Antigen-binding molecules and uses thereof

Abstract

The present disclosure relates generally to antigen-binding molecules, more specifically antigen-binding molecules that specifically bind to serine peptidase inhibitor, kunitz type 1 (SPINT1), assays and devices comprising said antigen-binding molecules, and uses thereof.

Description

ANTIGEN-BINDING MOLECULES AND USES THEREOFRELATED APPLICATIONS

[0001] This application claims priority to Australian Provisional Application No. 2024901760 entitled “Antigen-binding molecules and uses thereof’ filed 12 June 2024, the contents of which are incorporated herein by reference in their entiretyFIELD

[0002] The present disclosure relates generally to antigen-binding molecules, more specifically antigen-binding molecules that specifically bind to serine peptidase inhibitor, kunitz type 1 (SPINT1), assays and devices comprising said antigen-binding molecules, and uses thereof, including for diagnosing conditions associated with changes in SPINT1 protein expression, such as placental insufficiency and abnormal fetal weight.BACKGROUND

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] Reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0005] Fetal growth restriction (FGR), also known as intrauterine growth restriction (IUGR), represents a leading cause of post-natal ill health, is a leading cause of stillbirth in humans and leaves a lifelong legacy to children and family. FGR is a failure of a fetus to reach its genetically pre-determined growth potential. It represents a very serious complication of pregnancy and, as indicated above, is the biggest risk factor for stillbirth - a devastating tragedy that affects 1: 130 pregnancies in Australia. In addition, approximately 27,000 FGR or small for gestational age (SGA; birthweight <10thcentile) babies are born in Australia per year. Being FGR or SGA at birth are associated outcomes of placental insufficiency.

[0006] FGR is also a major determinant of perinatal morbidity, with low-birthweight infants experiencing poorer neurodevelopmental outcomes (Miller et al. (2016) The Journal ofPhysiology 594:807-823). Moreover, FGR is associated with adverse outcomes later in life: school-aged children who were growth restricted have higher rates of impaired cognition, memory, attention and gross motor proficiencies (Miller et al. (2016) supra). Its effects can persist lifelong - adults have a higher prevalence of major chronic diseases such as cardiovascular disease, stroke and diabetes (Barker and Osmond (1986) Lancet 7: 1077 -1081; Cooper et al. (1997) Annals of the Rheumatic Disease 56: 17-21; Hales and Barker (1992) Diabetologia 35:595-601; Barker (1998) Clinical Science 95: 115-128; Barker (2000) Theriogenology 53:555-574). Placental insufficiency can lead to chronic fetal hypoxemia and reduced nutrient supply to the fetus which ultimately leads to a slowing of fetal growth and poor brain development which itself can lead to neurological issues. These are all features of FGR and SGA babies.

[0007] Placental insufficiency arises when the placenta fails to provide adequate oxygen and nutrient exchange to the fetus (Mifsud and Sebire (2014) Fetal diagnosis and therapy 36: 117- 128). The fetus responds by decreasing its growth and redistributing resources to ensure survival; channelling blood to vital organs such as the brain. When these survival adaptations fail, stillbirth occurs. There is a need to be able to reliably detect poor fetal growth and placental function to enable timely delivery before stillbirth occurs. A major barrier to reducing the health burden of FGR is the inability to accurately identify SGA fetuses, where FGR cases are more common.

[0008] Current methods to detect FGR are surprisingly poor. The most commonly used screening test for FGR is to detect SGA fetuses in pregnancy using a tape measure. The maternal abdomen is measured to assess the size of the uterus and an ultrasound performed if the fetus is suspected of being small. However, this approach has sensitivity (i.e. detection rate) of only around 20% to detect SGA in women of normal weight, which falls even further among women who are overweight or obese. Offering ultrasound to all women in late pregnancy might be expected to reliably pick up all small babies, but this approach has been shown to have detection rates of only 46% (Fadigas et al. (2015) Ultrasound in obstetrics and genecology 45:559-565) - 57% (Sovio et al. (2015) Lancet 356:2089-2097) for SGA at birth, even in ideal research settings (Fadigas et al. (2015) supra). FGR is also sometimes defined as fetal weight <3rdcentile. Current clinical care, using the tape measure followed by selective ultrasound, is also generally poor at detecting babies in the <3rdcentile. The detection rate ofcurrent care is only 14% in US study reported by Sparks et al. (JMFM 2011;24(5):708-12), or 32% in a UK study reported by Sovio et al. (Lancet 2015; 356:2089-2097).Cost and access to universal ultrasound precludes widespread adoption, highlighting the clear need for new tools to better identify pregnancies affected by FGR.Ultrasound is still a valuable tool in the management of pregnancy such as in detecting fetal macrosomia, where a newborn has a significantly larger birth weight than average (i.e., a weight generally of more than 4kg), which can lead to birth complications and increased risk of injury to newborn.

[0009] The present inventors have previously shown that (i) circulating levels of SPINT1 in pregnant female subjects correlates with clinical parameters of placental insufficiency, such as uterine blood flow (uterine artery Doppler velocity) at the time of sampling, neonatal lean mass, placental weight and placental surface area; (ii) circulating levels of SPINT1 are significantly lower in pregnant female subjects who subsequently delivered a small-for-gestational-age (SGA) baby, including when measured from about 27 to 29 weeks gestation, as compared to pregnant female subjects who do not subsequently delivered an SGA baby; and (iii) circulating levels of SPINT1 in pregnant female subjects are indicative of the risk of low birthweight, noting that pregnant female subjects with a low level of SPINT1, as compared to controls, are at significantly greater risk of giving birth to a low birthweight (small for gestational age) neonate (see WO 2019 / 222812, the entire contents of which is incorporated herein by reference). However, there remains a need for improved tools for measuring SPINT1 protein, including in biological samples such as whole blood, plasma and serum.SUMMARY

[0010] Taught herein is an antigen-binding molecule that specifically bind to serine peptidase inhibitor, kunitz type 1 (SPINT1), wherein the antigen-binding molecule comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2 and a VH CDR3 comprising the amino acid sequence of SEQ ID NOG; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NOG, a VL CDR2comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 8:VH CDR1 TYTMG (SEQ ID NO: 1)VH CDR2 TISSGGDKTYYPDSVKG (SEQ ID NO:2)VH CDR3 YDDWDHGMDY (SEQ ID NOG)VL CDR1 SASSSVNYMH (SEQ ID NO:4)VL CDR1 RASKSVSTSGYSYMH (SEQ ID NOG)VL CDR2 TTSNLAS (SEQ ID NOG)VL CDR2 LVSNLES (SEQ ID NO:7)VL CDR3 HQWSSWT (SEQ ID NOG)

[0010] In an embodiment, the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NOG and a VH CDR3 comprising the amino acid sequence of SEQ ID NOG; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NOG, a VL CDR2 comprising the amino acid sequence of SEQ ID NOG, and a VL CDR3 comprising the amino acid sequence of SEQ ID NOG.

[0011] In an embodiment, the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NOG and a VH CDR3 comprising the amino acid sequence of SEQ ID NOG; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NOG, a VL CDR2 comprising the amino acid sequence of SEQ ID NOG, and a VL CDR3 comprising the amino acid sequence of SEQ ID NOG.

[0012] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NOG or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12 or anamino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence having at least 80% sequence identity to any of the foregoing; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% sequence identity thereto:VH FR1 EVMLVESGGGLVKPGGSLKLSCIASGFTFS (SEQ ID NO:9)VH FR2 WVRQSPEKRLEWVA (SEQ ID NO: 10)VH FR3 RFIISRDNAKNNLYLQMSSLKSEDTALYYCVK (SEQ ID NO: 11)VH FR4 WGQGTSVIVSS (SEQ ID NO: 12)VL FR1 QIVLTQSPAIMSASLGEEITLTC (SEQ ID NO: 13)VL FR1 DIVLTQSPASLAVSLGQRATISY (SEQ ID NO: 14)VL FR2 WYQQKSGTSPKLLIY (SEQ ID NO: 15)VL FR2 WNQQKPGQPPRLLIY (SEQ ID NO: 16)VL FR3 GVPSRFSGSGSGTFYSLTISSVEAEDVADYYC (SEQ ID NO: 17) VL FR3 GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO: 18) VL FR4 FGGGTKLEIK (SEQ ID NO: 19)

[0013] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR3 comprising the amino acid sequence of SEQ IDNO: 17 or an amino acid sequence having at least 80% sequence identity thereto; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% sequence identity thereto.

[0014] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:9, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 13, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 15, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 17; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19.

[0015] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 80% sequence identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having at least 80% sequence identity thereto.

[0016] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:20, and the VL comprises the amino acid sequence of SEQ ID NO:21.

[0017] In an embodiment, the VH is encoded by the nucleic acid sequence of SEQ ID NO:23, and the VL is encoded by the nucleic acid sequence of SEQ ID NO:24.

[0018] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 80% sequence identity thereto; and a VL FR4comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% sequence identity thereto.

[0019] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:9, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11, and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 14, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 16, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 18, and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19.

[0020] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 80% sequence identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:22 or an amino acid sequence having at least 80% sequence identity thereto.

[0021] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:20, and the VL comprises the amino acid sequence of SEQ ID NO:22.

[0022] In an embodiment, the VH is encoded by the nucleic acid sequence of SEQ ID NO:23, and the VL is encoded by the nucleic acid sequence of SEQ ID NO:25.

[0023] In an embodiment, the antigen -binding molecule is an antibody or a SPINT1 -binding fragment thereof.

[0024] In an embodiment, the SPINT1 -binding fragment is selected from the group consisting of an Fab fragment, an scFab, an Fab’, a single chain variable fragment (scFv) and a one-armed antibody.

[0025] In another aspect disclosed herein, there is provided an antigen-binding molecule that specifically bind to serine peptidase inhibitor, kunitz type 1 (SPINT1), wherein the antigenbinding molecule comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO:26, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:27 and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:27; and wherein the VL comprisesa complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO:29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:31:VH CDR1 GYNMN (SEQ ID NO:26)VH CDR2 NTDPYYDGITYNQKFKG (SEQ ID NO:27)VH CDR3 GGYGNYVMDY (SEQ ID NO:28)VL CDR1 RSSQSLETSNGNAYLN (SEQ ID NO:29)VL CDR2 RVSNRFS (SEQ ID NO: 30)VL CDR3 LQVTHVPFT (SEQ ID NOG 1)

[0026] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO:33 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR3 comprising the amino acid sequence of SEQ ID NO:38 or an amino acid sequence having at least 80% sequence identity to any of the foregoing, and a VL FR4 comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence having at least 80% sequence identity thereto:VH FR1 EVQLQQSGPEVEKPGASVKISCKASGYTFI (SEQ ID NO:32)VH FR2 WVRQINGKSLEWIG (SEQ ID NO: 33)VH FR3 KATMTVDKSSNTAYMQLESLTSEDSAVYYCAR (SEQ ID NO:34)VH FR4 WGQGTSVTVSS (SEQ ID NO:35)VL FR1 DVVMTQTPLSLPVSLGDQASISC (SEQ ID NO:36)VL FR2 WYVQKPGQSPQVLIY (SEQ ID NO: 37)VL FR3 GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO:38)VL FR4 FGSGTTLELK (SEQ ID NO: 39)

[0027] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO: 32, a VH FR2 comprising the amino acid sequence of SEQ ID NO:33, a VH FR3 comprising the amino acid sequence of SEQ ID NO:34; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO:36, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 37, a VL FR3 comprising the amino acid sequence of SEQ ID NO:38; and a VL FR4 comprising the amino acid sequence of SEQ ID NO:39.

[0028] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:40 or an amino acid sequence having at least 80% sequence identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:41 or an amino acid sequence having at least 80% sequence identity thereto.

[0029] In an embodiment, the VH is encoded by the nucleic acid sequence of SEQ ID NO:42 and the VL is encoded by the nucleic acid sequence of SEQ ID NO:43.

[0030] In an embodiment, the antigen -binding molecule is an antibody or a SPINT1 -binding fragment thereof.

[0031] In an embodiment, the SPINT1 -binding fragment is selected from the group consisting of an Fab fragment, an scFab, an Fab’, a single chain variable fragment (scFv) and a one-armed antibody.

[0032] The present disclosure also extends to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antigen-binding molecule described herein.

[0033] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:24 or SEQ ID NO:25, or a nucleic acid sequence having at least 80% sequence identity to any of the foregoing.

[0034] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:24, or a nucleic acid sequence having at least 80% sequence identity thereto.

[0035] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:25, or a nucleic acid sequence having at least 80% sequence identity thereto.

[0036] Also disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antigen-binding molecule described herein.

[0037] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:42, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:43, or a nucleic acid sequence having at least 80% sequence identity to any of the foregoing.

[0038] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:43.

[0039] The present disclosure also extends to an expression construct comprising a nucleic acid sequence encoding the antigen-binding molecule described herein, operably linked to one or more regulatory sequences.

[0040] The present disclosure also extends to a host cell comprising the expression construct disclosed herein.

[0041] Also disclosed herein is a vector comprising a nucleic acid sequence encoding the antigen-binding molecule described herein.

[0042] The present disclosure also contemplates a kit comprising the antigen-binding molecule described herein.

[0043] Also disclosed herein is a device comprising the antigen-binding molecule described herein.

[0044] In an embodiment, the device is a point of care device.

[0045] In an embodiment, the device is a lateral flow device.

[0046] In another aspect disclosed herein, there is provided a method of determining the state of placental health in a female subject, the method comprising determining the circulating level of SPINT1 in a biological sample from the female subject using the antigen-binding moleculedescribed herein, wherein a reduction in the circulating level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of placental insufficiency.

[0047] In yet another aspect disclosed herein, there is provided a method of determining whether a fetus of a pregnant female subject is at risk of abnormal fetal weight, the method comprising determining the circulating level of SPINT1 in a biological sample from the female subject using the antigen-binding molecule described herein, wherein an elevation in the circulating level of SPINT1 relative to a control or over time, or a change in ratio relative to a control or over time, is indicative of abnormal fetal weight, wherein an increase in the circulating level of SPINT1 relative to a control or over time, or a change in ratio relative to a control or over time, is indicative of the likelihood of macrosomia, and wherein a decrease in the circulating level of SPINT1 relative to a control or over time, or a change in ratio relative to a control or over time, is indicative of the likelihood of small-for-gestational age, fetal growth restriction, or a baby at risk of adverse neonatal outcomes.

[0048] In an embodiment, the method further comprises determining at least one other biomarker and / or physiochemical parameter and / or clinical risk factors.

[0049] In an embodiment, the at least one other biomarker is selected from the group consisting of placental growth factor (P1GF), soluble fms-like tyrosine kinase- 1 (sFlt), SYNDECAN-1 and vascular endothelial growth factor (VEGF).

[0050] In an embodiment, the physiochemical parameter is generated by ultrasound or physical measurement.

[0051] In an embodiment, the circulating level or ratio of levels of SPINT1 is subject to analysis with an algorithm or by analytics function or process or other data processing means.

[0052] In an embodiment, the method further comprises assessing the subject for one or more risk factors associated with increased risk of abnormal fetal weight.

[0053] In an embodiment, the sample is maternal whole blood, plasma or serum.

[0054] In an embodiment, the female subject is determined to have placental insufficiency or where the fetus is determined to be at risk of macrosomia, further comprising subjecting the fetus to an early delivery.

[0055] The present disclosure also extends to a clinical management protocol for a pregnant female subject, the protocol comprising determining the circulating level of SPINT1 in a sample from the female subject using the antigen-binding molecule described herein, wherein a decrease in the circulating level of SPINT1 over time compared to a control or a statistically validated level, or a change in ratio reflecting a decrease in the circulating level of SPINT1 is indicative of placental insufficiency and wherein the fetus is monitored or subject to early delivery.

[0056] In an embodiment, the protocol further comprises determining at least one other biomarker and / or physiochemical parameter.

[0057] In an embodiment, the at least one other biomarker is selected from the group consisting of placental growth factor (P1GF), soluble fms-like tyrosine kinase- 1 (sFlt), SYNDECAN-1 and vascular endothelial growth factor (VEGF).

[0058] In an embodiment, the physiochemical parameter is generated by ultrasound or physical measurement.

[0059] In an embodiment, the circulating level or ratio of levels of SPINT1 is subject analysis with an algorithm or by analytics function or process or other data processing means.

[0060] In an embodiment, the sample is maternal whole blood, plasma or serum.

[0061] Also disclosed herein is an assay for determining the state of placental sufficiency in a female subject, the assay comprising determining the circulating level of SPINT1 in a sample from a pregnant female using the antigen-binding molecule described herein; subjecting the level of SPINT1 to an algorithm or analytics function or process or other data processing means generated from a first knowledge base of data comprising the level of the same biomarkers from a subject or cohort of subjects of known status with respect to placental sufficiency, wherein the algorithm or analytics or data processing provides an index of probability of the subject having or not having placental insufficiency or placental sufficiency.

[0062] The present disclosure also extends to use of a knowledge base of training data comprising (i) circulating levels of SPINT1 as determined using the antigen-binding molecule described herein, and (ii) a level of at least one other biomarker from a pregnant female subject with known placental sufficiency status to generate an algorithm or analytics function or process or other data processing means which, upon input of a second knowledge base of datacomprising levels of the same biomarkers from a patient with an unknown placental sufficiency status, provides an index of probability that predicts the nature of the placenta.

[0063] In another aspect disclosed herein, there is provided a panel of reagents for detecting biomarkers associated with placental insufficiency or abnormal fetal weight in a pregnant female subject, wherein the panel of reagents comprises the antigen-binding molecule described herein.

[0064] Also contemplated herein is a method for monitoring the progression of a pregnancy in a female subject, the method comprising:(a) determining the circulating level of SPINT1 using the antigen -binding molecule described herein, and at least one other biomarker in a sample obtained from a pregnant female subject at a first time point,(b) subjecting the levels determined in step (a) to an algorithm or analytics function or process or other data processing means to provide an index of probability of the subject having a placental sufficiency or insufficiency;(c) repeating steps (a) and (b) for circulating level of SPINT1 and at least one other biomarker in a sample obtained from a pregnant female subject at a subsequent time point to obtain an index of probability of the subject having a placental sufficiency or insufficiency, and(d) comparing the index of probability provided in step (b) with the index of probability provided in step (c), wherein a difference in the index of probabilities between the first time point and the subsequent time point is indicative of the progression of the placental health.

[0065] In an embodiment, the circulatory maternal fluid comprises whole blood, plasma or serum.

[0066] In an embodiment, the method further comprises inputting physiochemical data generated by ultrasound or physical measurement.

[0067] In an embodiment, the method further comprises inputting one or more risk factors associated with increased risk of abnormal fetal weight.

[0068] A list of abbreviations used throughout the subject specification are provided in Table 1, below.Table 1 - AbbreviationsBRIEF DESCRIPTION OF THE FIGURE

[0069] Figure 1 shows the components of the composite outcome identified with a SPINT1 <10th centile cut-off.

[0070] Figure 2 shows a summary of the ELISA disclosed in Example 4.DETAILED DESCRIPTION

[0071] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method step or group of elements or integers or method steps.

[0072] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes a single biomarker, as well as two or more biomarkers; reference to "an assay" includes a single assay, as well as two or more assays; reference to "the disclosure" includes single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". Any variants and derivatives contemplated herein are encompassed by "forms" of the invention. All aspects and forms of the invention are enabled across the width of the claims.

[0073] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. In addition, the present invention extends to ratios of two or more markers providing a numerical value associated with a level of risk of placental insufficiency.Antigen-binding molecules

[0074] The present disclosure is predicated, at least in part, on the development of novel SPINT1 -binding molecules and uses thereof, including for determining circulating levels of SPINT1 in a biological sample. Such uses may advantageously provide as assessment of the risk of placental insufficiency and fetal weight abnormality, such as SGA and macrosomia. Illustrative examples of such uses are described in WO 2019 / 222812, the entire contents of which is incorporated herein by reference.

[0075] Thus, in an aspect disclosed herein, there is provided an antigen-binding molecule that specifically bind to serine peptidase inhibitor, kunitz type 1 (SPINT1), wherein the antigenbinding molecule comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2 and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence ofSEQ ID N0:4 or SEQ ID NO:5, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:8:VH CDR1 TYTMG (SEQ ID NO: 1)VH CDR2 TISSGGDKTYYPDSVKG (SEQ ID NO:2)VH CDR3 YDDWDHGMDY (SEQ ID NOG)VL CDR1 SASSSVNYMH (SEQ ID NO:4)VL CDR1 RASKSVSTSGYSYMH (SEQ ID NOG)VL CDR2 TTSNLAS (SEQ ID NO: 6)VL CDR2 LVSNLES (SEQ ID NOG)VL CDR3 HQWSSWT (SEQ ID NOG)

[0076] SPINT1 is a protease inhibitor that regulates cell surface and extracellular serine proteases involved in tissue remodelling (Tanaka et al. (2005) Mol Cell Biol 25 :5687 -5698). SPINT1 was originally identified as an inhibitor of hepatocyte growth factor activator. It is also known as HALL Its importance in placental development is highlighted by the fact that SPINT1 knockout mice have severe growth restriction and embryonic lethality, due to failed placental development and function (Tanaka et al. (2005) supra). SPINT1 knockout mice have impaired formation of the labyrinth layer - a layer critically important in matemal / fetal exchange.

[0077] The term SPINT1, as used herein, also includes modified forms or homologs thereof. Illustrative examples of modified forms and homologs include derivatives, polymorphic variants, truncated forms (truncates) and aggregated or multimeric forms or forms having expansion elements (e.g. amino acid expansion elements).

[0078] Modified forms or homologs of SPINT1 will be familiar to persons skilled in the art, illustrative examples of which are described in GenBank Accession Nos: NM_001032367.1 to NP_001027539.1 (isoform 2 precursor), NM_003710.3 to NP_003701.1 (isoform 2 precursor), and NM_181642.2 to NP_857593.1 (isoform 1 precursor), the contents of which are incorporated herein by reference in their entirety.

[0079] In an embodiment, SPINT1 comprises an amino acid sequence of SEQ ID NO: 44 (GenBank Accession No: AB000095), or an amino acid sequence having at least 70% sequence identity thereto.

[0080] Reference to "at least 70% sequence identity" includes 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% 79%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, for example, after optimal alignment or best fit analysis. Thus, in an embodiment, the amino acid sequence of SPINT1 comprises at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or more preferably 100% sequence identity to SEQ ID NO:44, as described herein.

[0081] The terms “identity”, “similarity”, “sequence identity”, “sequence similarity”, “homology”, “sequence homology” and the like, as used herein, mean that at any particular amino acid residue position in an aligned sequence, the amino acid residue is identical between the aligned sequences. The term “similarity” or “sequence similarity” as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for an isoleucine or valine residue. As noted elsewhere herein, this may be referred to as conservative substitution. In an embodiment, modified form or homolog of SPINT1 has an amino acid sequence that differs from SEQ ID NO:44 by one or more conservative substitution of any of the amino acid residues contained therein.

[0082] In an embodiment, sequence identity with respect to a peptide sequence relates to the percentage of amino acid residues in the candidate sequence which are identical with the residues of the corresponding peptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C- terminal extensions, nor insertions shall be construed as reducing sequence identity or homology. Methods and computer programs for performing an alignment of two or more amino acid sequences and determining their sequence identity or homology are well known to persons skilled in the art. For example, the percentage of identity or similarity of two amino acid sequences can be readily calculated using algorithms, for example, BLAST, FASTA, or the Smith- Waterman algorithm. Techniques for determining an amino acid sequence "similarity" are well known to persons skilled in the art. In general, "similarity" means an exact amino acidto amino acid comparison of two or more peptide sequences or at the appropriate place, where amino acids are identical or possess similar chemical and / or physical properties such as charge or hydrophobicity. A so-termed "percent similarity" then can be determined between the compared peptide sequences. In general, "identity" refers to an exact amino acid to amino acid correspondence of two peptide sequences. Two or more peptide sequences can also be compared by determining their "percent identity". The percent identity of two sequences may be described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff (Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA), and normalized by Gribskov (Nucl. Acids Res. 14(6):6745-6763, 1986). Suitable programs for calculating the percent identity or similarity between sequences are generally known in the art. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul etal., (1997, Nucl. Acids Res.25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15).

[0083] As described herein, modified forms or homologs of SPINT1 include non-human isoforms. Illustrative examples of non-human isoforms include SPINT1 isoforms native to primates, companion animals such as cats and dogs and the like, working animals such as horses, donkeys and the like, livestock animals such as sheep, cows, goats, pigs and the like, laboratory test animals such as rabbits, mice, rats, guinea pigs, hamsters and the like and captive wild animals such as those in zoos and wildlife parks, deer, dingoes and the like.

[0084] In an embodiment, the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising theamino acid sequence of SEQ ID NO:2 and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 8. Unless otherwise stated, all VH, VL, CDR and FR sequences disclosed herein are based on the Kabat numbering scheme, as described, for example, in Wu and Kabat (J Exp Med.., 1970; 132 (2): 211-25) and Kabat et al. (Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0085] In an embodiment, the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2 and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO:5, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0086] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence having at least 80% sequence identity to any of the foregoing; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% sequence identity thereto:VH FR1 EVMLVESGGGLVKPGGSLKLSCIASGFTFS (SEQ ID N0:9)VH FR2 WVRQSPEKRLEWVA (SEQ ID NO: 10)VH FR3 RFIISRDNAKNNLYLQMSSLKSEDTALYYCVK (SEQ ID NO: 11)VH FR4 WGQGTSVIVSS (SEQ ID NO: 12)VL FR1 QIVLTQSPAIMSASLGEEITLTC (SEQ ID NO: 13)VL FR1 DIVLTQSPASLAVSLGQRATISY (SEQ ID NO: 14)VL FR2 WYQQKSGTSPKLLIY (SEQ ID NO: 15)VL FR2 WNQQKPGQPPRLLIY (SEQ ID NO: 16)VL FR3 GVPSRFSGSGSGTFYSLTISSVEAEDVADYYC (SEQ ID NO: 17) VL FR3 GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO: 18) VL FR4 FGGGTKLEIK (SEQ ID NO: 19)

[0087] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 80% sequence identity thereto; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% sequence identity thereto.

[0088] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:9, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 13, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 15, a VL FR3 comprising the aminoacid sequence of SEQ ID NO: 17; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19.

[0089] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 80% sequence identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having at least 80% sequence identity thereto.

[0090] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:20, and the VL comprises the amino acid sequence of SEQ ID NO:21.

[0091] In an embodiment, the VH is encoded by the nucleic acid sequence of SEQ ID NO:23, and the VL is encoded by the nucleic acid sequence of SEQ ID NO:24.

[0092] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 80% sequence identity thereto; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% sequence identity thereto.

[0093] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:9, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11, and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 14, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 16, a VL FR3 comprising the aminoacid sequence of SEQ ID NO: 18, and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19.

[0094] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 80% sequence identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:22 or an amino acid sequence having at least 80% sequence identity thereto.

[0095] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:20, and the VL comprises the amino acid sequence of SEQ ID NO:22.

[0096] In an embodiment, the VH is encoded by the nucleic acid sequence of SEQ ID NO:23, and the VL is encoded by the nucleic acid sequence of SEQ ID NO:25.

[0097] In another aspect disclosed herein, there is provided an antigen-binding molecule that specifically bind to serine peptidase inhibitor, kunitz type 1 (SPINT1), wherein the antigenbinding molecule comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO:26, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:27 and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:27; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO:29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:31:VH CDR1 GYNMN (SEQ ID NO:26)VH CDR2 NTDPYYDGITYNQKFKG (SEQ ID NO:27)VH CDR3 GGYGNYVMDY (SEQ ID NO:28)VL CDR1 RSSQSLETSNGNAYLN (SEQ ID NO:29)VL CDR2 RVSNRFS (SEQ ID NO: 30)VL CDR3 LQVTHVPFT (SEQ ID NOG 1)

[0098] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO:33 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprisingthe amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR3 comprising the amino acid sequence of SEQ ID NO:38 or an amino acid sequence having at least 80% sequence identity to any of the foregoing, and a VL FR4 comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence having at least 80% sequence identity thereto:VH FR1 EVQLQQSGPEVEKPGASVKISCKASGYTFI (SEQ ID NO:32)VH FR2 WVRQINGKSLEWIG (SEQ ID NO: 33)VH FR3 KATMTVDKSSNTAYMQLESLTSEDSAVYYCAR (SEQ ID NO:34)VH FR4 WGQGTSVTVSS (SEQ ID NO:35)VL FR1 DVVMTQTPLSLPVSLGDQASISC (SEQ ID NO:36)VL FR2 WYVQKPGQSPQVLIY (SEQ ID NO: 37)VL FR3 GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO:38)VL FR4 FGSGTTLELK (SEQ ID NO: 39)

[0099] In an embodiment, the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO: 32, a VH FR2 comprising the amino acid sequence of SEQ ID NO:33, a VH FR3 comprising the amino acid sequence of SEQ ID NO:34; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO:36, a VL FR2 comprising the amino acid sequence of SEQ ID NO:37, a VL FR3 comprising the amino acid sequence of SEQ ID NO:38; and a VL FR4 comprising the amino acid sequence of SEQ ID NO:39.

[0100] In an embodiment, the VH comprises the amino acid sequence of SEQ ID NO:40 or an amino acid sequence having at least 80% sequence identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:41 or an amino acid sequence having at least 80% sequence identity thereto.

[0101] In an embodiment, the VH is encoded by the nucleic acid sequence of SEQ ID NO:42 and the VL is encoded by the nucleic acid sequence of SEQ ID NO:43.

[0102] In an embodiment, the antigen -binding molecule is an antibody or a SPINT1 -binding fragment thereof.

[0103] The term “antibody”, as used herein, is to be understood to mean any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that binds specifically to, or interacts specifically with, the target antigen (SPINT1). The term “antibody” encompasses full-length immunoglobulin molecules comprising two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (which may be abbreviated as HCVR, VH or VH) and a heavy chain constant region. The heavy chain constant region typically comprises three domains - CHI, CH2 and CH3. Each light chain comprises a light chain variable region (which may be abbreviated as LCVR, VL, VK, VK or VL) and a light chain constant region. The light chain constant region will typically comprise one domain (CLI). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, also referred to as framework regions (FR). Each VH and VL typically comprises three CDR and four FR, arranged from amino -terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FR of the antigen-binding molecules described herein may be identical to the FR of germline sequences of the species from which the CDR sequences were derived or generated. In some embodiments, the FR may be naturally or artificially modified. Whilst it is generally desirable that each of the FR sequences are identical to FR sequences derived from immunoglobulin molecules of the species from which the CDR sequences were derived or generated, in some embodiments, the antigen-binding molecule, or antigen-binding fragment thereof, may comprise one or more amino acid residues across one or more of its FR sequences that would be foreign at a corresponding position in one or more FR from that species (e.g., chimeric antibodies). Preferably, where the antigen-binding molecule, or antigen-binding fragment thereof, comprises one or more amino acid residues across one or more of its FR sequences that would be foreign at a corresponding position in the species from which the CDR sequences were derived or generated, that "foreign" amino acid residue will not adverselyimpact the binding specificity of the antigen-binding molecule or antigen-binding fragment thereof to SPINT1, including native SPINT1.

[0104] Suitable antibodies include IgG, and sub-classes thereof. The subunit structures and three-dimensional configurations of different classes of immunoglobulins will be well known to persons skilled in the art.

[0105] As used herein, the term “complementarity determining region” (CDR) refers to the region of an immunoglobulin variable domain that recognizes and binds to the target antigen. Each variable domain may comprises up to three CDR sequences, identified as CDR1, CDR2 and CDR3. The amino acid sequence of each CDR is often defined by Kabat numbering (e.g., about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) of the light chain variable domain and residues 31-35 (Hl), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain; Kabat el al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or by Chothia numbering (e.g., about residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) of the light chain variable domain and 26-32 (Hl), 53- 55 (H2) and 96-101 (H3) of the heavy chain variable domain; see Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0106] In an embodiment, the antigen-binding molecule, as described herein, is conjugated to another molecule or moiety, illustrative examples of which include detectable moiety such as fluorophores and radioisotopes. The antigen-binding molecule described herein may suitably be labelled with a detectable moiety. Thus, in an embodiment, the antigen-binding molecule, as described herein, is conjugated to a detectable moiety. In an embodiment, the detectable moiety is a fluorophore. In an embodiment, the detectable moiety is biotin. Where the detectable moiety is biotin, the presence of the antigen-binding molecule may conveniently be detected using streptavidin and / or avidin conjugated to a secondary detectable label, illustrative examples of which include fluorescein, rhodamine, AMCA, biological fluorophores such a green fluorescent protein, phycoerythrin, and allophycocyanin, and quantum dots. In an embodiment, the secondary detectable label is Eu3+. In an embodiment, the presence of the detectable moiety is measured using time-resolved fluorometry or dissociation-enhanced time- resolved fluorometry.

[0107] The present disclosure also extends to multivalent antigen-binding molecules, as appropriate, illustrative example of which include bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems [(scFv)2 fragments], diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e. leucine zipper or helix stabilized) scFv fragments. Antigen-binding molecules also include aptamers, as are described in the art. In an embodiment, the antigen-binding molecule is an aptamer. Suitable aptamers, including methods of making same, will be familiar to persons skilled in the art, illustrative examples of which are described elsewhere herein.

[0108] It is to be understood that modifications may be also made to the antigen-binding molecules described herein, including to the CDR sequences, without adversely impacting the binding specificity of the antigen-binding molecules described herein. Thus, the present disclosure extends to functional variants of the antigen-binding molecules disclosed herein. The term "functional variant", as used herein, is to be understood as meaning a SPINT1 -binding molecule comprising an amino acid sequence that differs from a parent or comparator sequences (e.g., VH and / or VL) by one or more (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21 and so on) amino acid deletions, insertions and / or substitutions, wherein said difference does not, or does not completely, abolish the ability of the variant to specifically bind to SPINT1. Suitable methods of determining whether a variant retains said function will be familiar to persons skilled in the art, illustrative examples of which include ELISA. A functional variant may comprise an amino acid sequence that differs from the comparator sequence (e.g., of any one of SEQ ID NOs: 1-22, and 26-41). In some embodiments, the functional variant may comprise amino acid substitutions that enhance the binding affinity of the antigen -binding molecule to SPINT1, as compared to the reference molecule to which the variation is compared (e.g., an antigen-binding molecule comprising the VH and VL of SEQ ID NOs: 20-22, 40 and 41). In an embodiment, the functional variant differs from the comparator by one or more conservative amino acid substitutions, as described elsewhere herein.

[0109] Reference to "at least 80% sequence identity" includes 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity the reference sequence (e.g., of any one of SEQ ID NOs: 1-22, and 26-41), for example, after optimal alignment or best fit analysis. Thus, in an embodiment, the functional variant of theantigen-binding molecule comprises, consists or consists essentially of an amino acid sequence that has at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or preferably 100% sequence identity to any one of SEQ ID Nos: 1-22, and 26- 41, for example, after optimal alignment or best fit analysis. In another embodiment, the CDR sequences of the functional variant of the antigen-binding molecule comprise, consist or consist essentially of amino acid sequences having at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or preferably 100% sequence identity to any one of SEQ ID NOs: 1-22, and 26-41, for example, after optimal alignment or best fit analysis.

[0110] The terms “antigen -binding fragment”, “SPINT1 -binding fragment”, “antigen-binding domain”, “antigen-binding site” and the like are used interchangeably herein to refer to a part of an antibody that retains the ability to bind to SPINT1. These terms include naturally occurring, enzymatically obtainable, synthetic or genetically engineered (recombinant) polypeptides and glycoproteins that specifically bind to SPINT1 to form a complex.

[0111] Antigen-binding fragments may be derived, for example, from naturally-derived immunoglobulin molecules using any suitable method known to persons skilled in the art, illustrative examples of which include proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of nucleic acid sequences encoding antibody variable and optionally constant domains. Suitable nucleic acid sequences are known and / or are readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The nucleic acid sequences may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0112] Non-limiting examples of suitable antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated CDR such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, one-armed antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), and small modular immunopharmaceuticals (SMIPs), are also encompassed by the term “antigen-binding fragment,” as used herein.

[0113] In an embodiment, the antigen-binding fragment comprises at least one immunoglobulin variable domain. The variable domain may comprise an amino acid sequence of any suitable length or composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. Where the antigen-binding fragment comprises a VH domain and a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0114] In another embodiment, the antigen-binding fragment comprises at least one variable domain covalently linked to at least one constant domain. Non-limiting configurations of variable and constant domains that may be found within an antigen-binding fragment include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3, (vi) VH-CH2- CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2, (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2- CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. In some embodiments, the antigen -binding fragment, as herein described, may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalentassociation with one another and / or with one or more monomeric VH or VL domains (e.g.. by disulfide bond(s)). A multispecific antigen-binding molecule will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antigenbinding molecule format, including bispecific antigen-binding molecule formats, may be adapted for use in the context of an antigen -binding fragment of an antibody of the present disclosure using routine techniques available in the art.

[0115] The term “variable region” or “variable domain” refers to the domain of an immunoglobulin heavy or light chain that is involved in binding to the target antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native immunoglobulin molecule will generally have similar structures, with each domain comprising four conserved framework regions and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0116] In an embodiment, the SPINT1 -binding fragment is selected from the group consisting of an Fab fragment, an scFab, an Fab’, a single chain variable fragment (scFv) and a one-armed antibody.

[0117] The terms “specifically binds”, “specific binding” and the like, as used herein, typically refer to a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times background molecular associations under physiological conditions. When using one or more detectable binding agents that are proteins, specific binding is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen-binding molecule binds to a particular antigenic determinant, thereby identifying its presence. Specific binding to an antigenic determinant under such conditions requires an antigen-binding molecule that is selected for its specificity to that determinant. This selection may be achieved by subtracting out antigen-binding molecules that cross-react with other molecules. A variety of immunoassay formats may be used to select antigen-binding molecules {e.g., immunoglobulins), such that they are specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Methods of determining binding affinity and specificity are also well known in the art (see, for example, Harlow and Lane, supra); Friefelder, “Physical Biochemistry: Applications to biochemistry and molecular biology” (W.H. Freeman and Co. 1976)).

[0118] The terms “affinity” or “binding affinity” typically refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antigenbinding molecule) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair e.g., an antigen -binding molecule. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (kOff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

[0119] Methods of making the antigen-binding molecules described herein, including antibodies, derivatives, analogs and fragments thereof, and aptamers, are well-known in the art. For example, polyclonal antibodies can be generated by immunization of an animal, and monoclonal antibodies can be prepared according to standard (hybridoma) methodology. Antibodies, derivatives, analogs, and fragments thereof, can suitably be prepared recombinantly by isolating a DNA fragment from DNA encoding a monoclonal antibody and subcloning the appropriate V regions into an appropriate expression vector according to standard methods. Phage display and aptamer technology is described in the literature and permit in vitro clonal amplification of the antigen-binding molecule described herein with very affinity low cross-reactivity. Phage display reagents and systems are available commercially, and include the Recombinant Phage Antibody System (RPAS), commercially available from Amersham Pharmacia Biotech, Inc. of Piscataway, New Jersey and the pSKAN Phagemid Display System, commercially available from MoBiTec, LLC of Marco Island, Florida.

[0120] Aptamer technology is described for example and without limitation in US Patent Nos. 5,270,163; 5,475,096; 5,840,867 and 6,544,776.Nucleic acid molecules

[0121] The present disclosure also extends to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antigen-binding molecule described herein.

[0122] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:24 or SEQ ID NO: 25, or a nucleic acid sequence having at least 80% sequence identity to any of the foregoing.

[0123] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:24, or a nucleic acid sequence having at least 80% sequence identity thereto.

[0124] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:25, or a nucleic acid sequence having at least 80% sequence identity thereto.

[0125] Also disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antigen-binding molecule described herein.

[0126] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:42, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:43, or a nucleic acid sequence having at least 80% sequence identity to any of the foregoing.

[0127] In an embodiment, the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:43.

[0128] The term “polynucleotide” or “nucleic acid” are used interchangeably herein to refer to a polymer of nucleotides, which can be mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0129] The present disclosure also extends to an expression construct comprising a nucleic acid sequence encoding the antigen-binding molecule described herein, operably linked to one or more regulatory sequences.

[0130] By “ control element”, “control sequence”, "regulatory sequence" and the like, as used herein, is meant a nucleic acid sequence e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell. The control sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a cis-acting sequence such as an operator sequence and a ribosome binding site. Control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.

[0131] Also disclosed herein is a vector comprising a nucleic acid sequence encoding the antigen-binding molecule described herein.

[0132] The present disclosure also extends to a host cell comprising the expression construct disclosed herein.

[0133] The terms “host”, “host cell”, “host cell line” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate the antigen binding molecules of the present invention. Host cells include cultured cells, e.g., mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plantcells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a CHO or HEK293 cell line.Kits and devices

[0134] The present disclosure also contemplates a kit comprising the antigen-binding molecule described herein.

[0135] Kits may suitably contain reagents for detecting the antigen-binding molecule described herein, including in accordance with the methods described herein. Kits for carrying out the methods described herein may include, in suitable container means, (i) the antigen-binding molecule described herein, (ii) optionally, a probe that specifically binds to the antigen-binding molecule described herein, (iii) optionally, a label for detecting the presence of the probe and (iv) optionally, instructions for how to detect SPINT1 in a sample. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and / or other container into which the antigen-binding molecule described herein may be placed and / or suitably aliquoted. Where a second and / or third and / or additional component is provided, the kit will also generally contain a second, third and / or other additional container into which this component may be placed. Alternatively, a container may contain a mixture of more than one reagent, when required. The kits will typically include means for containing the reagents (e.g., the antigen-binding molecule described herein, etc.) in close confinement for commercial sale. Such containers may include injection and / or blow-moulded plastic containers into which the desired vials are retained.

[0136] The kits may further comprise positive and negative controls, including a stock or reference solution comprising SPINT1 protein, as well as instructions for the use of kit components contained therein, including in accordance with the methods described herein.

[0137] The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtiter plate dilution buffers and the like. The kit can also feature various devices (e.g., one or more) and reagents (e.g., one or more) for performing the methods described herein.

[0138] Also disclosed herein is a device comprising the antigen-binding molecule described herein. Suitable devices will be familiar to persons skilled in the art, illustrative examples ofwhich include point of care devices, such as a lateral flow device. Thus, in an embodiment, the device is a point of care device.

[0139] In an embodiment, the device is a lateral flow device.

[0140] Unless stated otherwise, the terms "lateral flow assay", "LFA", "lateral flow immunoassay" and "LFIA" are used interchangeably herein to denote an assay format or device that includes a series of operably connected active regions or elements at which various components of the assay are located. A common type of lateral flow assay device includes a zone, area or region for receiving the liquid sample, a conjugate region, and a reaction or test region. These assay devices are commonly known as lateral flow test strips and typically employ a porous material, e.g., nitrocellulose, defining a path for fluid flow and capable of supporting capillary flow. Other illustrative examples of suitable test strip material include Polyethylene terephthalate (PET) fibers, such as Dacron™ fibers, nitrocellulose, polyester, nylon, cellulose acetate, polypropylene, glass fibers, and a combinations of any of the foregoing materials and their backings.

[0141] Suitable LFA formats and devices, including immunochromatographic LFA, will be well known to persons skilled in the art, illustrative examples of which are described in the literature; see, e.g., "Rapid Lateral Flow Test Strips. Considerations for Product Development," EMD Millipore 2013; Koczula, et al., 2016, "Lateral flow assays," Essays in Biochemistry 60: 111-120; Sharma, et al., 2015, "Point-of-Care Diagnostics in Low Resource Settings: Present Status and Future Role of Microfluidics," Biosensors 5: 577-601; Sajid, et al., 2014, "Designs, formats and applications of lateral flow assay: A literature review," J. Saudi Chem. Soc. 19:689-705; Holstein, et al., "Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers," 2016, Analytical and Bioanalytical Chemistry 408(5): 1335-46; US Patent No. 9,034,657, "Two step lateral flow assay methods and devices"; US Patent Nos. 4,313,734; 4,376,110; 4,435,504; 4,703,017; 4,855,240; 4,954,452; 5,028,535; 5,075,078; 5,654,162; WO 95 / 16207; EP 0810436; and US Patent No. 8,859,265, "Lateral flow immunoassay device with a more rapid and accurate test result", each document incorporated by reference herein in its entirety. Other illustrative examples of suitable LFA formats are described in US Patent Nos. 5,559,041, 5,714,389, 5,120,643, and 6,228,660 and international publication nos. WO 2003 / 103835, WO 2005 / 089082, WO 2005 / 118139, WO 2006 / 137785 and WO 2022 / 027088, each of which are incorporated herein by reference in their entirety.

[0142] Without being bound by theory or a particular mode of application, the general principle behind LFA can be described as a fluid sample (or an extract thereof) comprising the analyte(s) of interest flows (e.g., by capillary action) through various regions of a polymeric strip on which reagents are bound, immobilised or otherwise attached that can interact with the analyte(s) of interest. A typical lateral flow test strip may comprise overlapping membranes that are mounted on a substrate such as a backing card for better stability and handling. The fluid sample is applied at one end of the strip, on the adsorbent sample pad or application region, which is typically impregnated with buffer salts and surfactants that make the sample suitable for interaction with the detection system. The sample region ensures that the analyte(s) present in the sample are capable of binding to the capture reagents that are employed on the strip. The sample then flows or migrates through a conjugate release pad or conjugation region, which will comprise a binding moiety that is specific to the analyte(s) or interest. The binding moiety (also referred to herein as a tracer antigen) will suitably comprise a detectable moiety, typically a coloured or fluorescent molecule or particle, such as colloidal gold or latex microspheres. The sample, together with a complex comprising the binding moiety bound to the analyte(s) of interest, migrates along the strip towards the detection zone or test region. The detection zone / test region is typically a porous membrane (usually composed of nitrocellulose) comprising an immobilised capture probe (usually an antibody or a binding molecule). The capture probe is typically immobilised onto the test strip in a line configuration, although alternative configurations may be suitable. The immobilised capture probe reacts with the complex comprising the binding moiety and the analyte(s) of interest. Recognition of the sample analyte(s) results in a detectable response (e.g., change in colour) within the test region. An LFA device may optionally comprise a control region beyond the test region in the direction of flow. The control region will suitably comprise a binding agent that reacts with or binds to the binding moiety from the sample or the conjugation region, as an indicator that the device has performed properly, insofar as there has been sufficient flow of the sample and reagents from the sample application region across the strip to the control region. The read-out, typically represented by visible lines on the test strip, can be assessed by eye or by an automated reader. In an embodiment, the presence of SPINT21 in the sample is indicated by the absence of a detectable signal visible to the naked eye at the test region.

[0143] Where an automated reader is used to detect a signal at the test and / or control regions, the optical density (OD) values can be used in combination with a pre -determined standard curve (calibration curve) to determine the concentration of the analyte(s) of interest in the sample.

[0144] LFA can be characterised into two general formats: direct and competitive. A direct format is typically used for larger analytes such as the p24 antigen used in the human immunodeficiency virus (HIV) test, as well as analytes with multiple antigenic sites, such as human chorionic gonadotropin (hCG), as used in pregnancy tests. The hCG test is an example of a sandwich-based assay, where the target is immobilized between two complementary antibodies. In the direct test, the presence of a visible test region indicates a positive result and the control region usually contains species-specific anti-immunoglobulin antibodies, specific for the antibody in the conjugate. In the case of small molecules with single antigenic determinants, which are unable to bind to two antibodies simultaneously, competitive LFA formats are often used. In an embodiment, the LFA format is a competitive LFA format.

[0145] In an embodiment, the LFA device may be configured to test multiple analytes simultaneously, including under the same conditions. For example, additional test regions of capture antigens specific to different analytes can be immobilized in an array format on the device.

[0146] In an embodiment, multiple test regions comprising the same capture antigen in different amounts can be used for semi-quantitative analysis of the target analyte. The principle of this assay format is based on the stepwise capture of analyte-tracer antigen complexes by the immobilized capture antigen on each successive test region, where the number of detectable / visible regions appearing on the test strip is directly proportional to the concentration of the analyte in the sample.

[0147] As noted elsewhere herein, the sample will flow across the device, typically by capillary forces, from the sample application region to the control region. To maintain flow, an absorbent pad may be attached to the LFA device beyond the control region in the direction of flow, the purpose of which includes to wick any excess reagents and prevent backflow of the fluid along the device.

[0148] The term "direction of flow" is used herein to denote the flow of the fluid sample from the application region towards the control region of the LFA device. The flow is typicallyfacilitated by capillary forces. The capillary driven flow may be advantageously controlled by one or more of methods suitable for interrupting the capillary flow of the fluid sample, illustrative examples of which include opening or closing an external vent, imposition of a soluble membrane along the flow path, imposition of a non-soluble but removable membrane along the flow path, decreasing the capillary force by compressing the capillary bed and limiting the flow path. Such devices can be incorporated into the LFA device during manufacture. Illustrative examples of sample flow control that can be employed in the LFA devices described herein are shown in US Patent Nos: 5,620,657; 5,705,397; 6,901,963; 7,803,319 and US patent publication Nos: 2002 / 0119486; 2010 / 0159599; 2011 / 0306072, the contents of which are incorporated herein by reference in their entirety.

[0149] The LFA device can be of any suitable shape and / or dimension, such as one or a combination of square, round, oval, polygonal, hexagonal, and the like. In an embodiment, the LFA device has a substantially rectangular shape or configuration.

[0150] The LFA device may suitably comprise a substrate comprising, at least in part, any bibulous or non-bibulous material, such as nitrocellulose, nylon, paper, glass fiber, dacron, polyester, polyethylene, olefin, or other cast or thermoplastic materials such as polyvinyl chloride, polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride, polyamide, polycarbonate, polystyrene, etc. In an embodiment, at least one test strip material is nitrocellulose having a pore size of at least about 1 micron, more preferably of greater than about 5 microns, or about 8-12 microns. Suitable nitrocellulose sheets having a nominal pore size of up to approximately 12 microns, are available commercially from sources that will be known to persons skilled in the art, including, for example, Schleicher and Schuell GmbH.

[0151] The LFA device may optionally include indicia that can include a designation for the test to be performed using the test strip. Such indicia may be printed on the test strip material using methods known in the art. Alternatively, indicia may be on other thin members, such as plastic or paper that are attached to the test strip, such as by adhesives, tape or the like.

[0152] The LFA device may include one or more materials. If a device comprises more than one material, the two or more of the materials are preferably in fluid communication with one another. For example, one material of the device may be overlaid on another material of the device, such as, for example, filter paper overlaid onto nitrocellulose. Alternatively, or in addition, the device may include a region comprising one or more materials followed by aregion comprising one or more different materials. In this context, the regions will suitably be in fluid communication and may or may not partially overlap one another.

[0153] As noted elsewhere herein, the material or materials of the LFA device can be bound to a support or solid surface such as found, for example, in thin-layer chromatography and may have an absorbent pad either as an integral part or in liquid contact. For example, the device may comprise nitrocellulose sheet "backed", for example with a supporting sheet, such as a plastic sheet, to increase its handling strength. This can be manufactured by forming a thin layer of nitrocellulose on a sheet of backing material. The actual pore size of the nitrocellulose when backed in this manner will tend to be lower than that of the corresponding unbacked material. Alternatively, a pre-formed sheet of nitrocellulose and / or one or more other bibulous or non-bibulous materials can be attached to at least one supporting sheet, such as a sheet made of polymers (see, e.g., US Patent No. 5,656,503, the contents of which is incorporated herein by reference in its entirety). A supporting sheet can be transparent, translucent or opaque. Where the support sheet is transparent, the supporting sheet is preferably moisture impervious but can be moisture resistant or moisture pervious. In an embodiment, the device can be viewed through a window. In some embodiments, the window may comprise a transparent material such as glass, plastic or mylar.

[0154] The component parts of the LFA device may be present in a suitable housing, examples of which will be known to persons skilled in the art. The housing may suitably be configured to enclose the bibulous member and other assay components. The housing may be fabricated from any suitable material, where the material may be a material that is sufficiently rigid to maintain the integrity of the bibulous member and other components housed therein and will also be suitably inert to the various fluids and reagents that contact the housing during use. Suitable housing material includes plastics. The housing may include a port or analogous structure configured to allow sample application to the sample application region and one or more windows configured to allow viewing of the test and control regions. The housing may further comprise markings, such as test region and control region markings (e.g., "T" and "C"), etc.

[0155] The LFA device and methods disclosed herein may provide qualitative or quantitative results. Qualitative results typically include results that provide a simple "yes" or "no" determination of whether the analyte of interest (e.g., SPINT1) is present in the sample beingassayed. Qualitative results also include results that are positive if the amount of analyte in the sample exceeds a pre-determined threshold or reference value.

[0156] In an embodiment, where LFA device is configured to provide a qualitative result, such as where the analyte needs to be at a certain minimum concentration to be used in subsequent procedures, the assay device may be configured to have lower sensitivity than a comparable LFA device that is configured to detect the presence of the analyte(s) of interest at any concentration. Thus, in circumstances where a qualitative result in the format of an analyte simply being present in an amount that exceeds a pre-determined threshold is desired, the LFA device may be configured to have a sensitivity that is not sufficient to provide detection below the threshold. If the LFA device is too sensitive, there is a risk of a false positive result where an analyte that is too low in concentration to be useful nonetheless yields a positive result. This sensitivity can be set to any minimum amount of analyte in the sample. In some embodiments, multiple LFA devices (e.g., in the form of test strips) may be supplied (e.g., in the form of a kit) with different sensitivities depending on the necessary threshold for analyte utility. These types of qualitative embodiments are suitably distinguished from LFA devices that are configured to be sensitive for all levels of analyte in a given sample. The desired sensitivity may be provided in a given LFA device using any convenient protocol, such as by providing an appropriate amount of capture agent in the detection region, etc.

[0157] In contrast, quantitative results provide some measurement of how much of the analyte(s) of interest (e.g., SPINT1) is present in the sample. Accordingly, a quantitative result provides at least an approximation of the amount of the analyte(s) of interest that is present in the sample being assayed. To provide for quantitative results, the detection region may suitably include two or more distinct test regions that include the same or different amounts of the same capture antigen. As such, if the amount of analyte in the sample exceeds the amount of the analyte that can be captured in the first test region, the remaining free analyte will move to the second or subsequent test region. The resultant positive results from the first and second or subsequent test regions provide a quantitative measurement of the amount of analyte in the sample. By having a series of regions, which may be a gradient of two or more test regions each having differing (such as decreasing) amounts of capture antigen, a quantitative measurement of the analyte in the sample may be obtained. Alternatively, quantitativemeasurements can be obtained by densitometry, where only one capture region may be sufficient.

[0158] The present disclosure also extends to a multiplex LFA format in which the presence of two or more distinct (z.e., different) analytes in the sample is determined, either qualitatively or quantitatively. The number of distinct analytes that may be detected in a given multiplex assay may vary, ranging in some instances from 2 to at least 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and so on). To provide for multiplex analysis, the configuration of the lateral flow assay device may vary. For example, the lateral flow assay device may include a single sample application region and a test region that includes capture antigens for each of the two or more analytes, where the different capture antigens may be present in the same test region or in different test regions, which may depend on whether the detectable moieties employed for each analyte are distinguishable from each other. Accordingly, the lateral flow assay device may suitably include a single flow lane linking the sample application region to the test region. Another example of a suitable multiplex configuration includes a separate sample application region and test region for each of the two or more analytes of interest. Accordingly, the lateral flow assay device may include two or more distinct flow lanes, each having its own sample application region and test region. Other configurations include a configuration having multiple flow lanes extending from a single sample application region to multiple test regions, where a separate test region is provided for each analyte of interest. Additional details regarding multiplex configurations are described in US Patent No. 6,037,127, the disclosure of which is herein incorporated by reference in its entirety. Thus, in an embodiment disclosed herein, the LFA device disclosed herein comprises an array or a plurality (e.g., 2, 3, 4, 5 and so on) of test strips configured to detect a plurality of analytes of interest. For example, the LFA device may comprise a plurality of test strips configured to (i) receive the sample (whether via a single sample application region or a plurality of sample application regions) and (ii) move the sample by capillary action in the direction of flow from the sample application region(s) to each of the plurality of test strips, wherein at least two of the plurality of test strips each comprises a test region for detecting the presence of the analyte (e.g., SPINT1). The array or plurality of test strips may suitably be configured in the same cassette or housing, whether in a parallel configuration or otherwise, for ease of use. Illustrative examples of multiplex LFA devices are described in Anfossi et al. (2019, Biosensors (Basel)-, 9(1):2).

[0159] The present disclosure also extends to ECLIA, ELISA and Luminex LabMAP immunoassays as illustrative examples of suitable assays for detecting the level of SPINT1 using the antigen-binding molecules described herein. In an embodiment, a first SPINT1- binding molecule is immobilised to a surface and a second SPINT1 -binding molecule comprising a detectable moiety is configured to bind to SPINT1 when the SPINT1 is bound to or complexed with the immobilised first SPINT1 -binding molecule. Illustrative examples of suitable detectable moieties include fluorochromes, flurophores, enzymes, epitopes for binding a second binding reagent (e.g., when the second binding reagent / antibody is a mouse antibody, which is detected by a fluorescently-labelled anti-mouse antibody), for example an antigen or a member of a binding pair, such as biotin. The surface may be a planar surface, such as in the case of a typical grid-type array (for example, but without limitation, 96-well plates and planar microarrays) or a non-planar surface, as with coated bead array technologies, where each "species" of bead is labeled with, for example, a fluorochrome (such as the Luminex technology described in U. S. Patent Nos. 6,599, 331,6, 592,822 and 6,268, 222), or quantum dot technology (for example, as described in U. S. Patent No. 6,306. 610). Such assays may also be regarded as laboratory information management systems (LIMS).

[0160] Bead-type immunoassays are also contemplated herein, an illustrative example of which is the Luminex LabMAP system. The LabMAP system typically incorporates polystyrene microspheres that are dyed internally with two spectrally distinct fluorochromes. Using precise ratios of these fluorochromes, an array is created consisting of 100 different microsphere sets with specific spectral addresses. Each microsphere set can possess a different reactant on its surface. Because microsphere sets can be distinguished by their spectral addresses, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorochrome coupled to a reporter molecule quantifies the biomolecular interaction that has occurred at the microsphere surface. Microspheres are interrogated individually in a rapidly flowing fluid stream as they pass by two separate lasers in the Luminex analyzer. High-speed digital signal processing classifies the microsphere based on its spectral address and quantifies the reaction on the surface in a few seconds per sample.

[0161] As used herein, the term "immunoassay" refers to immune assays, typically, but not exclusively to sandwich assays, capable of detecting and quantifying the biomarker of interest, including SPINT1.

[0162] As indicated above, the antigen-binding molecule described herein may be used in any of a number of immunoassays which rely on the binding interaction between an antigenic determinant of the biomarker and the antigen-binding molecule. Examples of such assays are radioimmunoassay, enzyme immunoassays (e.g. ECLIA, ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination and histochemical tests. The antigen-binding molecule described herein may be used to detect and quantify the level of the biomarker in a sample in order to determine the level of placental sufficiency or insufficiency.

[0163] The antigen-binding molecule described herein or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies, etc. For example, the carrier or support may be nitrocellulose, or glass, polyacrylamides, gabbros, and magnetite. The support material may have any possible configuration including spherical (e.g., bead), cylindrical (e.g., inside surface of a test tube or well, or the external surface of a rod), or flat (e.g., sheet, test strip). Indirect methods may also be employed in which the primary antigenantibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against biomarker protein. By way of example, if the antigen-binding molecule described herein is a rabbit IgG antibody, the second antibody may be goat anti -rabbit gamma-globulin labeled with a detectable substance as described herein.

[0164] Where a radioactive label is used as a detectable substance, the biomarker may be localized by radioautography. The results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.Methods of use

[0165] The antigen-binding molecules described herein may suitably be used to improve pre- and post-natal health outcomes for fetuses, including fetuses developing in an environment of placental insufficiency, as previously described in WO / 2019 / 222812, the entire contents of which is incorporated herein by reference.

[0166] In an aspect disclosed herein, there is provided a method of determining the state of placental health in a female subject, the method comprising determining the circulating level of SPINT1 in a biological sample from the female subject using the antigen -binding molecule described herein, wherein a reduction in the circulating level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of placental insufficiency.

[0167] In yet another aspect disclosed herein, there is provided a method of determining whether a fetus of a pregnant female subject is at risk of abnormal fetal weight, the method comprising determining the circulating level of SPINT1 in a biological sample from the female subject using the antigen-binding molecule described herein, wherein an elevation in the circulating level of SPINT1 relative to a control or over time, or a change in ratio relative to a control or over time, is indicative of abnormal fetal weight, wherein an increase in the circulating level of SPINT1 relative to a control or over time, or a change in ratio relative to a control or over time, is indicative of the likelihood of macrosomia, and wherein a decrease in the circulating level of SPINT1 relative to a control or over time, or a change in ratio relative to a control or over time, is indicative of the likelihood of small-for-gestational age or fetal growth restriction.

[0168] In an embodiment, the method further comprises determining at least one other biomarker and / or physiochemical parameter and / or clinical risk factors.

[0169] In an embodiment, the at least one other biomarker is selected from the group consisting of placental growth factor (P1GF), soluble fms-like tyrosine kinase- 1 (sFlt), SYNDECAN-1 and vascular endothelial growth factor (VEGF).

[0170] In an embodiment, the physiochemical parameter is generated by ultrasound or physical measurement.

[0171] In an embodiment, the circulating level or ratio of levels of SPINT1 is subject to analysis with an algorithm or by analytics function or process or other data processing means.

[0172] In an embodiment, the method further comprises assessing the subject for one or more risk factors associated with increased risk of abnormal fetal weight.

[0173] In an embodiment, the sample is maternal whole blood, plasma or serum.

[0174] In an embodiment, the female subject is determined to have placental insufficiency or where the fetus is determined to be at risk of macrosomia, further comprising subjecting the fetus to an early delivery.

[0175] The present disclosure also extends to a clinical management protocol for a pregnant female subject, the protocol comprising determining the circulating level of SPINT1 in a sample from the female subject using the antigen-binding molecule described herein, wherein a decrease in the circulating level of SPINT1 over time compared to a control or a statistically validated level, or a change in ratio reflecting a decrease in the circulating level of SPINT1 is indicative of placental insufficiency and wherein the fetus is monitored or subject to early delivery.

[0176] In an embodiment, the protocol further comprises determining at least one other biomarker and / or physiochemical parameter.

[0177] In an embodiment, the at least one other biomarker is selected from the group consisting of placental growth factor (P1GF), soluble fms-like tyrosine kinase- 1 (sFlt), SYNDECAN-1 and vascular endothelial growth factor (VEGF).

[0178] In an embodiment, the physiochemical parameter is generated by ultrasound or physical measurement.

[0179] In an embodiment, the circulating level or ratio of levels of SPINT1 is subject analysis with an algorithm or by analytics function or process or other data processing means.

[0180] In an embodiment, the sample is maternal whole blood, plasma or serum.

[0181] Also disclosed herein is an assay for determining the state of placental sufficiency in a female subject, the assay comprising determining the circulating level of SPINT1 in a sample from a pregnant female using the antigen-binding molecule described herein; subjecting the level of SPINT1 to an algorithm or analytics function or process or other data processing means generated from a first knowledge base of data comprising the level of the same biomarkers from a subject or cohort of subjects of known status with respect to placental sufficiency, wherein the algorithm or analytics or data processing provides an index of probability of the subject having or not having placental insufficiency or placental sufficiency.

[0182] The present disclosure also extends to use of a knowledge base of training data comprising (i) circulating levels of SPINT1 as determined using the antigen-binding molecule described herein, and (ii) a level of at least one other biomarker from a pregnant female subject with known placental sufficiency status to generate an algorithm or analytics function or process or other data processing means which, upon input of a second knowledge base of data comprising levels of the same biomarkers from a patient with an unknown placental sufficiency status, provides an index of probability that predicts the nature of the placenta.

[0183] In another aspect disclosed herein, there is provided a panel of reagents for detecting biomarkers associated with placental insufficiency or abnormal fetal weight in a pregnant female subject, wherein the panel or reagents comprises the antigen-binding molecule described herein.

[0184] Also contemplated herein is a method for monitoring the progression of a pregnancy in a female subject, the method comprising:(a) determining the circulating level of SPINT1 using the antigen-binding molecule described herein, and at least one other biomarker in a sample obtained from a pregnant female subject at a first time point,(b) subjecting the levels determined in step (a) to an algorithm or analytics function or process or other data processing means to provide an index of probability of the subject having a placental sufficiency or insufficiency;(c) repeating steps (a) and (b) for circulating level of SPINT1 and at least one other biomarker in a sample obtained from a pregnant female subject at a subsequent time point to obtain an index of probability of the subject having a placental sufficiency or insufficiency, and(d) comparing the index of probability provided in step (b) with the index of probability provided in step (c), wherein a difference in the index of probabilities between the first time point and the subsequent time point is indicative of the progression of the placental health.

[0185] In an embodiment, the circulatory maternal fluid comprises whole blood, plasma or serum.

[0186] In an embodiment, the method further comprises inputting physiochemical data generated by ultrasound or physical measurement.

[0187] In an embodiment, the method further comprises inputting one or more risk factors associated with increased risk of abnormal fetal weight.

[0188] The term "assay" includes, but is not limited to, a method, protocol, step or series of steps and / or a process for determining the level or velocity of SPINT1 in a sample, including a biological sample. The level of SPINT1 may be compared to a control (e.g., a standardized control) or may be compared to a statistically validated pre-determined level. An example of a control is the level of circulating SPINT1 protein in a normal pregnant female subject, or a population of pregnant female subjects, of approximately the same age and physical characteristics. The velocity includes a rate or extent of increase or decrease in the level of SPINT1. The level or velocity of SPINT1 may be expressed as a concentration level in an amount of, but not limited to, picogram, nanogram, microgram or milligram per volume of circulating fluid (generally in millilitres although may be expressed in any volume amount). Alternatively, the level or velocity of the biomarkers is rationalized in the form of a ratio between each other and / or a ratio between one or other and another biomarker, such as placenta growth factor (P1GF) or soluble fms-like tyrosine kinase- 1 (sFlt) or SYNDECAN-1 or vascular endothelial growth factor (VEGF), amongst other markers.

[0189] The level or velocity of SPINT1 may also be subject to analysis by but not limited to a multivariate or monovariate algorithm or other analytics function to establish a value which is compared to a control or statistically validated pre-determined level.

[0190] The present disclosure teaches that a reduction in the level or velocity of SPINT1 is indicative of placental insufficiency in a pregnant female mammalian subject. Depending on what clinical intervention is initiated, the level of SPINT1 can be monitored throughout pregnancy and / or measured at pre-determined stages including at any time in the first, second or third trimester periods. For the avoidance of doubt, the assay may be performed at any gestational time point or window throughout the gestational time period. The ability to detect placental insufficiency via a biomarker or combination of biomarkers increases sensitivity and specificity to a far greater level than physical measurements, ultrasound, examination and / or birthweights or predicted fetal weights. These are encompassed by the term “physiochemical parameters”. Accordingly, enabled herein is an assay to determine the state of placental health in a female mammalian subject, the method comprising determining the circulating level of SPINT1, wherein a reduction in the level of SPINT1 relative to a control or over time or achange in ratio relative to a control or over time is indicative of placental insufficiency and an elevation in the level of SPINT-1 is a measure of placental sufficiency or an improvement in placental sufficiency.

[0191] As used herein, the term "indicative" (e.g., indicative of placental insufficiency or indicative of placental sufficiency) means a sign or indication or factor to be considered, as opposed to being definitive proof in and of itself, and generally refers to an increased likelihood of the presence of a particular condition. For example, a reduction in the level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time generally correlates with an increased likelihood of placental insufficiency. Likewise, an elevation in the level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time generally correlates with an increased likelihood of placental sufficiency.

[0192] In an embodiment, the assay determines the state of placental health in a female mammalian subject, the method comprising determining the circulating levels of SPINT1 wherein a reduction in the concentration of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of placental insufficiency and an elevation in SPINT-1 is a measure of placental sufficiency or an improvement in placental sufficiency.

[0193] In an embodiment, the assay determines the state of placental health in a female mammalian subject, the method comprising determining the circulating levels of SPINT1 and at least one other biomarker wherein a reduction in the concentration of SPINT1 and a change in at least one other biomarker relative to a control or over time or a change in ratio relative to a control or over time is indicative of placental insufficiency and an elevation in SPINT-1 and a change in at least one other biomarker is a measure of placental sufficiency or an improvement in placental sufficiency.

[0194] Also enabled herein is an assay to determine the state of placental health in a female mammalian subject, the method comprising determining the maternal circulating levels of SPINT1, wherein a reduction in the level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of placental insufficiency and an elevation in the level of SPINT-1 is a measure of placental sufficiency or an improvement in placental sufficiency.

[0195] Also taught herein is an assay to detect abnormal fetal weight such as in macrosomia in a female mammalian subject, the method comprising determining the maternal circulating level of SPINT1, wherein an elevation in the level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of potential macrosomia.

[0196] Also enabled herein is an assay to detect abnormal fetal weight in a female mammalian subject, said method comprising determining the maternal circulating level of SPINT1, wherein an elevation in the level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of potential high fetal weight and a reduction in the level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of potential low fetal weight or small-for-gestational age or fetal growth restriction.

[0197] The present invention extends to any mammalian subject, such as a human, non-human primate, a farm animal (e.g. a sheep, cow, horse, pig, alpaca, llama), a racing animal (e.g. a horse, camel or greyhound dog) or a domestic animal (e.g. dog or cat). A racing equine animal includes a quarter horse, thoroughbred, Arab and a warmblood horse. Hence, the present invention has application in human and veterinary clinical practice.

[0198] In an embodiment, the subject is a pregnant human female. In another embodiment, the mammalian subject is a pregnant mare. In yet another embodiment, the mammalian subject is a pregnant thoroughbred mare. The term "subject" includes a patient, pregnant mother and a female of child bearing age.

[0199] The results of the assay may be used in combination with one or more risk factors associated with the subject to further assist in making a diagnosis. The term “risk factors" is meant to include any factor that statistically increases the risk of abnormal fetal weight. Risk factors related to abnormal fetal weight include but are not limited to maternal age, pregestational body mass index, education, smoking, alcohol consumption, in vitro fertilization, anemia in pregnancy, preeclampsia, diabetes, gestational age, pregnancy weight gain, gender of the newborn, history of fetal macrosomia, history of low fetal weight or small- for-gestational age or fetal growth restriction.

[0200] Hence, taught herein is an assay to determine the state of placental health in a pregnant human female subject, the method comprising determining the circulating level of SPINT1, wherein a reduction in the level of SPINT1 relative to a control or over time or a change inratio relative to a control or over time is indicative of placental insufficiency and an elevation in the level of SPINT-1 is a measure of placental sufficiency or an improvement in placental sufficiency.

[0201] In an embodiment, the assay determines the state of placental health in a pregnant human subject, the method comprising determining the circulating levels of SPINT1 and at least one other biomarker wherein a reduction in the concentration of SPINT1 and a change in at least one other biomarker relative to a control or over time or a change in ratio relative to a control or over time is indicative of placental insufficiency and an elevation in SPINT-1 and a change in at least one other biomarker is a measure of placental sufficiency or an improvement in placental sufficiency.

[0202] Further enabled herein is a clinical management protocol for a pregnant human subject, the protocol comprising determining the level of circulating SPINT1, wherein a reduction over time in the level of SPINT1 compared to a control or a statistically validated level or a change in ratio reflecting a lowering of level of SPINT1 is indicative of placental insufficiency and wherein the fetus is monitored or subject to earlier delivery.

[0203] Also taught wherein is a clinical management protocol for macrosomia in a pregnant human subject, the protocol comprising determining the level of circulating SPINT1, wherein an elevation over time in the level of SPINT1 compared to a control or a statistically validated level or a change in ratio reflecting elevation of the level of SPINT1 is indicative of macrosomia wherein the fetus is monitored and potentially subject to earlier delivery.

[0204] The biomarkers are determined within maternal circulatory fluid which includes whole blood, plasma and serum. Other circulatory fluid such as lymph fluid, ascites or urine may also be assayed. The biomarker may be assayed alongside or in combination with physiochemical data obtained such as, by ultrasound.

[0205] The assay may be performed in any number of ways including direct measure of the biomarker or indirectly using the antigen-binding molecule described herein. Conveniently, the assay is an immunoassay such as an ELISA. The assay may be quantitated.

[0206] Hence, disclosed herein is a rapid, efficient and sensitive assay for the identification of placental insufficiency. The condition of placental insufficiency includes the effects caused directly or indirectly by placental insufficiency such as FGR and SGA babies, as well asadverse neonatal outcomes, as described elsewhere herein. In an embodiment, the assay enables early detection of placental insufficiency. Notwithstanding, the present disclosure is not limited to the early detection of placental insufficiency, since the assay may be used at any gestational stage of a pregnancy. In addition, elevated levels of SPINT1 or its ratios indicating an elevation in either or both is proposed to be an indicator of fetal macrosomia.

[0207] Also disclosed herein are ELISA protocols for the identification of placental insufficiency, which as discussed above, also includes the effects caused directly or indirectly by placental insufficiency such as macrosomia, FGR babies, SGA babies, and / or adverse neonatal outcomes, as described elsewhere herein. In an embodiment, the ELISA is a time- resolved fluorescent ELISA.

[0208] In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is about 25-75th centile is indicative of increased risk of placental insufficiency. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is about 25-75th centile is indicative of increased risk of macrosomia. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is about 25-75th centile is indicative of increased risk of FGR and SGA babies. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is about 25-75th centile is indicative of increased risk of adverse composite outcomes. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is about 10-24th centile is indicative of increased risk of placental insufficiency. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is about 10-24th centile is indicative of increased risk of macrosomia. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is about 10-24th centile is indicative of increased risk of FGR and SGA babies. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is about 10-24th centile is indicative of increased risk of adverse composite outcomes. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile is indicative of increased risk of placental insufficiency. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile is indicative of increased risk of macrosomia. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile is indicative of increased risk of FGR and SGA babies. In some embodiments, a circulating level of SPINT1 in a samplefrom a pregnant female that is < 10thcentile is indicative of increased risk of adverse composite outcomes.

[0209] In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile identifies at least about 60% of all babies under the 3rdcentile in a general obstetric population. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile identifies at least about 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51% or 50% of all babies under the 3rdcentile in a general obstetric population. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10thcentile identifies at least about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41% or 40% of all babies under the 3rdcentile in a general obstetric population. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile identifies at least about 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31% or 30% of all babies under the 3rdcentile in a general obstetric population. In an embodiment, a circulating level of SPINT1 in a sample from a pregnant female that is < 10* centile identifies at least about 50% of all babies under the 3rdcentile in a general obstetric population. In an embodiment, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile identifies at least about 45% of all babies under the 3rdcentile in a general obstetric population. In an embodiment, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile identifies at least about 42% of all babies under the 3rdcentile in a general obstetric population.

[0210] In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile is indicative of at least about 40% chance of a baby having composite adverse outcomes. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is < 10* centile is indicative of at least about 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31% or 30% chance of a baby having composite adverse outcomes. In an embodiment, a circulating level of SPINT1 in a sample from a pregnant female that is < 10* centile is indicative of at least about 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21% or 20% chance of a baby having composite adverse outcomes. In an embodiment, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile is indicative of at least about 30% chance of a baby having composite adverse outcomes. In an embodiment, a circulatinglevel of SPINT1 in a sample from a pregnant female that is < 10thcentile is indicative of at least about 26% chance of a baby having composite adverse outcomes.

[0211] The term “composite adverse outcomes” or “composite outcome” as used herein refers to a combined measure that includes multiple, related adverse events, including any one or more of the following (see Figure 1):• Perinatal death• Birthweight <3rdcentile• Apgar <7 at 5 minutes (Agpar is a numerical score assigned to newborns based on five factors: appearance (color), pulse (heart rate), grimace (reflex irritability), activity (muscle tone), and respiration (breathing)).• Resuscitation at birth with CPR and / or IPPV• Admitted to Special Care Nursery or Neonatal Intensive Care Unit for >48 hours, within 48 hours of birth. Cases where there was a specific diagnosis for the admission that is clearly unrelated to placental insufficiency - e.g., major congenital heart defect were excluded.• Requiring respiratory support in nursery (CPAP>4h / hi-flow 02 >4L / ventilation / IPPV)• Neonatal diagnosis of bronchopulmonary dysplasia, hypoxic ischaemic encephalopathy, necrotising enterocolitis, intraventricular haemorrhage.

[0212] In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile, has a positive predictive value of 56% of a baby at risk of having <20* centile birthweight. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile, has a positive predictive value of 39% of a baby at risk of having < 10* centile birthweight. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is < 10* centile, has a positive predictive value of 14% of a baby at risk of having <3rdcentile birthweight. In some embodiments, a circulating level of SPINT1 in a sample from a pregnant female that is <10* centile, has a positive predictive value of 26% of a baby at risk of having <3rdcentile birthweight and composite outcomes as described herein. Even when excluding <3rd centile, the predictive value of composite outcomes is still high for those with a SPINT1 <10th centile (18%), which suggests that a circulating level of SPINT 1 in a sample from a pregnant female that is < 10* centile is indicative of fetuses with likely placental insufficiency, even if the baby is over the 3rd centile.

[0213] Reference to the "algorithm" includes an algorithm which performs, but is not limited to, a multivariate or monovariate analysis function.

[0214] The determination of the concentrations or levels of the biomarkers enables establishment of a diagnostic rule based on the concentrations relative to controls. Alternatively, the diagnostic rule is based on the application of a statistical and machine learning algorithm. Such an algorithm uses relationships between biomarkers and state of placental sufficiency observed in training data (with known placental sufficiency status) to infer relationships which are then used to predict the status of patients with unknown status. An algorithm may be employed which provides an index of probability that a patient has placental insufficiency. As indicated above, the algorithm may perform but is not limited to, a multivariate or monovariate analysis function. Alternatively, the data is subject to analytics or analytical functions or processing.

[0215] Hence, in an embodiment, the present invention provides a diagnostic rule based on the application of a statistical and machine learning algorithm. Such an algorithm uses the relationships between biomarkers and placental sufficiency or insufficiency status observed in training data (with known placental sufficiency status) to infer relationships which are then used to predict the status of patients with unknown placental sufficiency status. Practitioners skilled in the art of data analysis recognize that many different forms of inferring relationships in the training data may be used without materially changing the present invention. Other analytics may also be employed to analyse data and identify associations between biomarkers and / or physiochemical parameters and placental insufficiency or its associated conditions such as FGR.

[0216] Hence, the present invention contemplates the use of a knowledge base of training data comprising the level of SPINT1, optionally with at least one other biomarker, in a sample from a subject with known placental sufficiency status to generate an algorithm or analytics function or process or other data processing means which, upon input of a second knowledge base of data comprising levels of the same biomarkers from a patient with an unknown placental sufficiency status, provides an index of probability that predicts the nature of the placenta. Other analytics functions may also be used.

[0217] The term "training data" includes knowledge of levels of biomarkers relative to a control. A "control" includes a comparison to levels of biomarkers in a subject with adequate placental sufficiency or with known placental insufficiency or may be a statistically determined level based on trials. A statistically determined level may be a statistically validated predetermined level or cut-off that has already been validated as being linked or associated with placental sufficiency or with placental insufficiency. The term "levels" also encompasses ratios of levels of biomarkers and their velocities.

[0218] The "training data" also includes the level of SPINT1. The data may comprise information on an increase or decrease in the level of SPINT1.

[0219] The present disclosure also contemplates a panel of reagents for detecting biomarkers associated with placental insufficiency or state of placental health or dysfunction in a subject, wherein the panel of reagents comprises the antigen-binding molecule described herein, optionally with at least one other agent for detecting a further biomarker to determine levels of the biomarkers and then optionally subjecting the levels to an algorithm or analytics function or process or other data processing means generated from a first knowledge base of data comprising the levels of the same biomarkers from a subject of known status with respect to the conditions wherein the algorithm provides an index of probability of the subject having or not having placental insufficiency.

[0220] The level of SPINT1 may provide the input test data referred to herein as a "second knowledge base of data". The second knowledge base of data either is considered relative to a control or is fed into an algorithm generated by a "first knowledge base of data" which comprise information of the level of the biomarker in a subject with a known placental sufficiency or insufficiency. The second knowledge base of data is from a subject of unknown status with respect to placental sufficiency or insufficiency. The output of the algorithm or analytics function or process or other data processing means is a probability or risk factor, referred to herein as an index of probability, of a subject having a particular level of placental sufficiency or insufficiency. The algorithm or analytics function or process or other data processing means may perform a multivariate or monovariate analysis function or other statistical operation.

[0221] The term "specifically binds" typically means that the antigen-binding molecule will bind preferentially to the target protein (SPINT1) over any non-specific binding to unrelated proteins. Illustrative examples of suitable antigen-binding molecules include antibodies, including chimeric, humanised variants, and antigen-binding fragments of any of the foregoing.

[0222] The present disclosure also provides a panel of reagents comprising the antigen-binding molecule described herein, preferably wherein the antigen-binding molecule is immobilized to a substrate. In an embodiment, the panel of reagents comprises a reagent capable of specifically binding to at least one other biomarker, illustrative examples of which are described elsewhere herein.

[0223] In an embodiment, the term "sample" means circulatory maternal fluid, such as whole blood, plasma or serum. In other embodiments, the sample may suitably be lymph fluid, urine, saliva or vaginal fluid / lavage.

[0224] Data generated from an assay to determine a circulatory level of SPINT1 can be used to determine the likelihood of or progression of the state of placental insufficiency in the subject, as described elsewhere herein. The input of data comprising the levels of the biomarkers is compared with a control or is put into the algorithm which provides a risk value of the likelihood that the subject has or will develop placental insufficiency.

[0225] In context of the present disclosure, "circulating level" includes a level of the biomarker (including of SPINT1) in whole blood or any blood fraction, for example serum or plasma, which can be analyzed according to the methods described herein. By measuring blood levels of a particular biomarker, it is meant that any appropriate blood fraction can be tested to determine blood levels and that data can be reported as a value present in that fraction.

[0226] The present disclosure also extends to a composition comprising a sample obtained from a pregnant female subject and the antigen-binding molecule described herein.

[0227] As described above, methods for diagnosing a state of placental health or dysfunction by determining levels of a specific biomarker and using this level as second knowledge base data in an algorithm generated with first knowledge base data or levels of the same biomarkers in patents with a known placental health. Also provided are methods of detecting placental dysfunction comprising determining the presence and / or velocity of specific identified biomarkers in a subject's sample.By "velocity" it is meant the change in the concentration of the biomarker in a patient's sample (the maternal circulatory fluid) over time.

[0228] In an embodiment, an increased index of probability of placental dysfunction developing at the later time point may indicate that the condition is progressing and that the treatment (if applicable) is not being effective. In contrast, a decreased index of probability at the later time point may indicate that placental sufficiency is improving and that the treatment (if application) is effective. The treatment may include early or earlier delivery of the baby.

[0229] As used herein, the terms “early delivery” and “earlier delivery” are used interchangeably herein to refer to delivery of a fetus before a pregnant subject undergoes natural labor and includes a delivery assistance intervention such as induction of labor. The earlier or earlier delivery may be pre-term (e.g., before 37 weeks of gestation), term (e.g., 37, 38, 39, 40, 41 or 42 weeks of gestation) or post-term (e.g., after 42 weeks of gestation) and includes vaginal, instrumental (forceps or vacuum birth), or Caesarean delivery. In specific embodiments, the terms “early delivery” and “earlier delivery” refer to a delivery assistance intervention (e.g., induction of labor) at 38, 38.5 or 39 weeks of gestation.

[0230] The methods described herein may be performed by utilizing pre-packaged diagnostic kits comprising the antigen-binding molecule described herein, including in a form that may be conveniently used, e.g. in clinical settings, to screen and diagnose patients and to screen and identify those individuals exhibiting a predisposition to developing placental dysfunction. The kit may also include detailed instructions for carrying out the methods described herein.

[0231] Reference to an "algorithm" or "algorithmic functions" as outlined above includes the performance of, but not limited to, a multivariate or monovariate analysis function. Other analytic functions may also or alternatively be performed. A range of different architectures and platforms may be implemented in addition to those described above. It will be appreciated that any form of architecture suitable for implementing the present invention may be used. One technique is the use of distributed architectures. This can increase the efficiency of the system by reducing data bandwidth costs and requirements, as well as ensuring that if one base station becomes congested or a fault occurs, other end stations could take over.This also allows load sharing or the like to ensure access to the system is available at all times.

[0232] In the above aspects, the term "data" means the levels or concentrations or velocities of a biomarker. This may be assayed alone or in combination with physical, chemical or physiochemical parameters such as data obtained from ultrasound or other physical testing procedures. The "communications network" includes the internet. When a server is used, it is generally a client server or more particularly a simple object application protocol (SOAP).

[0233] The present assay can be incorporated into present diagnostic architecture as an additional test during pregnancy or a stand-alone test. For example, the assay may be associated with ultrasound or physical measurements.

[0234] Once the information is available, the data can be used in a clinical management protocol for a pregnancy. This may include a decision to deliver a baby earlier than otherwise planned. Hence, a clinical management protocol for a pregnant mammalian subject, the protocol comprising determining the levels of circulating SPINT1 using the antigen -binding molecule described herein, wherein a reduction over time in the level of SPINT1 compared to a control or a statistically validated level or a change in ratio reflecting a lowering of level of SPINT1 is indicative of placental insufficiency and wherein the fetus is monitored or subject to earlier delivery.

[0235] In an embodiment, a clinical management protocol for a pregnant mammalian subject, the protocol comprising determining the level of circulating SPINT1 and at least one other biomarker wherein a reduction over time in the level of SPINT1 and a change in at least one other biomarker compared to a control or a statistically validated level or a change in ratio reflecting a lowering of the level of SPINT1 and a change in the at least one other biomarker is indicative of placental insufficiency and wherein the fetus is monitored or subject to earlier delivery.

[0236] Further enabled herein is a clinical management protocol for a pregnant mammalian subject, the protocol comprising determining the level of circulating SPINT1, wherein a reduction over time in the level of SPINT1 compared to a control or a statistically validated level or a change in ratio reflecting a lowering of the level of SPINT1 is indicative of placental insufficiency and wherein the fetus is monitored or subject to earlier delivery.

[0237] Further taught herein is a clinical management protocol for macrosomia in a pregnant mammalian subject, the protocol comprising determining the level of circulating SPINT1, wherein an elevation over time in the level of SPINT1 compared to a control or a statistically validated level or a change in ratio reflecting elevating placental insufficiency and macrosomia wherein the fetus is monitored or subject to earlier delivery.

[0238] Hence, in an embodiment, there is provided a clinical management protocol for a pregnant human female subject, the protocol comprising determining the level of circulating SPINT1, wherein a reduction over time in the level of SPINT1 compared to a control or a statistically validated level or a change in ratio reflecting a lowering of levels of SPINT1 is indicative of placental insufficiency and wherein the fetus is monitored or subject to earlier delivery.

[0239] In an embodiment, there is provided a clinical management protocol for a pregnant human female subject, the protocol comprising determining the level of circulating SPINT1 and at least one other bio marker, wherein a reduction over time in the level of SPINT1 and a change in the at least one other biomarker compared to a control or a statistically validated level or a change in ratio reflecting a lowering of the level of SPINT1 and a change in the level of the at least one other biomarker is indicative of placental insufficiency and wherein the fetus is monitored or subject to earlier delivery.

[0240] In certain embodiments, the subject is monitored using the assays, methods or protocols of the present invention to determine the presence or absence of placental insufficiency or its associated conditions such as FGR or SGA, or macrosomia, to provide an indicator as to whether an earlier delivery of the fetus may be required. The monitoring is often conducted by serial testing. In some instances, the pregnant subject is monitored as needed (e.g., on an as- needed basis) using the methods described herein. Alternatively, or in addition, the pregnant subject can be monitored weekly, monthly, or at any pre-specified intervals. In some instances, the pregnant subject is monitored at least once every 24 hours. In some instances the pregnant subject is monitored at least once every 1 day to 30 days. In some instances the pregnant subject is monitored at least once every at least 1 day. In some instances the pregnant subject is monitored at least once every at most 30 days. In some instances the pregnant subject is monitored at least (optionally on average) once every 1 day to 5 days, 1 day to 10 days, 1 day to 15 days, 1 day to 20 days, 1 day to 25 days, 1 day to 30 days, 5 days to 10 days, 5 days to15 days, 5 days to 20 days, 5 days to 25 days, 5 days to 30 days, 10 days to 15 days, 10 days to 20 days, 10 days to 25 days, 10 days to 30 days, 15 days to 20 days, 15 days to 25 days, 15 days to 30 days, 20 days to 25 days, 20 days to 30 days, or 25 days to 30 days. In some instances the pregnant subject is monitored at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 29, 30 or 31 days. In some instances, the pregnant subject is monitored at least once every 1, 2, or 3 months. In some instances, the pregnant subject is monitored via the methods described herein no more frequently than one week, 10 days, two weeks, three weeks, or one month. In other words, the predictive value of the some of the methods described herein can be of clinical use for at least one week, at least 10 days, at least two week, at least three weeks, or at least one month.

[0241] Without wishing to be bound by theory or mode of operation, it is proposed that early delivery of a fetus can be associated with greater risk that the baby will suffer complications in the near (e.g., respiratory distress) or long (e.g., IQ and attention deficit) term. In particular, it is known that delivery before 37 weeks gestation carries significantly increased risk and it is likely that a decision to intervene with a delivery assistance intervention (e.g., labour induction) would be taken on the basis of SPINT1 and optionally one or more other physiochemical parameters and / or risk factors. It is also known that delivery from 37-38 weeks comes with a manageable, but non-zero, risk and is likely to be considered on a case by case basis. By contrast, it is known that delivery at about 38.5-39.5 weeks is not associated with significant risk of complications to the fetus. Furthermore, clinical trials have shown induction is also not associated with an increased risk of medical interventions to the mother, such as caesarean section or instrumental assisted birth (Grobman et al. NEJM 2018, 379:513-523).

[0242] Accordingly, in specific embodiments, a pregnant subject is monitored periodically (as described for example above) using the assays, methods or protocols of the present invention from about 35-37 weeks gestation, and the fetus is subjected to early delivery between about 38 weeks of gestation and about 39 weeks of gestation, on the basis that the results of the assays, methods or protocols indicate placental insufficiency or fetal growth restriction or macrosomia. It is believed that such an intervention could decrease the likelihood of stillbirths (the risk of stillbirth increases significantly post 38 weeks gestation), fetal or maternal injury in pregnancies where there is suspected macrosomia (where the pregnancy isnot induced at term gestation and left to continue until spontaneous labour occurs (Boulvain et al. Lancet 2015;385 (9987):2600-2605) and also other complications that might otherwise occur post 39 weeks gestation. Conversely, if in these embodiments, the results of the assays, methods or protocols indicate placental sufficiency and / or expected weight within the normal range, the fetus is not subjected to early delivery and the pregnant subject is permitted to proceed to later gestations.

[0243] The level of SPINT1 as determined by the methods described herein may suitably be applied to a birthweight calculator, that is, to an apparatus or method of predicting the birthweight of a neonate by correlating the circulating level of SPINT1 in a pregnant female subject to predetermined circulating levels of SPINT1 of a female subject for which the birthweight of the neonate is known.

[0244] The level of SPINT1 as determined by the methods described herein may also suitably be applied to or applied in conjunction with different types of risk calculators (i.e. medical risk calculators incorporating various inputs, such as age, lifestyle factors, and medical history, to calculate a risk score in assisting individuals and healthcare providers make informed decisions); that is an apparatus or method that predicts or estimates the likelihood of a specific outcome, in this case, specific outcomes related to neonate health or placental health in a pregnant female subject, by correlating the circulating level of SPINT1 in a pregnant female subject to predetermined circulating levels of SPINT1 of a female subject for which the health outcome of the neonate is known. Illustrative examples of risk calculators relevant to neonatal health and placental health include, and are not limited to those provided by the Fetal Medicine Foundation i.e., Preeclampsia risk calculator, Small for Gestational Age risk calculator, Miscarriage Risk calculator, (see for example, Muin et al. PLoS ONE 2022; 17(l):e0260964; Figueird-Filho et al. Open Journal of Obstetrics and Gynecology 2012; 2, 298-303; Benitz and Achten Lancet Infect Dis 2021; 21:el34-40; Slagle et al. Am J Perinatol 2025; 42(05): 666- 673). It is to be understood that risk of particular outcomes can be expressed or presented in any number of ways. By the way of illustrative examples, risk could be expressed as a probability of an event occurring out of a total of defined possible outcomes (i.e. 1 in 7 risk, or 1 in 10 risk); relative risk; a relative increased risk or a relative increased risk, where the risk reduction or increase maybe expressed in percentage terms; or odds ratio (compares the odds of an event occurring in one group versus another).

[0245] The present disclosure also extends to an apparatus for predicting the birthweight of a neonate, the apparatus comprising (i) a receiver operative to receive biomarker information, wherein said biomarker information is a circulating level of SPINT1 in a sample from a pregnant female subject; and (ii) a birthweight determiner operative to employ said biomarker information to provide an output that represents a prediction of the birthweight of the neonate; wherein said predicted birthweight is based on a predetermined correlation between circulating levels of SPINT1 of a plurality of pregnant female subjects of the same species and neonatal birthweight.

[0246] In an embodiment, the predetermined correlation between (a) circulating levels of SPINT1 of a plurality of pregnant female subjects of the same species and (b) neonatal birthweight is defined by a regression coefficient (R2) of from about 0.020 and 0.060. In an embodiment, the predetermined correlation is defined by a regression coefficient of from about 0.026 to about 0.52. In an embodiment, the predetermined correlation is defined by a regression coefficient of about 0.52.

[0247] The present disclosure extends to an assay to determine the placental weight and / or the placental surface area in a pregnant female mammalian subject, the method comprising determining the maternal circulating levels of SPINT1, wherein a reduction in the level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of lower placental weight and lower placental surface area. Further enabled herein is a clinical management protocol for a pregnant mammalian subject, the protocol comprising determining the maternal circulating level of SPINT1, wherein a reduction in the level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of lower placental weight and lower placental surface area and wherein the fetus is monitored or subject to earlier delivery.

[0248] The present disclosure also extends to an assay to determine the lean mass of a neonate, the method comprising determining the circulating level of SPINT1 in a pregnant female mammalian subject, wherein a reduction in the level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of the neonate having a lower lean mass. Also enabled herein is a clinical management protocol for a pregnant mammalian subject, the protocol comprising determining the maternal circulating level of SPINT1, wherein a reduction in the level of SPINT1 relative to a control or over time or achange in ratio relative to a control or over time is indicative of the neonate having a lower lean mass and wherein the fetus is monitored or subject to earlier delivery and / or the subject is exposed to a treatment to enhance the lean mass of the neonate.Table 2 - Amino acid and nucleic acid sequences* The amino acid sequences of the VH, VL, CDR and FR are according to Kabat numbering

[0249] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0250] As used herein, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0251] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that may vary by as much 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0252] Throughout this specification and the statements 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.

[0253] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived fromit) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0254] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.EXAMPLES

[0255] Certain embodiments of the invention will now be described with reference to the following examples, which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.EXAMPLE 1Preparation and cloning of anti-SPINTl antibodiesMaterials and methods

[0256] Hybridoma Clones 11G3C10-2 and 2C11G7-1 cells producing anti-SPINTl monoclonal antibodies were prepared by Genscript USA Inc. (Piscataway, USA). Briefly, total RNA was isolated from the hybridoma cells following the technical manual of RNA-easy Isolation Reagent (Vazyme, Cat. No. : R701-01-AA). Total RNA was then reverse transcribed into cDNA using isotype-specific anti-sense primers or universal primers following the technical manual of SMARTScribe Reverse Transcriptase (TaKaRa, Cat. No.: 639536). The antibody fragments of VH and VL were amplified according to the standard operating procedure (SOP) of rapid amplification of cDNA ends (RACE) of GenScript USA Inc. Amplified antibody fragments were cloned into a standard cloning vector, separately. Colony PCR was performed to screen for clones with inserts of correct sizes. No less than five colonies with inserts of correct sizes were sequenced for each fragment.Sequences of Clone 11G3C10-2

[0257] Heavy chain variable region (VH) nucleic acid sequence (414 bp), showing the nucleic acid sequence encoding the signal peptide sequence (underlined). The nucleic acid sequences encoding the CDR sequences are highlighted in bold text, as determined by Kabat numbering:ATGAACTTTGGGCTGAGCCTGATTTTCCTTGTCCTAATTTTAAAAGGTGTCCAGTGTGAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTATAGCCTCTGGATTCACTTTCAGTACCTATACCATGGGTTGGGTTCGCCAGAGTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATCAGTAGTGGTGGTGATAAGACGTACTATCCAGACAGTGTGAAGGGTCGATTTATCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACGGCCTTGTATTACTGTGTAAAATATGACGACTGGGACCATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCATCGTCTCCTCA

[0258] Heavy chain variable region (VH) amino acid sequence (138 aa) showing the signal peptide sequence (underlined):MNFGLSLIFLVLILKGVQCEVMLVESGGGLVKPGGSLKLSCIASGFTFSTYTMGWVR QSPEKRLEWVATISSGGDKTYYPDSVKGRFIISRDNAKNNLYLQMSSLKSEDTALY YCVKYDDWDHGMDYWGQGTSVIVSS

[0259] Light chain variable region 1 (VL1) nucleic acid sequence (378 bp) showing the nucleic acid sequence encoding the signal peptide sequence (underlined). The nucleic acid sequences encoding the CDR sequences are highlighted in bold text, as determined by Kabat numbering:ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATTATGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATAATGTCTGCATCTCTAGGGGAGGAGATCACCCTAACCTGCAGTGCCAGCTCGAGTGTAAATTACATGCACTGGTACCAGCAGAAGTCAGGCACTTCTCCCAAACTCTTGATTTATACCACATCCAACCTGGCTTCTGGAGTCCCTTCTCGCTTCAGTGGCAGTGGGTCTGGGACCTTTTATTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGTTGCCGATTATTACTGCCATCAGTGGAGTAGTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0260] Light chain variable region 1 (VL1) amino acid sequence (126 aa), showing the signal peptide sequence (underlined):MDFOVOIFSFLLISASVIMSRGOIVLTOSPAIMSASLGEEITLTCSASSSVNYMHWYQO KSGTSPKLLIYTTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDVADYYCHQWSSWTF GGGTKLEIK

[0261] Light chain variable region 2 (VL2) nucleic acid sequence (378 bp) showing the nucleic acid sequence encoding the signal peptide sequence (underlined). The nucleic acid sequences encoding the CDR sequences are highlighted in bold text, as determined by Kabat numbering:ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCA CTGGTGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTA TAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCAT CTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGC AACCTATTACTGCCATCAGTGGAGTAGTTGGACGTTCGGTGGAGGCACCAAGC TGGAAATCAAA

[0262] Light chain variable region 2 (VL2) amino acid sequence (129 aa), showing the signal peptide sequence (underlined):METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGORATISYRASKSVSTSGYSYM HWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCHQ WSSWTFGGGTKLEIKSequences of Clone 2C11G7-1

[0263] Heavy chain variable region (VH) nucleic acid sequence (414 bp), showing the nucleic acid sequence encoding the signal peptide sequence (underlined). The nucleic acid sequences encoding the CDR sequences are highlighted in bold text, as determined by Kabat numbering:ATGGGATGGACCAGGATCTTTATTTTAACCCTGTCAGTAACTACAGGTGTCCACT CTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGGTGGAGAAGCCTGGCGCTTCAG TGAAGATATCCTGCAAGGCCTCTGGTTACACATTCATTGGCTACAATATGAACTGGGTGAGGCAGATCAATGGAAAGAGCCTTGAGTGGATTGGTAACACTGATCCT TATTATGATGGCATAACCTATAACCAGAAGTTCAAGGGCAAGGCCACAATGA CTGTAGACAAATCCTCCAATACAGCCTACATGCAGCTCGAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGGGGTATGGTAATTACGTGATGG ACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA

[0264] Heavy chain variable region (VH) amino acid sequence (138 aa) showing the signal peptide sequence (underlined):MGWTRIFILTLSVTTGVHSEVOLOQSGPEVEKPGASVKISCKASGYTFIGYNMNWVR QINGKSLEWIGNTDPYYDGITYNQKFKGKATMTVDKSSNTAYMQLESLTSEDSAVY YCARGGYGNYVMDYWGQGTSVTVSS

[0265] Light chain variable region (VL) nucleic acid sequence (393 bp) showing the nucleic acid sequence encoding the signal peptide sequence (underlined). The nucleic acid sequences encoding the CDR sequences are highlighted in bold text, as determined by Kabat numbering:ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAG CGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAACCAGTAATGGAAACGCCTATTTGAACTGGTACGTCCAGAAACCAGGCCAGTCTCCACAGGTTTTGATCTA CAGGGTTTCCAACCGATTTTCTGGGGTCCTAGACAGGTTCAGTGGTAGTGGATC AGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGCCTCCAAGTTACACATGTCCCATTCACGTTCGGCTCGGGGACAAC GTTGGAATTAAAA

[0266] Light chain variable region (VL) amino acid sequence (131 aa), showing the signal peptide sequence:MKLPVRLLVLMFWIPASSSDVVMTOTPLSLPVSLGDOASISCRSSOSLETSNGNAYLN WYVQKPGQSPQVLIYRVSNRFSGVLDRFSGSGSGTDFTLKISRVEAEDLGVYFCLQV THVPFTFGSGTTLELKEXAMPLE 2SPINT1 ELISA — Colorimetric vs Time Resolved Fluorescent Immunoassay

[0267] Time Resolved Fluorescent Immunoassay was performed using monoclonal antibody (mAb) Clones 11G3C10-2 and 2C11G7-1, and Eu-labeled Streptavidin (Delfia).A. Protocol1. Black "maxisorp" plates were coated with capture antibody Clone 11G3C10-2 diluted to Ipg / ml. Add lOOpl per well and incubate overnight at room temp;Next day, plates were washed x3 times using wash buffer (PBS with 0.1%Tween20); Blocking solution and diluent (1%BSA / PBS) was prepared and 230pl of the blocking solution was added to each well. The plates were then incubated at room temp for ~ 1 hour; Purified SPINT1 protein (Genscript USA Inc.) stock was prepared at 0.3mg / ml; SPINT1 stock was diluted in 1%BSA / PBS to 40,000pg / ml, then 2-fold serial dilutions were prepared (3ml + 3ml diluent), as outlined below: tubel 40000 ~8ml tube2 20000 3 ml Tube 1+3 ml diluent tube3 10000 3 ml Tube 2+3 ml diluent tube4 5000 3 ml Tube 3 +3 ml diluent tube5 2500 3ml Tube 4+3ml diluent tube6 1250 3ml Tube 5+3ml diluent tube? 625 3ml Tube 6+3ml diluent tube8 diluent Test samples were prepared by centrifugation (including after thawing) and then diluted in sample diluent - 1:6 dilution for plasma from pregnant subjects at 36 week gestation. lOOpl of SPINT1 stock (standard) or test sample were added per well as per plate layout, and incubate at room temp for ~2 hours; Plates were then washed x3 times using a plate washer; Biotinylated detection antibody Clone 2C11G7-1 was diluted to 500ng / ml and lOOpl was added to each, and incubated at room temp for ~2 hours; Plates were then wash x3 times using a plate washer;10. 115mL of lOOng / mL Eu- streptavidin was prepared in diluent (by adding 115pL of lOOpg / mL Eu-SA in 115ml of diluent) and added per well at lOOpl per well. Plates were then incubated for about 20-30 minutes (with slow shaking, ~300rpm) at room temperature;11. Plates were then washed x6 times;12. Enhancement Solution (Perkin Elmer) was dispensed into a clean tube - need enough for 200pl / well. About 200pl Enhancement Solution was then added to each well and incubate for at least 5 mins (~5-15mins) with slow shaking (~300rpm) at room temp; then incubate for a further 40mins.13. Time-resolved fluorescence (TRF) was then measured at 615 nm (setup at 337nm / 620- lOnm).B. Results

[0268] SPINT1 was measured in plasma samples obtained from a cohort of pregnant female subjects using the colorimetric and time-resolved fluorescent ELISA (127 small-for- gestational-age (SGA); 182 cohort / controls).

[0269] The colorimetric ELISA identified significantly reduced circulating SPINT1 protein in subjects with fetuses identified as being SGA, with p=4.03 x 10’5, and an area under the curve (AUC) of 0.64. In contrast, the time-resolved fluorescent ELISA identified significantly reduced circulating SPINT1 protein in subjects with fetuses identified as being SGA, with p=2.13x 1011, and a marked uplift in the AUC to 0.72 compared to the colorimetric ELISA results. This demonstrates that time-resolved fluorescent technology is superior to colorimetric ELISA for determining circulating levels of SPINT1 protein in plasma of pregnant female subjects.EXAMPLE 3SPINT1 at 36 weeks gestation to predict <3rdcentile birth, and adverse neonatal outcomes

[0270] The <3rdcentile is an appropriate indicator of placental insufficiency, as it incurs a 10+ fold increase in stillbirth risk (see Lees et al., 2022; Am J Obstet Gynecol', 226(3): 366-78).

[0271] Prediction at 36 weeks is an appropriate time, as stillbirth risk rises, and timing birth at 38 or 39 weeks is a sensible approach to mitigate the risk of stillbirth.

[0272] To detect <3rdcentile, selective ultrasound performs at only 14% (see Sparks et al., 2011; J Matern Fetal Neonatal Med', 24(5): 708-12) or 32% sensitivity (see Sovio et al., 2015; Lancet', 386(10008): 2089-97) at around 90% specificity.This study sought to provide an improvement over the level of performance provided by ultrasound alone by employing the time-resolved fluorescent ELISA as described in Examples 1 and 2, above.Results

[0273] A low circulating SPINT1 (<10thcentile, n=2900 with validation from UK cohort):Has a 48-55% sensitivity of babies destined to birth <3rdcentile birthweight4.8-5.5 increased risk of baby bom <3rdcentileA positive predictive value of 26% of poor neonatal outcomesA positive predictive value of 55% of poor neonatal outcomes IF the baby is also identified as small on ultrasoundA high SPINT1 was associated with reduced risks

[0274] These results suggest that SPINT1 may be used as a universal biomarker to help prevent stillbirth and neonatal adverse outcomes, and performs better than clinical tape measure and selective ultrasound alone.

[0275] SPINT1 levels measure at 36 weeks gestation was prioritised for the first generation test, noting the potential to of SPINT1 as an indicator of poor placental function more generally across pregnancy.Table 3 - SPINT < 10thcentile measured at 36 weeks gestation

[0276] Birthweight and estimated fetal weight percentiles were determined using GROW charts. The cohort included 2900 female subjects, with 6 missing observations for the Apgar variable.

[0277] InterpretationSPINT1 <10thcentile identifies 42% of all babies under the 3rdcentile in a general obstretic populationSPINT < 10thcentile confers a 26% chance of an adverse neonatal outcome (composite)In an analysis that looks for composite adverse outcomes that excludes identifying birthweight <3rdcentile, a low SPINT1 identifies those with a doubled risk of an adverse neonatal outcome (LR 2.05, 17.9% positive predictive value).Composite outcome (any of) — see Figure 1Perinatal death.Birthweight <3rdcentile.Apgar <7 at 5 minutes.Resuscitation at birth with CPR and / or IPPV.Admitted to Special Care Nursery or Neonatal Intensive Care Unit for >48 hours, within 48 hours of birth. We excluded cases where there was a specific diagnosis for the admission that is clearly unrelated to placental insufficiency - e.g., major congenital heart defect.Requiring respiratory support in nursery (CPAP>4h / hi-flow 02 >4L / ventilation / IPPV).Neonatal diagnosis of bronchopulmonary dysplasia, hypoxic ischaemic encephalopathy, necrotising enterocolitis, intraventricular haemorrhage.able 4: Detecting a birthweight <3rdpercentile at various circulating SPINT1 centile cut-offs

[0278] InterpretationThere was a ‘dose response’ in the positive likelihood ratio, where the chances of <3rdcentile baby drops with higher SPINT1 levelsRisk difference of a baby <3rdcentile between SPINT1 <10thvs <90this 21 foldThe positive likelihood ratio and general performance of SPINT1 increases when SPINT1 is under the 10th centile. This was considered a suitable cut-off point for a potential clinical test.A SPINT1 <75thcentile is very reassuring - the risk of having a baby <3rdreduces by 70-80% from baseline.able 5: Absolute risk of adverse outcomes at various circulating SPINT1 centile cut-offs at 36 weeks gestation (n=2900)

[0279] InterpretationSPINT1 <10* centile has a positive predictive value of 56% of a baby <20* centile. Compared to babies at 75% centile, those <20* still incur a double of perinatal mortality risk. Hence, even if not <3rdcentile, a baby has a high chance of becoming <10* or <20*The composite outcome excluding <3rdcentile is still elevated for those with a SPINT1 <10* centile. This means it captures fetuses with likely placental insufficiency, even if the baby is over the 3rdcentile.

[0280] InterpretationEFW <10thcentile and SPINT1 < 10lhare likely additive in predicting both birthweight <3rdcentile and the composite outcome.It is encouraging that as a lone biomarker, SPINT1 < 10lhappeared to be equal in identifying <3rdcentile; and trending towards being better at detecting composite (when excluding birthweight <3rdin the composite for that analysis).able 7: Cambridge POPS cohort SPINT + Ultrasound: SPINT <10thcentile and / or Ultrasound result of an Estimated fetal weight <10thcentile toetect a birthweight <3rdpercentilehe total number of women in the analysis at 36 wkGA was 115, including 36 cases of FGR and 79 controls. One control was excluded due to a missingalue in EFW. 10th percentile cut-off for SPINT1 was calculated in the random sub-cohort (n=80). Controls were weighted by the inverse of the randomub-cohort sampling fraction (= 3623 / 80 = 45.29) in the calculation of PPV and NPV. The proportions of screen positives, sensitivity, specificity, PPVnd NPV are given in percentages (%). TP, true positive; FP, false positive; TN, true negative, FN, false negative; ER, likelihood ratio; CI, confidencnterval; PPV, positive predictive value; NPV, negative predictive value; DOR, diagnostic odds ratio; EFW, estimated fetal weight.

[0281] InterpretationThis is strong validation of SPINT < 10lhcentile in a new cohort.SPINT1 aloneThe sensitivity was better than 42% in the cohort reported in Tables 3-6, but similar.And Positive LR was 5.5, vs 4.8 in the cohort reported in Tables 3-6.SPINT + UltrasoundThe positive likelihood ratio increases quite markedly, as does the direct odds ratio when both SPINT1 and EFW are both < 10thcentile.

[0282] InterpretationSensitivity of ultrasound is reduced significantly with high body mass index.SPINT1 aloneIn contrast, sensitivity is unaffected by BMI for SPINT1.Summary

[0283] SPINT1 <10th centile predicts <3rdcentile or a composite of adverse neonatal outcomes (see Table 3).

[0284] Predictive performance of SPINT1 was shown at various centile cut-offs (see Tables 4 and 5).

[0285] Circulating SPINT1 levels when combined with ultrasound results provide improved outcomes (see Tables 6 and 7). Table 7 also provides validation of the performance of circulating levels of SPINT1 alone.

[0286] Summary statistics of the Melbourne cohort is set out in Table 8. A sub-analysis of SPINT1 vs Ultrasound prediction, stratified to BMI, in set out in Table 9.

[0287] Low circulating SPINT1 levels (<10* centile, n=2900 with validation from UK cohort) provides:(a) a 48-55% sensitivity of babies destined to birth <3rdcentile birthweight;(b) a 4.8-5.5 increased risk of baby bom <3rdcentile;(c) a positive predictive value of 26% of poor neonatal outcomes;(d) a positive predictive value of 55% of poor neonatal outcomes IF the baby is also identified as small on US; and(e) a high SPINT1 is reassuring, with reduced risks.

[0288] These data show that SPINT1 has merit as an important biomarker for predicting stillbirth risk and neonatal adverse outcomes, and performs better than clinical tape measure (measurement from top of the symphysis pubis to the uterine fundus, done in the clinic) and selective ultrasound.EXAMPLE 4Optimised ELISA Assay for SPINT1 to predict <3rdcentile birth, and adverse neonatal outcomes in a large-scale trials

[0289] SPINT-1 standard formulations for the standard curve were set up using serial dilutions: 160000.000 pg / mL; 80000.000 pg / mL; 40000.000 pg / mL; 20000.000 pg / mL; 10000.000 pg / mL; 5000.000 pg / mL; 2500.000 pg / mL and 0.000 pg / mL, and were prepared in duplicate in the ELISA plate.

[0290] The ELISA plates were pre-coated with an anti-SPINTl antibody.

[0291] This kit was developed by Genscript for quantitative detection of Human SPINT1 Protein in (Cat No. U642H029G0).

[0292] lOOpL of the standard solutions and samples (in this case, plasma samples, which were diluted 1:2) were aliquoted into the wells of the plates. The plate was covered and incubated at 25+2 °C for 1 hour, without agitation.

[0293] The plate is then washed with 300 pL of IX wash buffer thrice. Residual buffer is removed using a paper towel.

[0294] lOOpL of biotin-labeled detection antibody solution was added to each well. The plate was covered and incubated at 25+2 °C for 1 hour, without agitation.

[0295] The plate was washed with 300 pL of IX wash buffer thrice. Residual buffer is removed using a paper towel.

[0296] lOOpL of Eu-streptavidin antibody solution (diluted to working concentration) was added to each well. The plate was covered and incubated at 25+2 °C for 20 minutes, at 400rpm shaking.

[0297] The plate was washed with 300 pL of IX wash buffer, six times. Residual buffer was removed using a paper towel.

[0298] 200 pl of Enhancement Solution was added to all the wells and the plate was incubated at 25+2 °C, at 400 rpm shaking for 15 minutes and protected from light.

[0299] The plate was read on a multifunctional microplate reader, using parameters set as Excitation: 320 / 75 nm, Emission: 615 / 8nm, Delay time: 400 ps , Emission time: 400 ps - 800ps.

[0300] Typical assay data is shown below.

[0301] An example of standard curve readings is shown below.Table 10: Example of typical standard curve readings

[0302] The intra-assay precision of this kit / protocol is less than or equal to 6.70%.Table 11: Intra-assay precision

[0303] The inter-assay precision of this kit / protocol is less than or equal to 6.16%.Table 12: Inter-assay precision

[0304] The calculated sensitivity (in buffer) is 2500.000pg / ml (Average of three times detection). The MRD in the matrix is 1:4, so the sensitivity in the matrix is 10000.000 pg / ml.Table 13: Assay sensitivity

[0305] Recovery data is shown in the table below.Table 14: Recovery data

Claims

CLAIMS:

1. An antigen-binding molecule that specifically binds to serine peptidase inhibitor, kunitz type 1 (SPINT1), wherein the antigen-binding molecule comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2 and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 8:VH CDR1 TYTMG (SEQ ID NO: 1)VH CDR2 TISSGGDKTYYPDSVKG (SEQ ID NO: 2)VH CDR3 YDDWDHGMDY (SEQ ID NO: 3)VL CDR1 SASSSVNYMH (SEQ ID NO: 4)VL CDR1 RASKSVSTSGYSYMH (SEQ ID NO: 5)VL CDR2 TTSNLAS (SEQ ID NO: 6) VL CDR2 LVSNLES (SEQ ID NO: 7) VL CDR3 HQWSSWT (SEQ ID NO: 8)2. The antigen-binding molecule of claim 1, wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2 and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

3. The antigen-binding molecule of claim 1, wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2 and a VH CDR3comprising the amino acid sequence of SEQ ID NO: 3; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 5, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

4. The antigen-binding molecule of any one of claims 1 to 3, wherein the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence having at least 80% sequence identity to any of the foregoing; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% sequence identity thereto:VH FR1 EVMLVESGGGLVKPGGSLKLSCIASGFTFS (SEQ ID NO: 9)VH FR2 WVRQSPEKRLEWVA (SEQ ID NO: 10)VH FR3 RFIISRDNAKNNLYLQMSSLKSEDTALYYCVK (SEQ ID NO: 11) VH FR4 WGQGTSVIVSS (SEQ ID NO: 12)VL FR1 QIVLTQSPAIMSASLGEEITLTC (SEQ ID NO: 13)VL FR1 DIVLTQSPASLAVSLGQRATISY (SEQ ID NO: 14)VL FR2 WYQQKSGTSPKLLIY (SEQ ID NO: 15)VL FR2 WNQQKPGQPPRLLIY (SEQ ID NO: 16)VL FR3 GVPSRFSGSGSGTFYSLTISSVEAEDVADYYC (SEQ ID NO: 17) VL FR3 GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO: 18) VL FR4 FGGGTKLEIK (SEQ ID NO: 19)5. The antigen-binding molecule of claim 4, wherein the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 80% sequence identity thereto; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% sequence identity thereto.

6. The antigen-binding molecule of claim 5, wherein the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO: 9, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 13, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 15, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 17; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19.

7. The antigen-binding molecule of claim 5 or claim 6, wherein the VH comprises the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 80% sequence identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having at least 80% sequence identity thereto.

8. The antigen-binding molecule of claim 7, wherein the VH comprises the amino acid sequence of SEQ ID NO:20, and the VL comprises the amino acid sequence of SEQ ID NO:21.

9. The antigen-binding molecule of claim 8, wherein the VH is encoded by the nucleic acid sequence of SEQ ID NO:23, and the VL is encoded by the nucleic acid sequence of SEQ ID NO:24.

10. The antigen-binding molecule of claim 4, wherein the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity thereto, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 80% sequence identity thereto; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% sequence identity thereto.

11. The antigen-binding molecule of claim 10, wherein the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO: 9, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 11, and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 14, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 16, a VL FR3 comprising the amino acidsequence of SEQ ID NO: 18, and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19.

12. The antigen-binding molecule of claim 10 or claim 11, wherein the VH comprises the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 80% sequence identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:22 or an amino acid sequence having at least 80% sequence identity thereto.

13. The antigen-binding molecule of claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO:20, and the VL comprises the amino acid sequence of SEQ ID NO:22.

14. The antigen-binding molecule of claim 8, wherein the VH is encoded by the nucleic acid sequence of SEQ ID NO:23, and the VL is encoded by the nucleic acid sequence of SEQ ID NO:25.

15. The antigen-binding molecule of any one of the claims 1 to 14, wherein the antigenbinding molecule is an antibody or a SPINT1 -binding fragment thereof.

16. The antigen-binding molecule of claim 15, wherein the SPINT1 -binding fragment is selected from the group consisting of an Fab fragment, an scFab, an Fab’, a single chain variable fragment (scFv) and a one-armed antibody.

17. An antigen-binding molecule that specifically binds to serine peptidase inhibitor, kunitz type 1 (SPINT1), wherein the antigen-binding molecule comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH comprises a complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27; and wherein the VL comprises a complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31:VH CDR1 GYNMN (SEQ ID NO: 26)VH CDR2 NTDPYYDGITYNQKFKG (SEQ ID NO: 27) VH CDR3 GGYGNYVMDY (SEQ ID NO: 28) VL CDR1 RSSQSLETSNGNAYLN (SEQ ID NO: 29) VL CDR2 RVSNRFS (SEQ ID NO: 30) VL CDR3 LQVTHVPFT (SEQ ID NO: 31)18. The antigen-binding molecule of claim 17, wherein the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having at least 80% sequence identity thereto, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 80% sequence identity thereto; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 80% sequence identity thereto; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 80% sequence identity to any of the foregoing, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 80% sequence identity to any of the foregoing, and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having at least 80% sequence identity thereto:VH FR1 EVQLQQSGPEVEKPGASVKISCKASGYTFI (SEQ ID NO: 32)VH FR2 WVRQINGKSLEWIG (SEQ ID NO: 33)VH FR3 KATMTVDKSSNTAYMQLESLTSEDSAVYYCAR (SEQ ID NO: 34)VH FR4 WGQGTSVTVSS (SEQ ID NO: 35)VL FR1 DVVMTQTPLSLPVSLGDQASISC (SEQ ID NO: 36)VL FR2 WYVQKPGQSPQVLIY (SEQ ID NO: 37)VL FR3 GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO: 38)VL FR4 FGSGTTLELK (SEQ ID NO: 39)19. The antigen-binding molecule of claim 18, wherein the VH comprises a framework region 1 (VH FR1) comprising the amino acid sequence of SEQ ID NO: 32, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 33, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 34; and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35; and wherein the VL comprises a framework region 1 (VL FR1) comprising the amino acid sequence of SEQ ID NO: 36, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 37, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 38; and a VL FR4 comprising the amino acid sequence of SEQ ID NO: 39.

20. The antigen-binding molecule of claim 18 or claim 19, wherein the VH comprises the amino acid sequence of SEQ ID NO:40 or an amino acid sequence having at least 80% sequence identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO:41 or an amino acid sequence having at least 80% sequence identity thereto.

21. The antigen-binding molecule of claim 20, wherein the VH is encoded by the nucleic acid sequence of SEQ ID NO: 42 and the VL is encoded by the nucleic acid sequence of SEQ ID NO:43.

22. The antigen-binding molecule of any one of the claims 1 to 21, wherein the antigenbinding molecule is an antibody or a SPINT1 -binding fragment thereof.

23. The antigen-binding molecule of claim 22, wherein the SPINT1 -binding fragment is selected from the group consisting of an Fab fragment, an scFab, an Fab’, a single chain variable fragment (scFv) and a one-armed antibody.

24. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antigen-binding molecule of any one of claims 1 to 16.

25. The isolated nucleic acid molecule of claim 24, wherein the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VLcomprises SEQ ID NO:24 or SEQ ID NO: 25, or a nucleic acid sequence having at least 80% sequence identity to any of the foregoing.

26. The isolated nucleic acid molecule of claim 25, wherein the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:24, or a nucleic acid sequence having at least 80% sequence identity thereto.

27. The isolated nucleic acid molecule of claim 25, wherein the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:25, or a nucleic acid sequence having at least 80% sequence identity thereto.

28. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antigen-binding molecule of any one of claims 17 to 21.

29. The isolated nucleic acid molecule of claim 28, wherein the nucleic acid sequence encoding the VH comprises SEQ ID NO:42, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:43, or a nucleic acid sequence having at least 80% sequence identity to any of the foregoing.

30. The isolated nucleic acid molecule of claim 29, wherein the nucleic acid sequence encoding the VH comprises SEQ ID NO:23, or a nucleic acid sequence having at least 80% sequence identity thereto, and wherein the nucleic acid sequence encoding the VL comprises SEQ ID NO:43.

31. An expression construct comprising a nucleic acid sequence encoding the antigenbinding molecule of any one of claims 1 to 23, operably linked to one or more regulatory sequences.

32. A host cell comprising the expression construct of claim 31.

33. A vector comprising a nucleic acid sequence encoding the antigen -binding molecule of any one of claims 1 to 23.

34. A kit comprising the antigen-binding molecule of any one of claim 1 to 23.

35. A device comprising the antigen-binding molecule of any one of claim 1 to 23.

36. The device of claim 35, wherein the device is a point of care device.

37. The device of claim 35 or claim 36, wherein the device is a lateral flow device.

38. A method of determining the state of placental health in a female subject, the method comprising determining the circulating level of SPINT1 in a biological sample from the female subject using the antigen-binding molecule of any one of claim 1 to 23, wherein a reduction in the circulating level of SPINT1 relative to a control or over time or a change in ratio relative to a control or over time is indicative of placental insufficiency.

39. A method of determining whether a fetus of a pregnant female subject is at risk of abnormal fetal weight, the method comprising determining the circulating level of SPINT1 in a biological sample from the female subject using the antigen-binding molecule of any one of claim 1 to 23, wherein an elevation in the circulating level of SPINT1 relative to a control or over time, or a change in ratio relative to a control or over time, is indicative of abnormal fetal weight, wherein an increase in the circulating level of SPINT1 relative to a control or over time, or a change in ratio relative to a control or over time, is indicative of the likelihood of macrosomia, and wherein a decrease in the circulating level of SPINT1 relative to a control or over time, or a change in ratio relative to a control or over time, is indicative of the likelihood of small-for- gestational age or fetal growth restriction.

40. The method of claim 39, further comprising determining at least one other biomarker and / or physiochemical parameter and / or clinical risk factors.

41. The method of claim 40, wherein the at least one other biomarker is selected from the group consisting of placental growth factor (P1GF), soluble fms-like tyrosine kinase- 1 (sFlt) and / or vascular endothelial growth factor (VEGF).

42. The method of claim 40, wherein the physiochemical parameter is generated by ultrasound or physical measurement.

43. The method of any one of claims 39 to 42, wherein the circulating level or ratio of levels of SPINT1 is subject to analysis with an algorithm or by analytics function or process or other data processing means.

44. The method of any one of claims 39 to 43, further comprising assessing the subject for one or more risk factors associated with increased risk of abnormal fetal weight.

45. The method of any one of claims 39 to 44, wherein the sample is maternal whole blood, plasma or serum.

46. The method of any one of claims 39 to 45, wherein, where the female subject is determined to have placental insufficiency or where the fetus is determined to be at risk of macrosomia, further comprising subjecting the fetus to an early delivery.

47. A clinical management protocol for a pregnant female subject, the protocol comprising determining the circulating level of SPINT1 in a sample from the female subject using the antigen-binding molecule of any one of claim 1 to 23, wherein a decrease in the circulating level of SPINT1 over time compared to a control or a statistically validated level, or a change in ratio reflecting a decrease in the circulating level of SPINT1 is indicative of placental insufficiency and wherein the fetus is monitored or subject to early delivery.

48. The protocol of claim 47, further comprising determining at least one other biomarker and / or physiochemical parameter.

49. The protocol of claim 48, wherein the at least one other biomarker is selected from the group consisting of placental growth factor (P1GF), soluble fms-like tyrosine kinase- 1 (sFlt) and vascular endothelial growth factor (VEGF).

50. The protocol of claim 49, wherein the physiochemical parameter is generated by ultrasound or physical measurement.

51. The protocol of any one of claims 47 to 50, wherein the circulating level or ratio of levels of SPINT1 is subject analysis with an algorithm or by analytics function or process or other data processing means.

52. The protocol of any one of claims 47 to 51, wherein the sample is maternal whole blood, plasma or serum.

53. An assay for determining the state of placental sufficiency in a female subject, the assay comprising determining the circulating level of SPINT1 in a sample from a pregnant female using the antigen-binding molecule of any one of claim 1 to 23; subjecting the level of SPINT1 to an algorithm or analytics function or process or other data processing means generated from a first knowledge base of data comprising the level of the same biomarkers from a subject or cohort of subjects of known status with respect to placental sufficiency, wherein the algorithm or analytics or data processing provides an index of probability of the subject having or not having placental insufficiency or placental sufficiency.

54. Use of a knowledge base of training data comprising (i) circulating levels of SPINT1 as determined using the antigen-binding molecule of any one of claim 1 to 23, and (ii) a level of at least one other biomarker from a pregnant female subject with known placental sufficiency status to generate an algorithm or analytics function or process or other data processing means which, upon input of a second knowledge base of data comprising levels of the same biomarkers from a patient with an unknown placental sufficiency status, provides an index of probability that predicts the nature of the placenta.

55. A panel of reagents for detecting biomarkers associated with placental insufficiency or abnormal fetal weight in a pregnant female subject, wherein the panel of reagents comprises the antigen-binding molecule of any one of claim 1 to 23.

56. A method for monitoring the progression of a pregnancy in a female subject, the method comprising:(e) determining the circulating level of SPINT1 using the antigen-binding molecule of any one of claim 1 to 23, and at least one other biomarker in a sample obtained from a pregnant female subject at a first time point,(f) subjecting the levels determined in step (a) to an algorithm or analytics function or process or other data processing means to provide an index of probability of the subject having a placental sufficiency or insufficiency;(g) repeating steps (a) and (b) for circulating level of SPINT1 and at least one other biomarker in a sample obtained from a pregnant female subject at a subsequent time point to obtain an index of probability of the subject having a placental sufficiency or insufficiency, and(h) comparing the index of probability provided in step (b) with the index of probability provided in step (c), wherein a difference in the index of probabilities between the first time point and the subsequent time point is indicative of the progression of the placental health.

57. The assay of claim 53, the use of claim 54, or the method of claim 56, wherein the circulatory maternal fluid comprises whole blood, plasma or serum.

58. The assay, use or method of any one of claims 53 to 57, further comprising inputting physiochemical data generated by ultrasound or physical measurement.

59. The assay, use or method of any one of claims 53 to 58, further comprising inputting one or more risk factors associated with increased risk of abnormal fetal weight.

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