutoDock Vina docking algorithm and plotted by UCSF Chimera. Crystal structure of PAI-1 (PDB 1 A7C) is shown. (B) Western blots showing target validation of Amentoflavone by thermal shifting assay. PAI-1 recombinant protein in active conformation was treated with DMSO, 800 pM Amentoflavone, TM5441 , or Napabucasin and then heat-shocked to induce protein denaturation at various temperatures, followed by western blots using anti-PAI-1 antibody. Active PAI-1 was stabilised by Amentoflavone and TM5441 but not Napabucasin. (C) Graph showing inhibition of PAI- 1 function by Amentoflavone as assessed by an enzymatic assay. Significant reduction in PAI-1 activity was observed for 150 pM TM5441 , 250 pM TM5441 , and 250 pM Amentoflavone as compared to the DMSO-treated sample, suggesting that TM5441 and Amentoflavone inhibit PAI-1 ’s ability to inhibit tPA. (D,E) PAI-1 in active conformation (CPAI, Molecular Innovation) was incubated with DMSO, 500 pM TM5441 (TM), Amentoflavone (AF), or Napabucasin (NP) at 37°C for 40 min, and then incubated with human tPA (HTPA-TC, Molecular Innovation) or human uPA (UPA-HTC, Molecular Innovation) at 37°C for 30 min. Proteins were separated in SDS-PAGE under non-reducing condition and stained with Coomassie Blue R-250. Complex formation of PAI-1/tPA and PAI-1/uPA were disrupted by TM5441 and Amentoflavone. (F) Graph showing amentoflavone inhibited proliferation of colorectal PM cells treated with ascites containing high levels of PAI-1 (light grey, n=4), ascites containing low levels of PAI-1 (dark grey, n=3) and Foetal Bovine Serum (FBS, black) in biological triplicates. (G) Graph showing that cancer cells treated with patient-derived ascites enriched with PAI-1 were more sensitive to Amentoflavone inhibition than FBS-treated cancer cells. (H) Ascites displaying differential sensitivity to Amentoflavone in inhibiting proliferation of PM cancer cells was confirmed by significantly lower IC50 value in ascites containing high levels of PAI-1 compared to that of ascites containing low levels of PAI-1 . Inhibitory efficiency by Amentoflavone is comparable to that of TM5441 . All statistical analyses were performed on biological triplicates using unpaired two-sided t-test with p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p > 0.05 (ns).

Figures 5A to 5D. Graphs showing the effect of various PAI-1 inhibitors (TM5441 , Tiplaxtinin, AZ3976, and CDE-096) on ascites-treated Colo-205 cells. Representative inhibitor dose-response curves of PAI-1 paracrine addicted (PPA) group (ascites with elevated levels of PAI-1 (> 20 ng/ml ), which relied heavily on PAI-1 to activate STAT3 signalling, represented in light grey), co-activators predominant (CAP) group (PAI-1 levels below 20 ng/ml but nevertheless activated STAT3 signalling in cells exposed to these ascites, represented in mid grey) and Foetal Bovine Serum (FBS, control, black) were plotted. A left shift was observed for TM5441 and Tiplaxtinin indicating responsiveness to PAI-1 inhibition. A right shift was observed for AZ3976 and CDE-096 indicating resistance to PAI-1 inhibition in the presence of ascites compared to FBS. All statistical analyses were performed on biological triplicates using unpaired two- sided t-test with p < 0.01 (**), p < 0.001 (***) and p > 0.05 (ns).

Figures 6A to 6C. Effect of commercial antibodies targeting varying PAI-1 epitopes on STAT3 suppression in ascites-treated SNU-C1 cells: (A) 5% PC124, (B) 5% PC085, (C) 5% PC383 ascites.

Figures 7A to 7D. Evaluation of STAT3 suppression in colorectal PM cell lines upon exposure with ascites pre-treated with varying concentrations of PAI-1 antibody (MA-33H1 F7). (A and B) SNU-C1 cells treated with 5% pre-treated (A) PC99 and (B) PC 124 ascites. (C and D) Colo-205 cells treated with 5% pre-treated (C) PC99 and (D) PC124 ascites. Figures 8A to 8D. Effect of STAT3 suppression in colorectal PM cell lines upon exposure to ascites pretreated with varying concentrations of PAI-1 antibody (#242816). (A and B) SNU-C1 cells treated with 5% pre-treated (A) PC99 and (B) PC124 ascites. (C and D) Colo-205 cells treated with 5% pre-treated (C) PC99 and (D) PC 124 ascites.

Figures 9A to 9C. Effect of STAT3 suppression in colorectal PM cell lines upon exposure to ascites pretreated with varying concentrations of PAI-1 antibody (#242816) or IgG control. SNU-C1 cells treated with 5% pre-treated (A) PC11 , (B) PC139, and (C) PC322 ascites.

Figure 10A to 10F. (A) Peptides selected for screening of custom antibodies are indicated in PAI-1 protein (PDB 1 A7C), plotted by UCSF Chimera. (B) Schematic showing location of peptides on the protein surface (C) Sequences of peptides selected for screening of custom antibodies are listed (SEQ ID NO: 271-276). (D) Peptides selected for screening of antibodies by Phage Display are highlighted in PAI- 1 protein (PDB 1 A7C), plotted by UCSF Chimera. (E) Protein surface is shown. (F) Peptide sequences are listed (SEQ ID NO: 264-266).

Figures 11A to 11B. Effect of custom-made PAI-1 antibodies on STAT3 suppression in ascites-treated colorectal PM cell lines. (A) SNU-C1 cells treated with 5% PC322 ascites and IgG control or PAI-1 antibody. (B) Colo-205 cells treated with 5% PC383 ascites and IgG control or PAI-1 antibody.

Figures 12A to 12E. Target validation and functional validation of the antibodies and nanobodies. (A) Recognition of PAI-1 proteins by 15 human antibodies assessed by western blots. Active form and latent form of recombinant PAI-1 proteins were blotted with 1 pg/mL unspecific human IgG (IgG, 15154, Sigma), or 1 pg/mL discovered antibodies, followed by 1 :5,000 anti-human IgG antibody (109-035-003, Jackson ImmunoResearch). For nanobodies, blots were incubated with 5 pg/mL VHH targeting GFP (VHH, GT250, ChromoTek), or 5 pg/mL discovered nanobodies, followed by 1 :10,000 Biotin-SP Anti-His Tag (300-065-240, Jackson ImmunoResearch) and 1 :5,000 Peroxidase-Streptavidin (016-030-084, Jackson ImmunoResearch). (B) Table showing summary of results of western blots. (C) Chart showing effects of 1 pg of unspecific human IgG, 1 pg of each discovered antibody, 2.5 pg of nanobody targeting GFP, and 2.5 pg of each discovered nanobody on PAI-1 activity in inhibiting tPA function was quantified by an enzymatic activity assay kit (#K2040, Biovision). (D) Chart showing effects of 1 pg of unspecific human IgG, 1 pg of each discovered antibody, 5 pg of nanobody targeting GFP, and 5 pg of each discovered nanobody on PAI-1 activity in inhibiting uPA function was quantified by an enzymatic activity assay kit (ECM610, Merck). Significant reduction in PAI-1 activity was observed for A5 compared to IgG control and also observed for D4 and E8 compared to VHH control for both assays. All statistical analyses were performed on biological triplicates using unpaired two-sided t-test with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). (E) Confirmation ELISA of D4 and E8. NC, negative control (coating with carriers alone: BSA + OVA + KLH); * Clones having 67% amino acid identity in VHH sequence; f Clones having 78% amino acid identity in VHH sequence; $ Clones having 66% amino acid identity in VHH sequence. Figures 13A and 13B. (A) BIAcore analysis of biologies binding to recombinant PAI-1 . Antibodies were coupled to CM5 censorships using amine coupling and a serial dilution of wildtype active PAI-1 , stable active PAI-1 , and latent PAI-1 in PBST buffer passed over the sensorchip surface. Association constants (ka) and dissociation constants (kd) were measured using BIAcore T200 (GE Healthcare) by single-cycle kinetics (SCK). (B) PAI-1 proteins were coupled to CM5 censorships using amine coupling and a serial dilution of the biologies were flowed over the sensorchip surface. Kinetics parameters and affinity were measured using BIAcore K8 (GE Healthcare) by multiple-cycle kinetics (MCK) and calculated using BIAevaluation software (GE Healthcare).

Figures 14A to 14G. Graphs showing inhibition of proliferation of PM cells treated with ascites in vitro by the biologies. 5,000 Colo-205 cells were seeded in each well and incubated with 10% FBS or 5% ascites. The cells were then treated with PBS, (A) 10 pg/mL VHH targeting GFP (VHH control), 10 pg/mL D4, (C) 7.5 pg/mL VHH control, 7.5 pg/mL E8, (E) 150 pg/mL human IgG, or 150 pg/mL A5 for 72 hours in biological triplicates. Cell viability was measured using CellTiter-Glo (Promega), and shown as percentage relative to the PBS treatment. Significance level was calculated against the corresponding VHH control or IgG control under each condition. Significant reduction of cell proliferation were observed for A5, D4, and E8 in the presence of PM patient ascites. (B,D,F) Ascites with high level of PAI-1 ( (>20 ng/mL, High) were more sensitive to the inhibition of D4 and E8 compared to ascites with low level of PAI- 1 (<20 ng/mL, Low), consistent with paracrine addiction18 published previously. However, this differential sensitivity was not found for the treatment of A5. Each dot represents average of biological triplicates of one ascites treatment. (G) Levels of PAI-1 in each ascites were quantified using ELISA. All statistical analyses were performed using unpaired two-sided t-test with p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p > 0.05 (ns).

Figures 15A to 15B. Graphs showing inhibition of STAT3 signalling activation in PM cells treated with ascites by the biologies. 8 x 105 Colo-205 cells were seeded in each well and treated with 5% ascites with high level of PAI-1 or 5% ascites with low level of PAI-1 . Cells were treated with PBS vehicle (0 pg/mL), (A) 150 pg/mL human IgG antibody (negative control for A5), 150 pg/mL A5, (B) 15 pg/mL nanobody targeting GFP (VHH control for D4 and E8), 15 pg/mL D4, or 15 pg/mL E8 for 16 hours in biological duplicates. Levels of pSTAT3 and total STAT3 in cell lysates were measured using ELISA in technical duplicates and pSTAT3/STAT3 ratio was shown as percentage relative to the PBS treatment in x-axis. STAT3 signalling activation upon the exposure to ascites was suppressed by A5, D4 and E8. An additional biological replicate will be performed for statistical analyses.

Figures 16A to 16H. In vivo efficacy of biologies on PM tumour growth in the presence of ascites. (A) Timeline. 5 x 106 of Colo-205 cells were co-injected with 10% FBS (control), 5% ascites with high level of PAI-1 or 5% ascites with low level of PAI-1 into the abdominal cavity of 6- to 8-week-old BALB/c nude mice (female, n = 5 mice/group) and treated with PBS vehicle control, 17.5 pg VHH targeting GFP (VHH control) control or E8 administered intraperitoneally. Ascites and drug treatment were performed by injecting 400 pl of 5% ascites or 10% FBS with the biologies in PBS intraperitoneally every 3 days for 21 days. Tumour burden was quantified based on a modified peritoneal carcinomatosis index (PCI) score and presented as total PCI score. Total PCI score was calculated based on the sum of score for each region and ranges from 0 to 39. (B) E8 showed a trend of decreasing tumour burden in PAI-1 -high ascites (PC383 As_2) but not in FBS and PAI-1 -low ascites (PC426). (C,D,E) Bodyweight of each mouse did not decrease drastically across the treatment, except for 1 mouse treated with VHH control under 10% FBS. (F) 5x10⁶ Colo-205 cells were co-injected with ascites or FBS into the abdominal cavity of BALB/c nude mice and treated with PBS, 20 pg control nanobody (control VHH) or 20 pg E8 by intraperitoneal (i.p.) injection (n = 10 mice/group). Ascites/FBS and biologies treatment were repeated every 3 days for 21 days. Tumour burdens were assessed by modified peritoneal cancer index (PCI). In the presence of ascites (PC383 As), mice treated with E8 had significantly lower tumour burden compared to mice treated with control VHH. (G) Representative images of peritoneal metastases formed in response to E8 inhibition or control nanobody (control VHH). Grey arrows indicate visible tumours. (H) Bodyweight of each mouse was measured every 3 days. E8 treatment did not lead to a decrease in bodyweight over 21 days. All statistical analyses were performed using unpaired two-sided t-test with p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Figures 17A to 17E. Safety profile of intravenous injection of the biologies. (A) Timeline. Varying doses of A5 (50 pg - 200 pg), D4 (50 pg - 150 pg), or E8 (20 pg - 150 pg) were administered into BALB/c nude mice (n = 2 females and 2 males for each dose) via intravenous injection. PBS vehicle served as control (0 pg) for the biologies (n = 3 females and 3 males). Bodyweight and behaviour were assessed daily. (B) Bodyweight did not drop significantly for all mice. (C) Haematology counts were performed after 16h- fasting (n = 50). Counts of white blood cells, red blood cells, and platelet were shown. (D) Clinical chemistry test was performed after 16h-fasting in mice serum (n = 24). Concentration of total protein, glucose and albumin were shown. (E) Representative sections of the brain, heart, lung, kidney, liver and spleen tissues of PBS- and E8-treated mice subjected to haematoxylin and eosin (H&E) staining. No significant changes were observed.

Figure 18. Graph showing reduction of plasma clot lysis time by A5 antibody. Platelet-poor plasma pooling from 2 healthy individuals was spiked with stable mutant PAI-1 , and then incubated with PBS control, 2 pg IgG control or 2 pg A5. Subsequently, CaCI2 was added to trigger clot formation and tPA was added to induce the clot lysis. The clot status was monitored by measuring the turbidity at 405 nm, 37 °C in technical duplicates. Plasma treated with A5 showed shorter clot lysis time.

Figure 19. Ex vivo efficacy of E8 and D4 on human explant tumour models. Tumour samples from two patients with Struma Ovarii and Colorectal PM cancer were collected. Upon arrival in the lab within one hour after excision, tumour samples were immediately processed without prior freezing. Tissue fragments of approximately 2x2x2 mm were sectioned, transferred to a 24-well plate and cultured in DMEM/F12 complete medium supplemented with ROCK inhibitor for four hours prior to overnight serum-starvation. Subsequently, explants were grown in DMEM/F12 serum-free media supplemented with 5% PAI-1 -high cell-free ascites overnight before measuring the baseline viability readings (Vo) with alamarBlue (A50101 , Invitrogen). Viability (V24h) was then assessed following 24-hour incubation with 20 pg/mL E8, 20 pg/mL D4, 20 pg/mL VHH control, PBS vehicle control, 250 pM TM5441 (a PAI-1 inhibitor) or DMSO vehicle control. Relative changes in viability (Normalized Readout) were calculated as (V24h - Vo) / Vo. At least 3 human explants were tested under each condition. All statistical analyses were performed using unpaired two-tailed t-test with p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (“**), and p>0.05 (ns).

Examples

Example 1 : Materials and Methods

1.1 Complex Formation

Complex formation was investigated by incubating PAI-1 in active conformation (CPAI, Molecular Innovation) was with DMSO, 500 pM TM5441 (TM), Amentoflavone (AF), or Napabucasin (NP) at 37°C for 40 min, followed by incubated with human tPA (HTPA-TC, Molecular Innovation) or human uPA (UPA- HTC, Molecular Innovation) at 37°C for 30 min. Proteins were separated in SDS-PAGE under nonreducing conditions and stained with Coomassie Blue R-250.

1 .2 Thermal Shifting Assay

PAI-1 recombinant protein in active conformation was treated with DMSO, 800 pM Amentoflavone, TM5441 , or Napabucasin and then heat-shocked to induce protein denaturation at various temperatures, followed by western blots using anti-PAI-1 antibody.

1 .3 STAT3 suppression in ascites-treated colorectal PM cell lines

SNU-C1 cells treated with 5% pre-treated (A) PC11 , (B) PC139, and (C) PC322 ascites and IgG control or PAI-1 antibody.

1 .4 Target validation and functional validation of the antibodies and nanobodies

Recognition of PAI-1 proteins by 15 human antibodies assessed by western blots. Active form and latent form of recombinant PAI-1 proteins were blotted with 1 pg/mL unspecific human IgG (IgG, 15154, Sigma), or 1 pg/mL discovered antibodies, followed by 1 :5,000 anti-human IgG antibody (109-035-003, Jackson ImmunoResearch). For nanobodies, blots were incubated with 5 pg/mL VHH targeting GFP (VHH, GT250, ChromoTek), or 5 pg/mL discovered nanobodies, followed by 1 :10,000 Biotin-SP Anti-His Tag (300-065-240, Jackson ImmunoResearch) and 1 :5,000 Peroxidase-Streptavidin (016-030-084, Jackson ImmunoResearch).

Example 2: Identification of tarqetable site on PAI-1 for functional neutralisation

It has been previously demonstrated that ligand inhibition of PAI-1 using small molecule inhibitors (TM5441 and Tiplaxtinin) led to significant reduction of tumour development of peritoneal metastases (PM) (see WO 2020/197505 A1 ). However, there is a lack of PAI-1 inhibitors approved in clinical use.

A starting point for discovery of novel PAI-1 antibodies and/or nanobodies as drug candidates for PM treatment was identification of targetable sites of PAI-1. The insertion of the reactive centre loop (RCL) into the centre of PAI-1 to form a -sheet (s4A) has been reported to be critical for PAI-1 functions. Hence, it was hypothesized that blocking of the RCL could be critical for inhibiting the function of PAI-1 , particularly with reference to the inhibitory effect on cancer cells that are oncogenically addicted to PAI-1 in PM.

To investigate the importance of the s4A groove in inhibiting the oncogenic potential of PAI-1 in PM, the structure of PAI-1 was analysed in detail.

Figure 1 demonstrates the structural insights into PAI-1 mechanism of action as described in Sillen and Declerck. Front Cardiovasc Med. (2020) 7:622473. Figure 2 shows the schematic models of active and latent conformations of PAI-1 with the s4A structure labelled as described in Xue et al., Struct Lond Engl (1998) 6(5):627-36. PAI-1 at active conformation possesses an exposed reactive centre loop (RCL) (Figure 2B). Insertion of a portion of the RCL is observed in both active-to-latent conversion and complex formation with plasminogen activator (see “PA” in Figure 1 ). RCL insertion forms a strand between strands 3 and 5 of the antiparallel sheet A, referred to as s4A. The s4A structure is shown as a dark grey sheet in Figure 2.

Therefore, considering the critical roles of RCL insertion and the resultant s4A sheet, the groove between strands 3 and 5 of the antiparallel sheet A in active PAI-1 was identified as a candidate for blocking. It was theorised that this would block RCL insertion and thus inhibit PAI-1 function.

To test the hypothesis that this is a potential critical site amenable for PAI-1 inhibition, three peptides were synthesized representing distinct regions of the RCL (Figure 3A). Peptide 3 is distinct from peptides 1 and 2 because it occupies the C-terminus of s4A. Peptide 3 was the only peptide that was able to inhibit PAI-1 function as assessed by an enzymatic assay (PAI-1 in active conformation was incubated with DMSO or 300 pM of each peptide at 37°C for 60 min; results shown in Figure 3B). Taken together with the absence of inhibitory functions of peptides 1 and 2, this suggests that the critical component of inhibitory function resides within the C terminus of the s4A site.

To further validate that s4A is a targetable site to inhibit PAI-1 , structure-based in silico virtual screening was performed to identify hit compounds that can fit into the s4A position from a library of 2,054 natural compounds (SelleckChem L1400) and 2,682 FDA-approved drugs (SelleckChem L1300). 114 hit compounds were identified that showed better binding affinity to the s4A position as compared to well- known PAI-1 inhibitors (TM5441 and Tiplaxtinin). Amentoflavone was found to bind (Figure 4A) and stabilize PAI-1 via a thermal shifting assay (Figure 4B) and to neutralize PAI-1 activity in inhibiting tPA (Figure 4C). The neutralization effects were achieved by disrupting complex formation between PAI-1 and its substrates, tPA (Figure 4D) and uPA (Figure 4E).

Furthermore, Amentoflavone was shown to inhibit in vitro colorectal cancer cell proliferation in the presence of ascites (Figure 4F). Cancer cells treated with patient-derived ascites enriched with PAI-1 were more sensitive to Amentoflavone inhibition than FBS-treated cancer cells (Figure 4G) and Amentoflavone’s efficacy was comparable to efficacious PAI-1 inhibitors (Figure 4H). These observations further validate that the C terminus end of s4A is a promising site to be targeted for PAI-1 inhibition and PM treatment.

Four known PAI-1 inhibitors with varying efficacies (TM5441 , Tiplaxtinin, AZ3976, and CDE-096) were then tested for their ability to inhibit proliferation of cancer cells. As shown in Figure 5, TM5441 (Figure 5A) and Tiplaxtinin (Figure 5B) are able to more efficiently inhibit cellular growth in the presence of ascites as compared to a Foetal Bovine Serum (FBS) control, in contrast to AZ3976 (Figure 5C) and CDE-096 (Figure 5D). Computational analysis using AutoDock Vina demonstrated that the binding affinity to this s4A pocket is higher for TM5441 (-10.2 kcal/mol) and Tiplaxtinin (-10.9 kcal/mol), which are efficacious in inhibiting cancer cell both in vitro and in vivo in the presence of PAI-1 -enriched ascites, and relatively lower for AZ3976 (-8.3 kcal/mol) and CDE-096 (-9.7 kcal/mol), which are not able to inhibit PM cancer cell proliferation efficiently in the presence of ascites. This finding supports the hypothesis that the inhibitory functions on cancer cells exposed to ascites in vitro could be due to the binding affinity of inhibitors to the s4A site.

Due to their small sizes, small molecule inhibitors may have the potential to cross barriers and membranes (La Manna et al., Int J Mol Sci (2018) 19(9):2714). The use of PAI-1 small molecule inhibitors could be absorbed systematically and perturb the fibrinolytic pathway leading to (uncontrolled) bleeding. Thus, it was investigated whether larger molecules such as antibodies/nanobodies could have a higher therapeutic index with lower risk for toxicity as the rate of systemic absorption through the peritoneal membrane will be lower with larger molecules as compared to small molecule inhibitors.

To this end, 10 commercially available anti-PAI-1 antibodies were tested to determine the efficacy of these antibodies in inhibiting STAT3 activation, as it has been previously demonstrated that inhibition of PAI-1 inhibits downstream activation of STAT3 (Hendrikson et al., Cell Rep Med (2022) (2): 100526). Surprisingly, none of these antibodies were able to recapitulate the phenotype conferred by PAI-1 inhibition in the presence of ascites (see Figure 6, Figure 7, Figure 8 and Figure 9).

Next, custom anti-PAI-1 antibodies were generated. The target protein sequence of interest was subjected to a detailed analysis using i-DNA’s propriety algorithm (https://www.i-dna.sg/). An “epitope score” was assigned to each residue based on structural features, sequence conservation, hydrophobicity, solvent exposure and numerous other criteria. Six, 12-residue fragments with highest overall scores as epitopes were chosen for generation of monoclonal antibodies against PAI-1 (Figure 10A, Figure 10B and Figure 10C). Ten antibodies were subsequently selected for further testing.

None of the 10 custom antibodies were able to inhibit STAT3 activation in the presence of ascites and therefore recapitulate the phenotype conferred by PAI-1 inhibition (Figure 11A and Figure 11 B).

It was hypothesized that these antibodies did not exhibit the expected phenotype as they do not specifically bind to the critical groove within s4A, which is necessary for oncogenic inhibition of PAI-1 in the presence of ascites. It was therefore necessary to identify antibodies and/or nanobodies with the potential to inhibit the critical groove necessary for PAI-1 inhibition.

Example 3: Discovery, validation and characterisation of novel PAI-1 antibodies and nanobodies

Antibodies capable of binding to PAI-1 and competing with PAI-1 small molecule inhibitors (50%-50% mix of TM5441 and Tiplaxitinin) were obtained.

Nanobodies have a much smaller size of 15 kDa compared to conventional antibody at 150 kDa and possess an elongated CDR3 domain. Thus, nanobodies capable of binding to PAI-1 and competing with PAI-1 small molecule inhibitors (50%-50% mix of TM5441 and Tiplaxitinin) were also obtained using phage display peptides (Figure 10D, Figure 10E, and Figure 10F).

Fifteen antibodies and 2 nanobodies were selected for further validation. Sequence features of the antibodies and nanobodies are summarised in Tables A to D.

The target of 15 antibodies was validated by western blots (Figure 12A). Three antibodies (A5, C11 and D6) recognized both active and latent forms of PAI-1 protein, and 8 antibodies (A10, B6, D7, E2, F4, F8, F10 and G11 ) recognized only the latent form of PAI-1 . The remaining 4 antibodies (A7, A9, B1 and F5) did not recognise either form in western blots at a concentration of 1 pg/mL (Figure 12B). Human IgG unspecific to any targets served as a negative control for antibodies, and a nanobody targeting green fluorescent protein (GFP) served as negative control for nanobodies. For nanobodies, both D4 and E8 recognised both forms. A confirmatory ELISA showed D4 and E8 exhibited binding to phage display peptide 2 (SEQ ID NO:265) (Figure 12E).

After validating the targets of 11 antibodies and 2 nanobodies by western blots, the neutralising activity of these antibodies and nanobodies on PAI-1 functions was evaluated. One antibody (A5) and 2 nanobodies (D4 and E8) were shown to supress PAI-1 function in inhibiting both tPA (Figure 12C) and uPA activities (Figure 12D). Human IgG unspecific to any targets served as a negative control for antibodies and a nanobody targeting GFP served as a negative control for nanobodies. Therefore, 1 PAI-1 neutralising antibody (A5) and 2 PAI-1 neutralising nanobodies (D4 and E8) were successfully discovered.

Subsequently, surface plasmon resonance (SPR) was performed to determine binding affinities of the identified biologies to different conformations of PAI-1 . A5 displayed high affinity to wildtype active PAI-1 , stable active PAI-1 (stable quadruple mutant of PAI-1 with N150H, K154T, Q319L and M354I mutations) and latent PAI-1 with an equilibrium constant (KD) of 0.126 nM, 0.282 nM and 1 .60 nM, respectively (Figure 13A). SPR was also performed on antibodies, C11 and D6, which recognized both forms of PAI-1 in western blots. The SPR assessments of nanobodies, D4 and E8, were also performed.

Further SPR analysis using BIAcore were performed to assess binding of A5, D4 and E8 to recombinant PAI-1 . PAI-1 proteins were coupled to CM5 censorships using amine coupling and a serial dilution of the biologies were flowed over the sensorchip surface. Kinetics parameters and affinity were measured using BIAcore K8 (GE Healthcare) by multiple-cycle kinetics (MCK) and calculated using BIAevaluation software (GE Healthcare). Each of A5, D4 and E8 displayed high affinity to naturally occurring active and latent PAI-1 , and good affinity to stable active PAI-1 (stable quadruple mutant of PAI-1 with N150H, K154T, Q319L and M354I mutations) (Figure 13B).

Example 4: Application of novel PAI-1 antibodies and nanobodies in the presence of ascites for PM treatment

4.1 In vitro proliferation

The utility of the biologies in inhibiting PM cell proliferation in the presence of ascites was investigated in vitro by utilising a cell line model representative of colorectal PM, Colo-205. Endotoxin removal process was performed on antibody and nanobodies used for in vitro and in vivo tests, to reach < 1 EU/mL by ProteoGenix. Colo-205 cells were treated with varying doses of each biologic and its corresponding control in the presence of Foetal Bovine Serum (FBS), 3 PM patient ascites containing high levels of PAI- 1 , and 4 PM patient ascites containing low levels of PAI-1 . Nanobodies D4 and E8 were shown to reduce proliferation of cells treated with PM patient ascites compared to the VHH control (Figure 14A and Figure 14C), and E8 was more efficacious in supressing cancer cell proliferation. At 10 pg/mL and 7.5 pg/mL, ascites enriched with PAI-1 were more sensitive to inhibition compared to ascites with low levels of PAI-1 for D4 and E8, respectively (Figure 14B and Figure 14D), demonstrating differential sensitivity of ascites to 2 nanobodies based on the levels of PAI-1 present in the ascites. Nanobody targeting GFP serving as a negative control for the 2 discovered nanobodies did not show differential sensitivity between ascites containing varying levels of PAI-1 (Figure 14B and Figure 14D). At 150 pg/mL, A5 was also shown to reduce proliferation of cells treated with PM patient ascites compared to IgG control (Figure 14E), but there was no differential sensitivity between ascites containing varying levels of PAI-1 (Figure 14F), possibly due to relatively low efficacy compared to the nanobodies. Human IgG served as the negative control for A5. Levels of PAI-1 protein in each ascites were plotted in Figure 14G.

4.2 In vitro inhibition of STAT3 signalling activation

Building upon these findings, the effects of the identified biologies on the downstream pathways activated by PAI-1 in ascites was further investigated. Treatment of A5, D4 and E8 was found to reduce the pSTAT3/STAT3 ratio in PM cancer cells treated with ascites as a demonstration of inhibition of STAT3 signalling activation (Figure 15A and Figure 15B). These observations suggest that these 3 biologies are promising candidates to inhibit PM cancer cell proliferation in the presence of ascites.

4.3 In vivo efficacy on PM tumour growth

Building upon the promising findings in vitro, efficacy of E8 in supressing tumour growth was investigated via intraperitoneal (i.p.) administration in the presence of FBS, ascites with high PAI-1 , and ascites with low PAI-1 (Figure 16A). Lesser tumour burden was observed in some mice when treated with 17.5 pg E8 in the presence of PAI-1 -high ascites (Figure 16B). Bodyweight of each mouse did not decrease drastically across the treatment (Figure 16C, Figure 16D and Figure 16E). The efficacy of E8 in supressing tumour growth using an optimised dose (20 pg) was investigated. 10 mice were used in each treatment group. In the presence of ascites, significantly less tumour burden was observed in mice that were treated with E8 as compared to PBS and VHH control (Figure 16F, Figure 16G). Mice treated with E8 in the presence of ascites also had significantly less tumour burden as compared to mice treated with E8 in the presence of 10% FBS, suggesting better efficacy in the clinically- relevant ascites environment (Figure 16F, Figure 16G). Furthermore, the bodyweight of mice i.p. injected with E8 did not decrease throughout the 7 doses (Figure 16H).

4.4 Efficacy in human explant PM models

A perturbation experiment was performed in two human explant models of Struma Ovarii and colorectal PM cancer with the co-treatment of PAI-1 -high cell-free ascites. Upon arrival in the lab within one hour after excision, tumour samples were immediately processed without prior freezing. Tissue fragments of approximately 2x2x2 mm were sectioned, transferred to a 24-well plate and cultured in DMEM/F12 complete medium for the subsequent biologies treatment.

Tumour viability was significantly reduced upon 24-hour treatment of 20 pg/ml E8 and D4 (equivalent to 1 .2 pM) while a VHH control did not decrease tumour viability (Figure 19). These results demonstrate that E8 and D4 are efficacious in human tumours in their original states.

Example 5: In vivo systematic safety profile

A5 (50 pg, 100 pg, 150 pg, 200 pg), D4 (50 pg, 100 pg, 150 pg), or E8 (20 pg, 50 pg, 100 pg, 150 pg) were administered into BALB/c nude mice (n = 2 females and 2 males for each dose) via intravenous (i.v.) injection. Bodyweight and behaviour were assessed on a daily basis, followed by 16-hour fasting from Day 13 before the final assessment of haematology count, clinical chemistry profiles, and pathology evaluation (Figure 17A). Bodyweight of each mouse did not drop across 14 days upon i.v. administration (Figure 17B). Counts of white blood cells (WBC), red blood cells (RBC), and platelets did not vary much, except for WBC count of 1 male with 150 pg D4, platelets count of 1 male with 100 pg D4, 2 males with 150 pg D4, 1 male with 20 pg E8, and 1 female with 50 pg E8 (Figure 17C). Clinical chemistry profiles of total protein, glucose and albumin are shown in Figure 17D. These results suggest that these biologies are safe.

Pathology evaluation of the main organs of the mice such as brain, heart, lung, kidney, liver and spleen were also performed. No significant pathological changes were observed (Figure 17E). No histopathological changes such as inflammation, infiltration of inflammatory cells, degeneration/ necrosis were observed in these organs, further indicating that the biologies are not toxic. Taken together, presence of E8 in the systemic circulation does not result in overt side effects in NCr nude mice.

Example 6: Application of novel PAI-1 antibodies and nanobodies in other indications

Beyond PM pathogenesis, elevated PAI-1 has also been reported to be implicated in various pathologies including cancer (Placencio et al., Cancer Res (2015) 75(15):2969- 74), obesity, inflammation, metabolic syndrome (Cesari et al. Cardiovasc Ther. (2010) 28(5):e72-91 , and senescence (Vaughan et al., Arterioscler Thromb Vase Biol. (2017) 37(8): 1446-52). Therefore, preliminary investigations were performed on the utility of our PAI-1 biologies in haematology, as PAI-1 plays a critical role in the regulation of fibrinolysis and tissue remodelling.

Antibody A5 greatly reduced the clot lysis time of plasma (Figure 18). This observation was consistent with A5’s neutralization function on PAI-1 , which inhibits tissue plasminogen activator (tPA), a thrombolytic agent that breaks down blood clots.

References

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Claims

Claims:

1 . An antigen-binding molecule, optionally isolated, that binds to PAI-1 .

2. The antigen-binding molecule according to claim 1 , wherein the antigen-binding molecule comprises:

(a)

(b)

(c)

(d)

(e)

(f)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

(ii) a light chain variable (VL) region incorporating the following CDRs:

(g)

(h)

(i)

0)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:1 10 HC-CDR2 having the amino acid sequence of SEQ ID NO:1 1 1 HC-CDR3 having the amino acid sequence of SEQ ID NO:112; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:116 LC-CDR2 having the amino acid sequence of SEQ ID NO:117 LC-CDR3 having the amino acid sequence of SEQ ID NO:118; or

(k)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:122 HC-CDR2 having the amino acid sequence of SEQ ID NO:123 HC-CDR3 having the amino acid sequence of SEQ ID NO:124; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NQ:105 LC-CDR2 having the amino acid sequence of SEQ ID NO:129 LC-CDR3 having the amino acid sequence of SEQ ID NQ:130; or

(l)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:79 HC-CDR2 having the amino acid sequence of SEQ ID NQ:80 HC-CDR3 having the amino acid sequence of SEQ ID NO:134; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:135 LC-CDR2 having the amino acid sequence of SEQ ID NO:39 LC-CDR3 having the amino acid sequence of SEQ ID NO:136; or

(m)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:31 HC-CDR2 having the amino acid sequence of SEQ ID NO:32 HC-CDR3 having the amino acid sequence of SEQ ID NO:139; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:141 LC-CDR2 having the amino acid sequence of SEQ ID NQ:106 LC-CDR3 having the amino acid sequence of SEQ ID NO:142; or

(n)

(I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:147 HC-CDR2 having the amino acid sequence of SEQ ID NO:148 HC-CDR3 having the amino acid sequence of SEQ ID NO:149; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:152 LC-CDR2 having the amino acid sequence of SEQ ID NO:153 LC-CDR3 having the amino acid sequence of SEQ ID NO:154; or (O)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

(ii) a light chain variable (VL) region incorporating the following CDRs:

(P)

(q)

3. The antigen-binding molecule according to claim 1 or claim 2, wherein the antigen-binding molecule comprises:

(b) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15; and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule binds to active PAI-1 and binds to latent PAI-1 .

5. The antigen-binding molecule according to any one of claims 1 to 4, wherein the antigen-binding molecule binds to, or in proximity to, the s4A groove of PAI-1 .

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule exhibits competitive binding to PAI-1 with an agent known to bind to the s4A groove of PAI-1 , optionally wherein the agent is TM5441 and/or Tiplasinin.

7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule binds to PAI-1 via contact with one or more amino acid residues of the region shown in SEQ ID NO:265.

8. The antigen-binding molecule according to any one of claims 1 to 7, wherein the antigen-binding molecule is a multispecific antigen-binding molecule, and wherein the antigen-binding molecule further comprises an antigen-binding domain which binds to an antigen other than PAI-1 .

9. The antigen-binding molecule according to any one of claims 1 to 8, wherein the antigen-binding molecule is conjugated to a drug moiety or a detectable moiety.

10. A chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to any one of claims 1 to 9.

11 . A nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule according to any one of claims 1 to 9, or a CAR according to claim 10.

12. An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to claim 11 .

13. A cell comprising an antigen-binding molecule according to any one of claims 1 to 9, a CAR according to claim 10, a nucleic acid or a plurality of nucleic acids according to claim 11 , or an expression vector or a plurality of expression vectors according to claim 12.

14. A method comprising culturing a cell according to claim 13 under conditions suitable for expression of an antigen-binding molecule or CAR by the cell.

15. A composition comprising an antigen-binding molecule according to any one of claims 1 to 9, a CAR according to claim 10, a nucleic acid or a plurality of nucleic acids according to claim 11 , an expression vector or a plurality of expression vectors according to claim 12, or a cell according to claim 13, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

16. An antigen-binding molecule according to any one of claims 1 to 9, a CAR according to claim 10, a nucleic acid or a plurality of nucleic acids according to claim 1 1 , an expression vector or a plurality of expression vectors according to claim 12, or a cell according to claim 13, or a composition according to claim 15, for use in a method of medical treatment or prophylaxis.

17. An antigen-binding molecule according to any one of claims 1 to 9, a CAR according to claim 10, a nucleic acid or a plurality of nucleic acids according to claim 1 1 , an expression vector or a plurality of expression vectors according to claim 12, or a cell according to claim 13, or a composition according to claim 15, for use in a disease or condition in which PAI-1 is pathologically-implicated.

18. Use of an antigen-binding molecule according to any one of claims 1 to 9, a CAR according to claim 10, a nucleic acid or a plurality of nucleic acids according to claim 1 1 , an expression vector or a plurality of expression vectors according to claim 12, or a cell according to claim 13, or a composition according to claim 15, in the manufacture of a medicament for treating or preventing a disease or condition in which PAI-1 is pathologically-implicated.

19. A method of treating or preventing a disease or condition in which PAI-1 is pathologically-implicated, comprising administering to a subject a therapeutically- or prophylactically-effective amount of an antigenbinding molecule according to any one of claims 1 to 9, a CAR according to claim 10, a nucleic acid or a plurality of nucleic acids according to claim 1 1 , an expression vector or a plurality of expression vectors according to claim 12, or a cell according to claim 13, or a composition according to claim 15.

20. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use according to claim 17, the use according to claim 18, or the method according to claim 19, wherein the disease or condition is cancer.

21 . The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use, the use, or the method according to claim 120 wherein the cancer is associated with ascites characterised by the presence of PAI-1 .

22. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use, the use, or the method according to claim 20 or claim 21 , wherein the cancer is selected from: ovarian cancer, endometrial cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, pancreatic cancer, hepatobiliary cancer and peritoneal metastasis.

23. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use, the use, or the method according to claim 22, wherein the peritoneal metastasis is selected from: colorectal peritoneal metastasis, small bowel peritoneal metastasis, mesothelioma, endometrial peritoneal metastasis, gastric peritoneal metastasis, ovarian peritoneal metastasis, appendiceal peritoneal metastasis, pancreatic peritoneal metastasis, urothelial peritoneal metastasis, Pseudomyxoma peritonei (PMP), breast peritoneal metastasis, esophageal peritoneal metastasis, lung peritoneal metastasis, hepatobilliary peritoneal metastasis, peritoneal metastasis of unknown origin, and primary peritoneal carcinoma.

24. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use, the use, or the method according to any one of claims 20 to 23, wherein the cancer is characterised by:

(I) high STAT3 activation;

(II) high PAI-1 ;

(ill) high STAT3 activation and high PAI-1 ; or

(iv) high STAT3 activation and low PAI-1 .

25. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use according to claim 17, the use according to claim 18, or the method according to claim 19, wherein the disease or condition is characterised by coagulation.

26. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use, the use, or the method according to claim 25, wherein the disease or condition is selected from: thrombosis, e.g. deep vein thrombosis (DVT), portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, cerebral venous sinus thrombosis, thrombotic stroke; myocardial infarction; antiphospholipid syndrome (APS); disseminated intravascular coagulation (DIC); activated protein C resistance, e.g. Factor V Leiden; and cancer

27. An in vitro complex, optionally isolated, comprising an antigen-binding molecule according to any one of claims 1 to 9 bound to PAI-1 .

28. A method for detecting PAI-1 in a sample, comprising contacting a sample containing, or suspected to contain, PAI-1 with an antigen-binding molecule according to any one of claims 1 to 9, and detecting the formation of a complex of the antigen-binding molecule with PAI-1 .

29. A method of selecting or stratifying a subject for treatment with a P Al -1 -targeted agent, the method comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to any one of claims 1 to 9, and detecting the formation of a complex of the antigen-binding molecule with PAI-1 .

30. Use of an antigen-binding molecule according to any one of claims 1 to 9 as an in vitro or in vivo diagnostic or prognostic agent.