Binding molecules

Abstract

The present disclosure relates to binding molecules that bind to pregnancy-associated plasma protein A (PAPPA). The disclosure also relates to corresponding polynucleotides, vectors, host cells, methods of production, compositions, and methods of treating PAPPA-mediated diseases or disorders, including autosomal dominant polycystic kidney disease (ADPKD).

Description

[0001] BINDING MOLECULES

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to binding molecules that bind to pregnancy-associated plasma protein A (PAPP A). The disclosure also relates to corresponding polynucleotides, vectors, host cells, methods of production, compositions, and methods of treating PAPPA-mediated diseases or disorders, including autosomal dominant polycystic kidney disease (ADPKD).

[0004] BACKGROUND

[0005] Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder which results in the development and growth of multiple cysts within the kidney. The only approved treatment for ADPKD is tolvaptan. However, tolvaptan has limited efficacy and has a boxed warning for potentially fatal liver toxicity and causes polyuria. As a result, 70% of eligible patients choose not to use tolvaptan in clinical practice. Accordingly, there is a need for improved treatments for ADPKD.

[0006] Pregnancy-associated plasma protein A (“Pappalysin-1”, “PAPPA”, “PAPP- A”, “PAPPA-1”, “PAPPA1”, or “insulin- like growth factor binding protein-4 protease”) is a potential target for treating ADPKD. PAPPA is a secreted metalloproteinase which activates insulin-like growth factor (IGF) signalling by cleaving inhibitory insulin-like growth factor binding proteins (IGFBPs). PAPPA is highly expressed in ADPKD kidneys resulting in overactivation of IGF and cyst proliferation. In mouse models of ADPKD, PAPPA knockout has been shown to slow cyst progression. Accordingly, PAPPA inhibition may be beneficial in the treatment of certain PAPPA-mediated diseases or disorders, such as ADPKD.

[0007] SUMMARY OF THE DISCLOSURE

[0008] The disclosure relates to binding molecules that bind to PAPPA.

[0009] In one aspect, there is provided a binding molecule that binds to PAPPA and comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein:

[0010] (a) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 108 or 122 (e.g., SEQ ID NO: 108), HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 110, and wherein theVL region amino acid sequence comprises LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113;

[0011] (b) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 1 or 121 (e.g., SEQ ID NO: 121), HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6;

[0012] (c) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 9 or 121 (e.g., SEQ ID NO: 121), HCDR2 of SEQ ID NO: 10 and HCDR3 of SEQ ID NO: 11, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13 and LCDR3 of SEQ ID NO: 14;

[0013] (d) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 17, HCDR2 of SEQ ID NO: 18 and HCDR3 of SEQ ID NO: 19, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21 and LCDR3 of SEQ ID NO: 22;

[0014] (e) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 25, HCDR2 of SEQ ID NO: 26 and HCDR3 of SEQ ID NO: 1 and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 29 and LCDR3 of SEQ ID NO: 30;

[0015] (f) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 33, HCDR2 of SEQ ID NO: 34 and HCDR3 of SEQ ID NO: 35, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 37 and LCDR3 of SEQ ID NO: 38;

[0016] (g) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 41, HCDR2 of SEQ ID NO: 42 and HCDR3 of SEQ ID NO: 43, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 44, LCDR2 of SEQ ID NO: 45 and LCDR3 of SEQ ID NO: 46; or

[0017] (h) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 49, HCDR2 of SEQ ID NO: 50 and HCDR3 of SEQ ID NO: 51, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 52, LCDR2 of SEQ ID NO: 53 and LCDR3 of SEQ ID NO: 54.

[0018] In some instances, the binding molecule comprises:

[0019] (a) the VH region amino acid sequence comprising SEQ ID NO: 114, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0020] the VL region amino acid sequence comprising SEQ ID NO: 115, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto

[0021] (b) the VH region amino acid sequence comprising SEQ ID NO: 7, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprising SEQ ID NO: 8, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0022] (c) the VH region amino acid sequence comprising SEQ ID NO: 15, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0023] the VL region amino acid sequence comprising SEQ ID NO: 16, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0024] (d) the VH region amino acid sequence comprising SEQ ID NO: 23, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0025] the VL region amino acid sequence comprising SEQ ID NO: 24, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0026] (e) the VH region amino acid sequence comprising SEQ ID NO: 31, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0027] the VL region amino acid sequence comprising SEQ ID NO: 32, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0028] (f) the VH region amino acid sequence comprising SEQ ID NO: 39, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0029] the VL region amino acid sequence comprising SEQ ID NO: 40, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0030] (g) the VH region amino acid sequence comprising SEQ ID NO: 47, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0031] the VL region amino acid sequence comprising SEQ ID NO: 48, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; or

[0032] (h) the VH region amino acid sequence comprising SEQ ID NO: 55, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0033] the VL region amino acid sequence comprising SEQ ID NO: 56, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[0034] In some instances, the binding molecule comprises:

[0035] (a) the VH region amino acid sequence comprising SEQ ID NO: 114 and the VL region amino acid sequence comprising SEQ ID NO: 115;

[0036] (b) the VH region amino acid sequence comprising SEQ ID NO: 7 and the VL region amino acid sequence comprises SEQ ID NO: 8;

[0037] (c) the VH region amino acid sequence comprising SEQ ID NO: 15 and the VL region amino acid sequence comprising SEQ ID NO: 16;(d) the VH region amino acid sequence comprising SEQ ID NO: 23 and the VL region amino acid sequence comprising SEQ ID NO: 24;

[0038] (e) the VH region amino acid sequence comprising SEQ ID NO: 31 and the VL region amino acid sequence comprising SEQ ID NO: 32;

[0039] (f) the VH region amino acid sequence comprising SEQ ID NO: 39 and the VL region amino acid sequence comprising SEQ ID NO: 40;

[0040] (g) the VH region amino acid sequence comprising SEQ ID NO: 47 and the VL region amino acid sequence comprising SEQ ID NO: 48; or

[0041] (h) the VH region amino acid sequence comprising SEQ ID NO: 55 and the VL region amino acid sequence comprising SEQ ID NO: 56.

[0042] In some instances, the binding molecule does not bind to PAPPA-2.

[0043] In some instances, the binding molecule binds to human PAPPA. In some instances, the binding molecule binds to cynomolgus PAPPA.

[0044] In some instances, the binding molecule comprises an Fc region. In some instances, the binding molecule comprises an Fc region and wherein the binding molecule comprises a L234F / L235E / P331S triple mutation in the Fc region. In some instances, the binding molecule comprises an Fc region and wherein the binding molecule comprises a L234F / L235E / P331S triple mutation in the Fc region and M252Y / S254T / T256E mutations in the Fc region.

[0045] In some instances, the binding molecule is an anti-PAPPA antibody, or an antigen-binding fragment thereof. In some instances, the anti-PAPPA antibody, or antigen-binding fragment thereof, is a human antibody, or an antigen-binding fragment thereof. In some instances, the anti-PAPPA antibody, or antigen-binding fragment thereof, is a monoclonal antibody, or an antigen-binding fragment thereof.

[0046] In some instances, the binding molecule is an IgG immunoglobulin or an antigen-binding fragment thereof. In some instances, the binding molecule is an IgGl immunoglobulin or an antigen-binding fragment thereof.

[0047] In another aspect, there is provided an isolated polynucleotide encoding the binding molecule of the disclosure.In another aspect, there is provided a vector comprising the polynucleotide of the disclosure. In another aspect, there is provided a vector comprising a polynucleotide encoding a VH region of the disclosure, and a polynucleotide encoding a VL region of the disclosure.

[0048] In another aspect, there is provided a host cell comprising a polynucleotide of the disclosure. In another aspect, there is provided a host cell comprising a vector of the disclosure.

[0049] In another aspect, there is provided a method of producing a binding molecule of the disclosure, the method comprising expressing a polynucleotide of the disclosure or a vector of the disclosure in a host cell, e.g., a host cell of the disclosure.

[0050] In another aspect, there is provided a pharmaceutical composition comprising a binding molecule of the disclosure, and a pharmaceutically acceptable carrier or diluent.

[0051] In another aspect, there is provided a method of treating a PAPPA mediated disease, disorder or condition in a subject, the method comprising administering to the subject a binding molecule of the disclosure, or a pharmaceutical composition of the disclosure. In some instances, the PAPPA mediated disease, disorder or condition is autosomal dominant polycystic kidney disease (ADPKD).

[0052] BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1: A-D demonstrate that the ADPKD mouse model exhibits upregulated and cyst-enriched murine PAPPA expression in kidneys, along with increased murine PAPPA activity. Figure A displays the mRNA expression of murine PAPPA in kidney tissues from healthy and ADPKD mice as measured by qPCR, while Figure B shows the RNAscope detection of murine PAPPA in kidney sections, and Figure C illustrates the quantification of the number of murine PAPPA mRNA divided by the total nucleus. Figure D shows the plasma level of cleaved endogenous Igfbp4 in healthy and ADPKD mice. Statistical analysis was performed using a t-test; **P<0.01, ***P<0.001, ****P<0.0001.

[0053] FIGURE 2: A-E demonstrate that treatment with a tool PAPPA mAb ameliorates ADPKD progression in mice at all doses. The tool PAPPA mAb is a modified version of mAB-PA 1 / 41 disclosed in W02020198166 and has a murine variable region and a human constant region. Figure A illustrates the study timeline, with Pkdl deletion induced at post-natal day (PND) 18-20, andfrom PND42 onwards, ADPKD mice receiving weekly subcutaneous injections of either an isotype control antibody or tool PAPPA mAb at doses of 1, 3, 10, and 30 mg / kg. Figure B presents the mouse kidney volume measured by ultrasound at postnatal day 96, while Figure C displays the blood urea levels assayed at postnatal day 109-112. Figure D shows representative images of H&E staining on kidney sections from isotype control or tool PAPPA mAb 1 mg / kg treated mice. Figure E illustrates the cystic index, calculated by dividing the cyst area by the kidney section area based on H&E staining. Statistical analysis was performed using ANOVA; *P<0.05, **P<0.01, *** <0.001, tool PAPPA mAb vs isotype Ctrl; Dunnett’s post-hoc correction.

[0054] FIGURE 3: A-D demonstrate that treatment with the tool PAPPA mAb in ADPKD mouse model exhibits a dose-dependent effect without inducing polyuria, unlike tolvaptan. ADPKD mice received weekly subcutaneous injections of either an isotype control antibody or tool PAPPA mAb at doses of 0.1, 0.3, and 3 mg / kg. Additionally, ADPKD mice were treated with tolvaptan by daily gavage at 75 mg / kg in combination with 0.1% tolvaptan in food, and a diet control group of ADPKD mice were handled in a similar manner without tolvaptan. Figure A presents the mouse kidney volume measured by ultrasound at postnatal day 98, while Figure B displays the blood urea levels assayed at postnatal day 109-112. Figure C shows the kidney-to-body-weight ratio at termination, and Figure D illustrates the urine osmolality measured for urine collected from mice one day before termination. Tolvaptan dose (75 mg / kg p.o. daily + 0.1% in food) was scaled from the highest approved dose used in clinical practice; PND: post-natal day. **adj. P<0.01, ****adj. PO.OOO 1 by MCP-mode analysis of dose-dependent response. 7M).000l. treatment vs control; Dunnett’s post-hoc correction.

[0055] FIGURE 4: A-B confirm the target engagement of tool PAPPA mAb in the ADPKD mouse model. Figure A illustrates the plasma levels of endogenous murine PAPPA protein in healthy mice, control ADPKD mice and mice treated with tool PAPPA mAb at various doses or tolvaptan at termination, while Figure B displays the plasma levels of cleaved endogenous Igfbp4. Figure C illustrates the plasma levels of endogenous IGF1 protein in healthy mice and ADPKD mice treated with either isotype control or tool PAPPA mAb at two different doses. **P<0.01, ***P<0.001 ****P<0.0001 One-way ANOVA and Brown-Forsythe and Welch ANOVA if not equal variance

[0056] FIGURE 5: A-B demonstrate that tool PAPPA mAb inhibits Igf signalling and cyst proliferation in an ADPKD mouse model. Figure A displays the phosphorylation of murine Erk, Akt, and S6k in kidney tissues from ADPKD mice treated with either isotype control or tool PAPPA mAb at a doseof 3 mg / kg, while Figure B shows representative images of PCNA immunofluorescence staining on kidney sections, and illustrates the percentage of PCNA-positive nuclei to total nuclei. *P<0.05, ** <0.01, ****P<0.0001, treatment vs control; Dunnett’s post-hoc correction

[0057] FIGURE 6: A-E demonstrate that tool PAPPA mAh dose-dependently downregulates mRNA expression of inflammation, fibrosis, and injury markers in ADPKD mouse kidneys. Figure A shows the mRNA expression of Ccl2 in kidneys from healthy mice, ADPKD control mice, and mice treated with tool PAPPA mAb at various doses or tolvaptan, while Figure B-E show expression of Tnf, Tgfbl, Collal, and Havcrl, respectively. *P<0.05, **P<0.01. Median with 95% CI. Diet Ctrl vs tolvaptan: unpaired parametric two-tailed t test, or unpaired t test with Welch’s correction if not equal variance. Treatment effects vs Isotype Ctrl: Ordinary one-way ANOVA and Dunnett’s multiple comparisons test, or Brown-Forsythe and Welch ANOVA if not equal variance

[0058] FIGURE 7 : A and B demonstrate that tool PAPPA mAb treatment reversed the aberrantly regulated proteins in kidneys of ADPKD mouse model. Figure A shows a volcano plot of significantly regulated proteins in proteomics analysis of kidneys from ADPKD isotype control mice compared to healthy mice. Each dot represents an individual protein and black dots represent proteins with adjusted p-value <0.05 and absolute log fold change > 0.5. Figure B shows the negative correlation on fold change of significantly regulated proteins between tool PAPPA mAb and isotype control treatment (adjusted p-value < 0.05) with significantly regulated proteins between ADPKD isotype control and healthy mice (adjusted p-value < 0.05). Each dot represents an individual protein, red dots represent proteins that are regulated in both comparisons.

[0059] FIGURE 8: Exposure-response relationship for tool PAPPA mAb in ADPKD mice. Relationship between the tool PAPPA mAb plasma concentration and change in blood urea from baseline (A), cleaved IGFBP4 formation (B), and total murine PAPPA levels in plasma (C).

[0060] FIGURE 9: Schematic of the PAPPA and PAPPA-2 constructs used for generation of PAPPA proteins.

[0061] FIGURE 10: A and C. Non-specific binding of control and PAPPA antibodies to HEK293 cells. The results demonstrate that none of the PAPPA antibodies bind to HEK293 cells. B and D. Selfassociation of control and PAPPA antibodies. The results demonstrate that none of the PAPPA antibodies have a risk of self-association.FIGURE 11: Risk of fast in vivo clearance of control and PAPPA antibodies. A. Results for AB1, AB2, AB3, AB10, AB8, AB5 and AB16. B. Results for AB34 TM. The results demonstrate that none of the PAPPA antibodies have a risk of fast in vivo clearance due to non-specific binding to baculovirus particles.

[0062] FIGURE 12: IGF-1 signalling in MCF-7 cells. The results demonstrate that IGF-1 signals via both phosphorylation of Ser473 and Thr308 on human AKT and can be inhibited with PAPPA mAbs.

[0063] FIGURE 13: AB1 shows target engagement in cynomolgus monkey. (A) Reduction of free PAPPA levels in plasma upon administration of AB1 to cynomolgus monkey. (B) Exposure-response relationship between AB 1 plasma concentration and free PAPPA levels in cynomolgus monkey.

[0064] FIGURE 14: Cryo-EM structure model of PAPPA [82-1183] (grey) and AB2 (VH in black, VL in white). The model shows the binding epitope of AB2, located in the LG domain of PAPPA.

[0065] FIGURE 15: A. BLI results for binding of AB1 and tool PAPPA mAb to PAPPA [82-324], B. BLI results for binding of AB34 TM, AB34 TM YTE, and tool PAPPA mAb to PAPPA LGD.

[0066] FIGURE 16: A. BLI results for binding of AB1 and tool PAPPA mAb to the PAPPA-STC-1 complex. B. BLI results for binding of AB1, AB34 TM, AB34 TM YTE, a benchmark PAPPA mAb, and tool PAPPA mAb to the PAPPA-STC-1 complex.

[0067] FIGURE 17: MSD assay results for inhibition of IGFBP4 cleavage by AB1, AB34 TM, AB34 TM YTE, and the tool PAPPA mAb in human ADPKD cystic cell culture.

[0068] DETAILED DESCRIPTION CDRs of a binding molecule that binds to PAPPA

[0069] This disclosure provides a binding molecule that binds to PAPPA. Accordingly, in one aspect, there is provided a binding molecule that binds to pregnancy-associated plasma protein A (PAPPA), wherein the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3.Table 1 provides CDR sequences for exemplary binding molecules of the disclosure. In some instances, the binding molecule comprises a set of six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of any binding molecule illustrated in Table 1. In some instances, the binding molecule comprises a set of six CDRs as illustrated in Table 1. For example, the binding molecule may comprise all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as indicated for any one of the binding molecules illustrated in Table 1.

[0070] Thus, in some instances, the binding molecule comprises a heavy chain variable region (VH) which comprises HCDR1, HCDR2, HCDR3 as indicated for any one of the binding molecules illustrated in Table 1, and a light chain variable region (VL) which comprises LCDR1, LCDR2 and LCDR3 as indicated for the same binding molecule illustrated in Table 1.

[0071] Table 1: Amino acid sequences of the CDRs of exemplary binding molecules.

[0072] Binding CDR Amino acid sequence SEQ CDR Amino acid sequence SEQ molecule ID ID AB1 HCDR1 ELSMY 121 LCDR1 RASQGISSYLA 4

[0073] HCDR2 GFDPEDGETLYVQKFQG 2 LCDR2 AASTLQS 5 HCDR3 GGAVALFDY 3 LCDR3 QHLYSFPIT 6 AB2 HCDR1 ELSMY 121 LCDR1 RASQGISSYLA 12

[0074] HCDR2 GFDPEDGETLYVQKFQG 10 LCDR2 AASTLQS 13 HCDR3 GGAVALFDY 11 LCDR3 QHLYSFPIT 14 AB3 HCDR1 SYSMN 17 LCDR1 RASQSISSYLN 20

[0075] HCDR2 SISSSSSYIYYADSVKG 18 LCDR2 AASSLQS 21 HCDR3 EGVLFFDY 19 LCDR3 QQSSITPRT 22 AB10 HCDR1 SFAMS 25 LCDR1 RASQGISSWLA 28

[0076] HCDR2 AISVSGDITYYADSVKG 26 LCDR2 VASSLQS 29 HCDR3 NGDVAMVYFDY 27 LCDR3 QQANSFPFT 30 AB8 HCDR1 SGSYYWT 33 LCDR1 KSSQSVLYSSNNKNYLA 36

[0077] HCDR2 YIYYSGSTNYNPSLKS 34 LCDR2 WASTRES 37 HCDR3 YSSSWVFDY 35 LCDR3 QQYYSTPLT 38 AB5 HCDR1 PYYWS 41 LCDR1 KSSQSVLYRSNNKNYLA 44

[0078] HCDR2 YIYHSGSTNYNPSLKS 42 LCDR2 WASTRES 45 HCDR3 GPEPFDN 43 LCDR3 QQYYSTPYT 46 AB16 HCDR1 RYAMS 49 LCDR1 QASQDISNYLN 52

[0079] HCDR2 VISGSGGYTYYADSVKG 50 LCDR2 DASNLET 53 HCDR3 FRGYSYGSFDY 51 LCDR3 QQYDNFPLT 54

[0080]

[0081] AB34 HCDR1 ELEMY 108 LCDR1 RASQGIYQYLA 111 HCDR2 GFDPEDGETLYVQKFQG 109 LCDR2 AASALQS 112 HCDR3 GGAVALFDY 110 LCDR3 QHLYSFPIT 113

[0082]

[0083] In some instances, the amino acid sequences of the CDRs recited in Table 1 are defined using the Kabat numbering system. In any of the aspects and instances described herein, the amino acid sequence of SEQ ID NO: 121 (e.g., the amino acid sequence of the H-CDR1 of AB1 or AB2) may further comprise the amino acid sequence TLT at its N terminus. Thus, in any of the instances and aspects described herein, the amino acid sequence of the H-CDR1 of AB1 may be TLTELSMY (SEQ ID NO: 1). In any of the instances and aspects described herein, the amino acid sequence of the H-CDR1 of AB2 may be TLTELSMY (SEQ ID NO: 9). In any of the aspects and instances described herein, the amino acid sequence of SEQ ID NO: 108 (e.g., the amino acid sequence of the H-CDR1 of AB34) may further comprise the amino acid sequence HLE at its N terminus. Thus, in any of the instances and aspects described herein, the amino acid sequence of the H-CDR1 of AB34 may be HLEELEMY (SEQ ID NO: 122).

[0084] In some instances, the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein:

[0085] (a) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 108 or 122 (e.g., SEQ ID NO: 108), HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 110, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113;

[0086] (b) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 1 or 121 (e.g., SEQ ID NO: 121), HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6;

[0087] (c) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 9 or 121 (e.g., SEQ ID NO: 121), HCDR2 of SEQ ID NO: 10 and HCDR3 of SEQ ID NO: 11, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13 and LCDR3 of SEQ ID NO: 14;

[0088] (d) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 17, HCDR2 of SEQ ID NO: 18 and HCDR3 of SEQ ID NO: 19, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21 and LCDR3 of SEQ ID NO: 22;(e) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 25, HCDR2 of SEQ ID NO: 26 and HCDR3 of SEQ ID NO: 1 and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 29 and LCDR3 of SEQ ID NO: 30;

[0089] (f) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 33, HCDR2 of SEQ ID NO: 34 and HCDR3 of SEQ ID NO: 35, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 37 and LCDR3 of SEQ ID NO: 38;

[0090] (g) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 41, HCDR2 of SEQ ID NO: 42 and HCDR3 of SEQ ID NO: 43, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 44, LCDR2 of SEQ ID NO: 45 and LCDR3 of SEQ ID NO: 46; or

[0091] (h) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 49, HCDR2 of SEQ ID NO: 50 and HCDR3 of SEQ ID NO: 51, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 52, LCDR2 of SEQ ID NO: 53 and LCDR3 of SEQ ID NO: 54.

[0092] In preferred instances, the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6. In some instances, the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 121, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6.

[0093] In preferred instances, the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 110, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113. In some instances, the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, whereinthe VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 122, HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 110, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113.

[0094] In some instances, the binding molecule has one or more of the functional properties as disclosed herein. For example, in some instances, the binding molecule has a binding specificity as disclosed herein. In some instances, the binding molecule binds to one or more PAPPA sequences or domains as disclosed herein. In some instances, the binding molecule binds to the LG domain of PAPPA as described herein. In some instances, the binding molecule has inhibitory activity against PAPPA as disclosed herein. In some instances, the binding molecule has a binding affinity as disclosed herein. In some instances, the binding molecule has a non-specific binding propensity and / or a selfassociation propensity as disclosed herein. In some instances, the binding molecule has an in vivo clearance as disclosed herein.

[0095] In other instances, the binding molecule shows sequence variation at one or more CDRs when compared to the corresponding CDR sequences of a reference binding molecule. In such instances, the binding molecule retains the ability to bind PAPPA, such as to bind specifically to PAPPA.

[0096] In some instances, the reference binding molecule has one or more of the functional properties as disclosed herein. For example, in some instances, the reference binding molecule has a binding specificity as disclosed herein. In some instances, the reference binding molecule binds to one or more PAPPA sequences or domains as disclosed herein. In some instances, the reference binding molecule binds to the LG domain of PAPPA as described herein. In some instances, the reference binding molecule has inhibitory activity against PAPPA as disclosed herein. In some instances, the reference binding molecule has a binding affinity as disclosed herein. In some instances, the reference binding molecule has a non-specific binding propensity and / or a self-association propensity as disclosed herein. In some instances, the reference binding molecule has an in vivo clearance as disclosed herein.

[0097] In some instances, the binding molecule has one or more of the same functional properties as the reference binding molecule. The functional property or properties may comprise any functional property as disclosed herein. For example, in some instances, the binding molecule has the same binding specificity as the reference binding molecule. In some instances, the binding moleculebinds to the same PAPPA sequence(s) or domain(s) as the reference binding molecule. In some instances, the binding molecule binds to the same epitope as the reference binding molecule. In some instances, the binding molecule has the same inhibitory activity against PAPPA as the reference binding molecule. In some instances, the binding molecule has the same binding affinity as the reference binding molecule. In some instances, the binding molecule has the same nonspecific binding propensity and / or self-association propensity as the reference binding molecule. In some instances, the binding molecule has the same in vivo clearance as the reference binding molecule.

[0098] In some instances, the binding molecule has one or more of the same functional properties as the reference binding molecule and binds to the same epitope as the reference binding molecule. For example, in some instances, the binding molecule has the same binding specificity as the reference binding molecule and binds to the same epitope as the reference binding molecule. In some instances, the binding molecule binds to the same PAPPA domain(s) as the reference binding molecule and binds to the same epitope as the reference binding molecule. In some instances, the binding molecule has the same inhibitory activity against PAPPA as the reference binding molecule and binds to the same epitope as the reference binding molecule. In some instances, the binding molecule has the same binding affinity as the reference binding molecule and binds to the same epitope as the reference binding molecule. In some instances, the binding molecule has the same non-specific binding propensity and / or self-association propensity as the reference binding molecule and binds to the same epitope as the reference binding molecule. In some instances, the binding molecule has the same in vivo clearance as the reference binding molecule and binds to the same epitope as the reference binding molecule.

[0099] In some instances, the binding molecule does not bind to the same epitope as the reference binding molecule. In some instances, the binding molecule has one or more of the same functional properties as the reference binding molecule but does not bind to the same epitope as the reference binding molecule. The functional property or properties may comprise any functional property as disclosed herein. For example, in some instances, the binding molecule has the same binding specificity as the reference binding molecule and does not bind to the same epitope as the reference binding molecule. In some instances, the binding molecule binds to the same PAPPA domain(s) as the reference binding molecule and does not bind to the same epitope as the reference binding molecule. In some instances, the binding molecule has the same inhibitory activity against PAPPA as the reference binding molecule and does not bind to the same epitope as the reference binding molecule.In some instances, the binding molecule has the same binding affinity as the reference binding molecule and does not bind to the same epitope as the reference binding molecule. In some instances, the binding molecule has the same non-specific binding propensity and / or selfassociation propensity as the reference binding molecule and does not bind to the same epitope as the reference binding molecule. In some instances, the binding molecule has the same in vivo clearance as the reference binding molecule and does not bind to the same epitope as the reference binding molecule.

[0100] In some instances, the reference binding molecule comprises a set of CDRs as illustrated in Table 1, i.e. all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as indicated for any one of the binding molecules illustrated in T able 1. In some instances, the reference binding molecule comprises the VH and VL sequences as indicated for any one of the binding molecules illustrated in Table 2. In some instances, the reference binding molecule comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of AB16. In some instances, the reference binding molecule comprises a VH region comprising the amino acid sequence of SEQ ID NO: 55 and a VL region comprising the amino acid sequence of SEQ ID NO: 56. In preferred instances, the reference binding molecule comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of AB1. In preferred instances, the reference binding molecule comprises a VH region comprising the amino acid sequence of SEQ ID NO: 7 and a VL region comprising the amino acid sequence of SEQ ID NO: 8.

[0101] In some instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the reference binding molecule, wherein any one or more of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the binding molecule comprises 1, 2, 3, 4 or 5 amino acid differences compared to the respective CDR sequences of the reference binding molecule. In some instances the binding molecule comprises: a light chain CDR1 having at most 1, 2, 3, 4 or 5 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 1, 2, 3, 4 or 5 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR3 having at most 1, 2, 3, 4 or 5 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR1 having at most 1, 2, 3, 4 or 5 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1, 2, 3, 4 or 5 amino acid differences when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 having at most 1, 2, 3, 4 or 5 amino acid differences when compared to acorresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule, and preferably exhibits the same antigen cross-reactivity as the reference binding molecule.

[0102] In some instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the reference binding molecule, wherein any one or more of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the binding molecule comprises 1, 2, or 3 amino acid differences compared to the respective CDR sequences of the reference binding molecule. In some instances the binding molecule comprises: a light chain CDR1 having at most 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR3 having at most 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR1 having at most 1, 2, or 3, amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 having at most 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule, and preferably exhibits the same antigen cross-reactivity as the reference binding molecule.

[0103] In some instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the reference binding molecule, wherein any one or more of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the binding molecule comprises 1, or 2 amino acid differences compared to the respective CDR sequences of the reference binding molecule. In some instances the binding molecule comprises: a light chain CDR1 having at most 1 or 2 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 1 or 2 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR3 having at most 1 or 2 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR1 having at most 1 or 2 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1 or 2 amino acid differences when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 having at most 1 or 2 amino acid differences when compared to a corresponding reference CDR sequence; wherein the bindingmolecule binds to the same target antigen as the reference binding molecule, and preferably exhibits the same antigen cross-reactivity as the reference binding molecule.

[0104] In some instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the reference binding molecule, wherein any one or more of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the binding molecule comprises 1 amino acid difference compared to the respective CDR sequences of the reference binding molecule. In some instances the binding molecule comprises: a light chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a light chain CDR3 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule, and preferably exhibits the same antigen cross-reactivity as the reference binding molecule.

[0105] In some instances the binding molecule comprises: a light chain CDR1 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 5 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR3 identical to the corresponding reference CDR sequence; a heavy chain CDR1 having at most 3 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 identical to the corresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule, and preferably exhibits the same antigen cross-reactivity profile as the reference binding molecule.

[0106] In some instances the binding molecule comprises: a light chain CDR1 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a light chain CDR3 identical to the corresponding reference CDR sequence; a heavy chain CDR1having at most 3 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 identical to the corresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule, and preferably exhibits the same antigen cross-reactivity profile as the reference binding molecule.

[0107] In some instances the binding molecule comprises: a light chain CDR1 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a light chain CDR3 identical to the corresponding reference CDR sequence; a heavy chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 identical to the corresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule, and preferably exhibits the same antigen cross-reactivity profile as the reference binding molecule.

[0108] In some instances the binding molecule comprises: a light chain CDR1 identical to the corresponding reference CDR sequence; a light chain CDR2 identical to the corresponding reference CDR sequence; a light chain CDR3 identical to the corresponding reference CDR sequence; a heavy chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 identical to the corresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule, and preferably exhibits the same antigen cross-reactivity profile as the reference binding molecule.

[0109] In some instances, the binding molecule has at most 8, 7, 6, 5, 4, 3 or 2 amino acid differences total in the CDRs thereof when compared to a corresponding reference binding molecule. In some instances, the binding molecule has at most 8, 7, 6, 5, 4, 3 or 2 amino acid differences total in the CDRs thereof when compared to a corresponding reference binding molecule, with the proviso that there are at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid differences per CDR. In some instances, the binding molecule has at most 6, 5, 4, 3 or 2 amino acid differences total in theCDRs thereof when compared to a corresponding reference binding molecule. In some instances, the binding molecule has at most 6, 5, 4, 3 or 2 amino acid differences total in the CDRs thereof when compared to a corresponding reference binding molecule, with the proviso that there are at most 5, at most 4, at most 3, at most 2 or at most 1 amino acid differences per CDR. In some instances, the binding molecule has at most 6, 5, 4, 3 or 2 amino acid differences total in the CDRs thereof when compared to a corresponding reference binding molecule, with the proviso that there are at most 3, at most 2 or at most 1 amino acid differences per CDR. In some instances, the binding molecule has at most 3 or 2 amino acid differences total in the CDRs thereof when compared to a corresponding reference binding molecule. In some instances, the binding molecule has at most 3 or 2 amino acid differences total in the CDRs thereof when compared to a corresponding reference binding molecule, with the proviso that there are at most 3, at most 2, or at most 1 amino acid differences per CDR. In some instances, the binding molecule has at most 3 amino acid differences total in the CDRs thereof when compared to a corresponding reference binding molecule, with the proviso that there are at most 2 or at most 1 amino acid differences per CDR. In some instances, the binding molecule has at most 2 amino acid differences total in the CDRs thereof when compared to a corresponding reference binding molecule. In some instances, the binding molecule has at most 2 amino acid differences total in the CDRs thereof when compared to a corresponding reference binding molecule, with the proviso that there is at most 1 amino acid difference per CDR.

[0110] In some instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of a reference binding molecule, wherein:

[0111] (a) the HCDR1 of the binding molecule comprises 1 amino acid difference compared to the HCDR1 sequence of the reference binding molecule, and the HCDR2 of the binding molecule comprises 1 amino acid difference compared to the HCDR2 sequence of the reference binding molecule;

[0112] (b) the HCDR1 of the binding molecule comprises 3 amino acid differences compared to the HCDR1 sequence of the reference binding molecule;

[0113] (c) the LCDR1 of the binding molecule comprises 2 amino acid differences compared to the LCDR1 sequence of the reference binding molecule, and the LCDR2 of the binding molecule comprises 1 amino acid difference compared to the LCDR2 sequence of the reference binding molecule;

[0114] (d) the LCDR2 of the binding molecule comprises 5 amino acid differences compared to the LCDR2 sequence of the reference binding molecule;(e) the HCDR1 of the binding molecule comprises 3 amino acid differences compared to the HCDR1 sequence of the reference binding molecule, the LCDR1 of the binding molecule comprises 2 amino acid differences compared to the LCDR1 sequence of the reference binding molecule, and the LCDR2 of the binding molecule comprises 1 amino acid difference compared to the LCDR2 sequence of the reference binding molecule; or

[0115] (f) the HCDR1 of the binding molecule comprises 3 amino acid differences compared to the HCDR1 sequence of the reference binding molecule, and the LCDR2 of the binding molecule comprises 5 amino acid differences compared to the LCDR2 sequence of the reference binding molecule.

[0116] Without being bound by theory, it is believed that a binding molecule which comprises a VH region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the HCDR3 of the binding molecule is the HCDR3 of AB1 (i.e., an HCDR3 of SEQ ID NO: 3) and wherein the LCDR3 of the binding molecule is the LCDR3 of AB1 (i.e., an LCDR3 of SEQ ID NO: 6) will exhibit the same or similar properties to AB1, e.g., the same or similar binding specificity as AB1 as described herein and / or the same or similar ability to bind to the LG domain of PAPPA as described herein. In particular, it is believed that a binding molecule having the HCDR3 of AB1 and the LCDR3 of AB1 will bind to the LG domain of PAPPA as described herein. Thus, in some instances, the binding molecule comprises a VH region having an HCDR3 of SEQ ID NO: 3 and a VL region having an LCDR3 of SEQ ID NO: 6. In some instances, the HCDR3 and LCDR3 sequences of the binding molecule are identical to the respective HCDR3 and LCDR3 sequences of AB 1.

[0117] In some instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of AB1, wherein any one or more of said HCDR1, HCDR2, LCDR1, and LCDR2 sequences of the binding molecule comprises 1, 2, 3, 4 or 5 amino acid differences compared to the respective CDR sequences of AB 1. In some instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of AB1, wherein any one or more of said HCDR1, HCDR2, LCDR1, and LCDR2 sequences of the binding molecule comprises 1, 2, or 3 amino acid differences compared to the respective CDR sequences of ABE In some instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of AB1, wherein any one or more of said HCDR1, HCDR2, LCDR1, and LCDR2 sequences of the binding molecule comprises 1, or 2 amino acid differences compared to the respective CDR sequences of ABE Insome instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of AB1, wherein any one or more of said HCDR1, HCDR2, LCDR1, and LCDR2 sequences of the binding molecule comprises 1 amino acid difference compared to the respective CDR sequences of AB 1.

[0118] In some instances, the binding molecule comprises all six of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of AB1, wherein:

[0119] (a) the HCDR1 of the binding molecule comprises 1 amino acid difference compared to the HCDR1 sequence of AB1, and the HCDR2 of the binding molecule comprises 1 amino acid difference compared to the HCDR2 sequence of AB1;

[0120] (b) the HCDR1 of the binding molecule comprises 3 amino acid differences compared to the HCDR1 sequence of AB1;

[0121] (c) the LCDR1 of the binding molecule comprises 2 amino acid differences compared to the LCDR1 sequence of AB1, and the LCDR2 of the binding molecule comprises 1 amino acid difference compared to the LCDR2 sequence of AB1;

[0122] (d) the LCDR2 of the binding molecule comprises 5 amino acid differences compared to the LCDR2 sequence of AB1;

[0123] (e) the HCDR1 of the binding molecule comprises 3 amino acid differences compared to the HCDR1 sequence of AB1, the LCDR1 of the binding molecule comprises 2 amino acid differences compared to the LCDR1 sequence of AB1, and the LCDR2 of the binding molecule comprises 1 amino acid difference compared to the LCDR2 sequence of AB1; or

[0124] (f) the HCDR1 of the binding molecule comprises 3 amino acid differences compared to the HCDR1 sequence of AB1, and the LCDR2 of the binding molecule comprises 5 amino acid differences compared to the LCDR2 sequence of AB1.

[0125] An amino acid difference may be an amino acid substitution, insertion or deletion. In preferred instances the amino acid difference is a substitution, e.g., conservative amino acid substitution, such as conservative amino acid substitution as described herein.

[0126] VH / VL regions of a binding molecule that binds to PAPPA

[0127] In some instances, the binding molecule comprises a heavy chain variable (VH) region and a light chain variable (VL) region. Table 2 provides the amino acid sequences of VH and VL regions for exemplary binding molecules of the disclosure. In some instances, the binding molecule comprises VH and VL region amino acid sequences of any binding molecule illustrated in Table 2. A bindingmolecule of the disclosure may comprise the heavy chain variable region (VH) and the light chain variable region (VL) of any binding molecule of Table 2.

[0128] Table 2: Amino acid sequences of VH and VL regions of exemplary binding molecules

[0129] Binding VH / VL amino acid sequence SEQID molecule

[0130] AB1 VH QVQL VQS GAE VKK PGAS VKVS CKVSGYTLTEL SMYWVRQAP GKGL E WMGGF 7

[0131] DPEDGETLYVQKFQGRVTMTEDTSTDTAYMDLSSLRSEDTAVYYCATGGAV AL FD YWGQGTL VT VS S VL DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAA 8 STLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLYSFPITFGQGT RLEIK AB2 VH QVQL VQS GAE VRK PGAS VKVS CKVSGYTLTEL SMYWVRQAP GKGL E WMGGF 15

[0132] DPEDGETLYVQKFQGRVTMTEDTSTDTAYMDLSSLRSEDTAVYYCATGGAV AL FD YWGQGTL VT VS S VL DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAA 16 STLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLYSFPITFGQGT RLEIK AB3 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSS YSMNWVRQAPGKGLEWVSS I 23

[0133] SSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGVL FFDYWGQGTLVTVSS VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWFQQKPGKAPKLLIYAA 24 SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSS ITPRTFGQGT KVEIK AB10 VH EVQWE S GGDL VQ PGGS LRL S CAAS GFT F S S FAMS WVRQAPGKGLEWVS Al 31

[0134] SVSGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNGDV AMVYFDYWGQGTLVTVS S VL DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKVLIYVA 32 SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGT KVDIK AB8 VH QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWTWIRQPPGKGLEWIG 39

[0135] YIYYSGSTNYNPSLKSRVTISVDTSRNQFSLKLSSVTAADTAVYYCAKYSS SWVFDYWGQGTLVTVS S VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPK 40 LLIYWASTRESGVPDRFSGSGSGTEFTLTISSLQAEDVAVYYCQQYYSTPL TFGGGT KVEIK

[0136]

[0137] AB5 VH QVQLQESGPGLVKPSETLSLTCTVSGASITPYYWSWIRQPPGKGLEWIGYI 47 YHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTRGPEPF DNWGQGTLVTVSS VL DIVMTQSPDSLTVSLGERATINCKSSQSVLYRSNNKNYLAWYQQKPGQAPK 48 LLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVEVYYCQQYYSTPY TFGQGTKLEIK AB16 VH EVQLVE S GGGL VQ PGGS LRL S CAAS GFT F S RYAMS GVRQAPGKGLEWVS VI 55

[0138] SGSGGYTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKFRGY SYGSFDYWGQGTLVTVSS VL DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKVPKLLIYDA 56 SNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNFPLTFGGGT KVETK AB34 VH QVQL VQS GAE VKK PGAS VKVS CKVSGYHLEEL EMYWVRQAP GKGL EWMGGF 114 DPEDGETLYVQKFQGRVTMTEDTSTDTAYMDLSSLRSEDTAVYYCATGGAV AL FD YWGQGTL VT VS S VL DIQLTQSPSFLSASVGDRVTITCRASQGIYQYLAWYQQKPGKAPKLLIYAA 115 SALQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLYSFPITFGQGT RLEIK

[0139]

[0140] In some instances, the binding molecule comprises:

[0141] (a) a VH region amino acid sequence comprising SEQ ID NO: 114 and a VL region amino acid sequence comprising SEQ ID NO: 115;

[0142] (b) a VH region amino acid sequence comprising SEQ ID NO: 7 and a VL region amino acid sequence comprising SEQ ID NO: 8;

[0143] (c) a VH region amino acid sequence comprising SEQ ID NO: 15 and a VL region amino acid sequence comprising SEQ ID NO: 16;

[0144] (d) a VH region amino acid sequence comprising SEQ ID NO: 23 and a VL region amino acid sequence comprising SEQ ID NO: 24;

[0145] (e) a VH region amino acid sequence comprising SEQ ID NO: 31 and a VL region amino acid sequence comprising SEQ ID NO: 32;

[0146] (f) a VH region amino acid sequence comprising SEQ ID NO: 39 and a VL region amino acid sequence comprising SEQ ID NO: 40;

[0147] (g) a VH region amino acid sequence comprising SEQ ID NO: 47 and a VL region amino acid sequence comprising SEQ ID NO: 48; or

[0148] (h) a VH region amino acid sequence comprising SEQ ID NO: 55 and a VL region amino acid sequence comprising SEQ ID NO: 56.In preferred instances, the binding molecule comprises a VH region amino acid sequence comprising SEQ ID NO: 7 and a VL region amino acid sequence comprising SEQ ID NO: 8. In preferred instances, the binding molecule comprises a VH region amino acid sequence comprising SEQ ID NO: 114 and a VL region amino acid sequence comprising SEQ ID NO: 115.

[0149] In some instances, the binding molecule comprises:

[0150] (a) the VH region amino acid sequence comprising SEQ ID NO: 114, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0151] the VL region amino acid sequence comprising SEQ ID NO: 115, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0152] (b) the VH region amino acid sequence comprising SEQ ID NO: 7, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0153] the VL region amino acid sequence comprising SEQ ID NO: 8, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0154] (c) the VH region amino acid sequence comprising SEQ ID NO: 15, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0155] the VL region amino acid sequence comprising SEQ ID NO: 16, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0156] (d) the VH region amino acid sequence comprising SEQ ID NO: 23, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0157] the VL region amino acid sequence comprising SEQ ID NO: 24, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0158] (e) the VH region amino acid sequence comprising SEQ ID NO: 31, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0159] the VL region amino acid sequence comprising SEQ ID NO: 32, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0160] (f) the VH region amino acid sequence comprising SEQ ID NO: 39, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0161] the VL region amino acid sequence comprising SEQ ID NO: 40, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0162] (g) the VH region amino acid sequence comprising SEQ ID NO: 47, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprising SEQ ID NO: 48, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; or

[0163] (h) the VH region amino acid sequence comprising SEQ ID NO: 55, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0164] the VL region amino acid sequence comprising SEQ ID NO: 56, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[0165] In preferred instances, the binding molecule comprises a VH region amino acid sequence comprising SEQ ID NO: 7, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and a VL region amino acid sequence comprising SEQ ID NO: 8, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. In preferred instances, the binding molecule comprises a VH region amino acid sequence comprising SEQ ID NO: 114, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and a VL region amino acid sequence comprising SEQ ID NO: 115, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[0166] In some instances, where the binding molecule comprises a VH region amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a reference VH region amino acid sequence; and a VL region amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a reference VL region amino acid sequence, the CDRs of such a binding molecule are identical to the CDRs of the reference VH and VL region amino acid sequences, and the differences all he outside those CDRs, e.g. the differences between the binding molecule and the reference VH and VL are in the framework regions of the VH and VL sequences.

[0167] For example, in some instances, the binding molecule comprises a VH region and a VL region, wherein the VH region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a VH region amino acid sequence of a reference binding molecule as set out in Table 2, and the VL region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the VL region amino acid sequence of the same binding molecule as set out in Table 2, and the VH region comprises a HCDR1, a HCDR2 and a HCDR3, and the VL region comprises a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have amino acid sequences as set out for the same binding molecule in Table 1.In some instances, the binding molecule comprises a VH region and a VL region, wherein the VH region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 7, and the VH region comprises a HCDR1 of SEQ ID NO: 1, a HCDR2 of SEQ ID NO: 2 and a HCDR3 of SEQ ID NO: 3; and wherein the VL region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 8, and the VL region comprises a LCDR1 of SEQ ID NO: 4, a LCDR2 of SEQ ID NO: 5 and a LCDR3 of SEQ ID NO: 6. In some instances, the binding molecule comprises a VH region and a VL region, wherein the VH region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 7, and the VH region comprises a HCDR1 of SEQ ID NO: 121, a HCDR2 of SEQ ID NO: 2 and a HCDR3 of SEQ ID NO: 3; and wherein the VL region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 8, and the VL region comprises a LCDR1 of SEQ ID NO: 4, a LCDR2 of SEQ ID NO: 5 and a LCDR3 of SEQ ID NO: 6.

[0168] In some instances, the binding molecule comprises a VH region and a VL region, wherein the VH region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 114, and the VH region comprises a HCDR1 of SEQ ID NO: 108, a HCDR2 of SEQ ID NO: 109 and a HCDR3 of SEQ ID NO: 110; and wherein the VL region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 115, and the VL region comprises a LCDR1 of SEQ ID NO: 111, a LCDR2 of SEQ ID NO: 112 and a LCDR3 of SEQ ID NO: 113. In some instances, the binding molecule comprises a VH region and a VL region, wherein the VH region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 114, and the VH region comprises a HCDR1 of SEQ ID NO: 122, a HCDR2 of SEQ ID NO: 109 and a HCDR3 of SEQ ID NO: 110; and wherein the VL region amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 115, and the VL region comprises a LCDR1 of SEQ ID NO: 111, a LCDR2 of SEQ ID NO: 112 and a LCDR3 of SEQ ID NO: 113.

[0169] In some instances, when the binding molecule comprises a VH region amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a reference VH region amino acid sequence; and a VL region amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a reference VL region amino acid sequence, the binding molecule is a bindingmolecule as described herein that shows sequence variation at one or more CDRs when compared to the corresponding CDR sequences of a reference binding molecule, such as a reference binding molecule as illustrated in Table 1.

[0170] Fc region

[0171] In some instances, the binding molecule comprises an Fc region. The term “Fc region” refers to the region of a binding molecule that normally interacts with cell surface receptors called Fc receptors and some proteins of the complement system.

[0172] In some instances, the Fc region is an IgG Fc region, an IgA Fc region, an IgD Fc region, an IgE Fc region, or an IgM Fc region. In some instances, the Fc region is an IgG Fc region. In some instances, the IgG Fc region is an IgGl Fc region, an IgG2 Fc region, an IgG3 Fc region, or an IgG4 Fc region. In some instances, the IgG Fc region is an IgGl Fc region.

[0173] In some instances, the Fc region is a wild-type Fc region. In some instances, the Fc region is a wildtype IgG, IgA, IgD, IgE, or IgM Fc region. In some instances, the Fc region is a wild-type IgG Fc region. In some instances, the IgG Fc region is a wild-type IgGl, IgG2, IgG3, or IgG4 Fc region. In some instances, the IgG Fc region is a wild-type IgGl Fc region.

[0174] In some instances, the Fc region is a human Fc region, such as a human Ig Fc region of any Ig type as disclosed herein, such as a human IgG Fc region of any IgG type as disclosed herein. In some instances, the human Fc region is a wild-type human Fc region, such as a wild-type human Ig Fc region of any Ig type as disclosed herein, such as a wild-type human IgG Fc region of any IgG type as disclosed herein. In some instances, the Fc region is a human IgG Fc region, such as wild-type human IgG Fc region. In some instances, the Fc region is a human IgGl Fc region, such as wildtype human IgGl Fc region.

[0175] In some instances, the Fc region comprises one or more modifications from a wild-type Fc region, such as from any wild-type Fc region as disclosed herein. In some instances, the Fc region comprises one or more modifications from a wild-type Fc region, where the modification(s) reduce effector function of the binding molecule compared to the unmodified sequence. In some instances, the modifications reduce binding affinity of the Fc region to an Fc receptor. In some instances, the modifications reduce binding affinity of the Fc region to one or more Fc receptors. In some instances, the modifications reduce binding affinity of the Fc region to an IgG Fc receptor. In someinstances, the modifications reduce binding affinity for one or more of FcyRI, FcyRII, and FcyRIII. In some instances, the binding affinity is reduced to an undetectable level. In some instances, the reduced binding affinity is a lack of binding affinity, i.e. the binding molecule is unable to bind to the relevant Fc receptor(s). In some instances, the Fc region is an Fc silent region. As used herein, the term “Fc silent region” refers to an Fc region which does not induce antibody directed cytotoxicity (ADCC). ADCC is the process by which binding molecules, such as antibodies or antigen-binding fragments thereof, bind to Fc receptors on immune effectors cells and activate the immune effector cells. A binding molecule comprising an Fc region which has reduced effector Junction, or an Fc silent region, may have reduced ability to induce ADCC, and thereby have reduced off-target effects and minimise bystander killing. In some instances, the binding molecule has low immunogenic effect.

[0176] In some instances, the Fc region comprises one or more modifications from a wild-type Fc region, where the modification(s) increase the serum half-life of the binding molecule compared to the unmodified sequence. In some instances, the Fc region comprises one or more modifications from a wild-type Fc region, where the modification(s) increase the serum half-life of the binding molecule by approximately four times compared to the unmodified sequence. In some instances, the Fc region comprises one or more modifications from a wild-type Fc region, where the modification(s) increase binding affinity of the Fc region to an FcRn. In some instances, the Fc region comprises one or more modifications from a wild-type Fc region, where the modification(s) increase binding affinity of the Fc region to human FcRn.

[0177] In some instances, the Fc region comprises a combination of modifications described herein. For example, in some instances, the Fc region comprises: (a) one or more modifications from a wildtype Fc region where the modification(s) reduce effector function of the binding molecule compared to the unmodified sequence; and (b) one or more modifications from a wild-type Fc region where the modification(s) increase the serum half-life of the binding molecule compared to the unmodified sequence. In some instances, the Fc region comprises: (a) one or more modifications from a wildtype Fc region where the modification(s) reduce effector function of the binding molecule compared to the unmodified sequence; and (b) one or more modifications from a wild-type Fc region where the modification s) increase binding affinity of the Fc region to an FcRn. In some instances, the Fc region comprises: (a) one or more modifications from a wild-type Fc region where the modification(s) reduce binding affinity of the Fc region to one or more Fc receptors; and (b) one or more modifications from a wild-type Fc region where the modification(s) increase the serum half-life of the binding molecule compared to the unmodified sequence. In some instances, the Fc regioncomprises: (a) one or more modifications from a wild-type Fc region where the modification(s) reduce binding affinity of the Fc region to one or more Fc receptors; and (b) one or more modifications from a wild-type Fc region where the modification(s) increase binding affinity of the Fc region to an FcRn. In some instances, the Fc region is an Fc silent region which comprises one or more modifications from a wild-type Fc region where the modification(s) increase the serum half-life of the binding molecule compared to the unmodified sequence. In some instances, the Fc region is an Fc silent region which comprises one or more modifications from a wild-type Fc region where the modification s) increase binding affinity of the Fc silent region to an FcRn.

[0178] In some instances, the modifications are amino acid substitutions, insertions or deletions. In some instances, the modifications are amino acid substitutions, insertions or deletions of one or more amino acids. In some instances, the Fc region is a fragment of a wild-type Fc region. In some instances, the Fc region is a truncated form of a wild-type Fc region.

[0179] In some instances, the Fc region is an IgGl TM Fc region. IgGl TM is a IgGl triple mutant, which contains 3 point mutations (L234F / L235E / P331S) in the Fc region that reduce the binding affinity of the Fc region to Fc-gamma receptors (FcyRs) and C Iq (Oganesyan et al. Acta Crystallogr D Biol Crystallogr, (2008) 64: 700-704). The IgGl TM Fc region may therefore have reduced immune cell activation via reduced binding to Fc-gamma receptors, and reduced complement activation via reduced binding to Clq. In some instances, the binding molecule comprises a L234F / L235E / P33 IS triple mutation in the Fc region. In some instances, the binding molecule comprises a L234F / L235E / P33 IS triple mutation in the Fc region numbered based on Rabat numbering. In some instances, the Fc region is a human IgGl TM Fc region.

[0180] In some instances, the Fc region is an IgGl YTE Fc region. IgGl YTE is an IgGl mutant which contains 3 point mutations (M252Y / S254T / T256E) in the Fc region that result in an increase in the binding of the Fc region to human FcRn and improves the serum half-life of the binding molecule having the mutations. The YTE mutant has been shown to increase the serum half-life of antibodies by approximately four times as compared to wild-type versions of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006); Robbie et al., (2013) Antimicrob. Agents Chemother.

[0181] 57, 6147-6153). Accordingly, in some instances, the binding molecule comprises M252Y / S254T / T256E mutations in the Fc region. In some instances, the binding molecule comprises M252Y / S254T / T256E mutations in the Fc region numbered based on Rabat numbering. In some instances, the Fc region comprises M252Y / S254T / T256E mutations. In some instances, the Fc region is a human IgGl YTE Fc region.In some instances, the Fc region is an IgGl TM-YTE Fc region. IgGl TM-YTE is an IgGl mutant which contains L234F / L235E / P331S and M252Y / S254T / T256E mutations in the Fc region. Accordingly, in some instances, the binding molecule comprises a L234F / L235E / P331S triple mutation in the Fc region and M252Y / S254T / T256E mutations in the Fc region. In some instances, the binding molecule comprises a L234F / L235E / P331S triple mutation in the Fc region and M252Y / S254T / T256E mutations in the Fc region numbered based on Kabat numbering. In some instances, the binding molecule comprises L234F / L235E / M252Y / S254T / T256E / P331S mutations in the Fc region. In some instances, the binding molecule comprises L234F / L235E / M252Y / S254T / T256E / P331S mutations in the Fc region numbered based on Kabat numbering. In some instances, the Fc region is a human IgGl TM-YTE Fc region.

[0182] Heavy chain and light chain

[0183] In some instances, the binding molecule comprises a heavy chain and / or a light chain. In some instances, the binding molecule comprises a heavy chain and a light chain. In some instances, the binding molecule comprises a fragment of a heavy chain and / or a fragment of a light chain. In some instances, the binding molecule comprises a fragment of a heavy chain and a fragment of a light chain.

[0184] Any of the fragments disclosed herein may be functional fragments. In some instances, a functional fragment of a binding molecule binds to the same target antigen(s) as the full length binding molecule, and preferably exhibits the same antigen cross-reactivity profile as the full length binding molecule.

[0185] In some instances, the binding molecule comprises an antigen binding fragment of a heavy chain and / or an antigen binding fragment of a light chain. In some instances, the binding molecule comprises a fragment of a heavy chain and / or a fragment of a light chain, and the binding molecule retains the ability to bind to PAPPA. In some instances, the binding molecule comprises a heavy chain, or a fragment thereof, which comprises a VH region as disclosed herein, and a light chain, or a fragment thereof, which comprises a VL region as disclosed herein. In some instances, the heavy chain or fragment thereof, and the light chain or fragment thereof, comprise a VH region and a VL region, respectively, of a binding molecule as shown in Table 2. In some instances, the heavy chain or fragment thereof, and the light chain or fragment thereof, comprise a VH region and a VL region, respectively, wherein the VH and VL comprise a set of six CDRs as shown for any binding molecule in Table 1.In some instances, the binding molecule comprises two heavy chains and two light chains. In some instances, the binding molecule comprises two copies of a heavy chain or fragment thereof as described herein and two copies of a light chain or fragment thereof as described herein. In some instances, the two heavy chains and two light chains of the binding molecule form an immunoglobulin structure, such as an antibody or an antigen binding fragment thereof. In some instances, the two heavy chains of the binding molecule form an Fc region, such as any Fc region as described herein.

[0186] There are five major classes of heavy chain constant region, classified as IgA, IgG, IgD, IgE and IgM, each with characteristic effector functions designated by isotype. For example, IgG is separated into four subclasses known as IgGl, IgG2, IgG3, and IgG4. In some instances, the binding molecule is any isotype, i.e. IgA, IgD, IgE, IgG or IgM, or is a synthetic multimer of the four-chain immunoglobulin (Ig) structure. In preferred instances, the binding molecule is of an IgG isotype. The binding molecules can be any IgG subclass, for example IgGl, IgG2, IgG3, or IgG4 isotype. In preferred instances, the binding molecule is of an IgGl isotype. In some instances, the binding molecule has a human isotype, such as human IgG, or human IgGl.

[0187] In some instances, the binding molecule comprises a heavy chain constant region that is of IgG isotype. In some instances, the binding molecule comprises a portion of a heavy chain constant region that is of IgG isotype. In some instances, the IgG constant region or portion thereof is an IgGl, IgG2, IgG3, or IgG4 constant region. In some instances, the IgG constant region or portion thereof is an IgGl constant region. In some instances, the IgG constant region or portion thereof is a human IgG constant region or portion thereof. In some instances, the IgGl constant region or portion thereof is a human IgGl constant region or portion thereof.

[0188] Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and FcyRIII. The important sequences for the binding of IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains. In some instances, the binding molecule comprises a heavy chain constant region comprising one or more modifications from a wild-type heavy chain constant region, where the modification(s) reduce effector function of the binding molecule compared to the unmodified sequence. In some instances, the modification(s) are in the CH2 domain and / or the CH3 domain of the heavy chain. In some instances, the modificationsreduce binding affinity of the binding molecule to an Fc receptor. In some instances, the modifications reduce binding affinity of the binding molecule to one or more Fc receptors. In some instances, the modifications reduce binding affinity of the binding molecule to an IgG Fc receptor. In some instances, the modifications reduce binding affinity for one or more of FcyRI, FcyRII, and FcyRIII. In some instances, the binding affinity is reduced to an undetectable level. In some instances, the reduced binding affinity is a lack of binding affinity, i.e. the binding molecule is unable to bind to the relevant Fc receptor(s).

[0189] In preferred instances, the binding molecule comprises a heavy chain constant region that forms an Fc region disclosed herein. For example, in some instances, the binding molecule comprises a heavy chain constant region that forms an Fc region which is a human IgGl TM Fc region. In some instances, the binding molecule comprises a heavy chain constant region that forms an Fc region which is a human IgGl YTE Fc region. In some instances, the binding molecule comprises a heavy chain constant region that forms an Fc region which is a human IgGl TM-YTE Fc region.

[0190] In some instances, the binding molecule comprises a kappa light chain.

[0191] In some instances, the binding molecule comprises all or part of a heavy chain constant region amino acid sequence comprising SEQ ID NO: 90. In some instances, the binding molecule comprises a heavy chain constant region amino acid sequence comprising SEQ ID NO: 90, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. In some instances, the binding molecule comprises a heavy chain constant region amino acid sequence comprising SEQ ID NO: 90. In some instances, the binding molecule comprises all or part of a heavy chain constant region amino acid sequence comprising SEQ ID NO: 119. In some instances, the binding molecule comprises a heavy chain constant region amino acid sequence comprising SEQ ID NO: 119, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. In some instances, the binding molecule comprises a heavy chain constant region amino acid sequence comprising SEQ ID NO: 119. In some instances, the binding molecule comprises all or part of a light chain constant region amino acid sequence comprising SEQ ID NO: 91. In some instances, the binding molecule comprises a light chain constant region amino acid sequence comprising SEQ ID NO: 91, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. In some instances, the binding molecule comprises a light chain constant region amino acid sequence comprising SEQ ID NO: 91.SEQ ID NO: 90 - Heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0192] SEQ ID NO: 119 - Heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLYI TREPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0193] SEQ ID NO: 91 - Light chain constant region RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0194] In some instances, the binding molecule comprises all or part of a heavy chain constant region amino acid sequence comprising SEQ ID NO: 90 and all or part of a light chain constant region amino acid sequence comprising SEQ ID NO: 91. In some instances, the binding molecule comprises a heavy chain constant region amino acid sequence comprising SEQ ID NO: 90, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto and a light chain constant region amino acid sequence comprising SEQ ID NO: 91, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. In preferred instances, the binding molecule comprises a heavy chain constant region amino acid sequence comprising SEQ ID NO: 90, and a light chain constant region amino acid sequence comprising SEQ ID NO: 91.

[0195] In some instances, the binding molecule comprises a heavy chain constant region of SEQ ID NO: 90, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto and a light chain constant region of SEQ ID NO: 91, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, and further comprises a VH region comprising a HCDR1, a HCDR2 and a HCDR3, and a VL region comprises a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have amino acid sequences as set out for the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of a binding molecule in Table 1. In some instances, the binding molecule comprises a heavy chain constant region of SEQ ID NO: 90, and a light chain constant region of SEQ ID NO: 91, and further comprises a VH region comprising a HCDR1, a HCDR2 and a HCDR3, and a VL region comprises a LCDR1, aLCDR2, and a LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have amino acid sequences as set out for the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of a binding molecule in Table 1.

[0196] In some instances, the binding molecule comprises a heavy chain constant region of SEQ ID NO: 90, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto and a light chain constant region of SEQ ID NO: 91, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, and further comprises a VH region and a VL region as disclosed herein, e.g., the VH and VL sequences of any of the binding molecules of Table 2. In some instances, the binding molecule comprises a heavy chain constant region of SEQ ID NO: 90 and a light chain constant region of SEQ ID NO: 91, and further comprises a VH region and a VL region as disclosed herein, e.g. the VH and VL sequences of any of the binding molecules of Table 2. The heavy chain constant region includes three domains, CHI, CH2 and CH3. The light chain constant region is comprised of one domain, CL.

[0197] In some instances, the binding molecule comprises all or part of a heavy chain constant region amino acid sequence comprising SEQ ID NO: 119 and all or part of a light chain constant region amino acid sequence comprising SEQ ID NO: 91. In some instances, the binding molecule comprises a heavy chain constant region amino acid sequence comprising SEQ ID NO: 119, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto and a light chain constant region amino acid sequence comprising SEQ ID NO: 91, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. In preferred instances, the binding molecule comprises a heavy chain constant region amino acid sequence comprising SEQ ID NO: 119, and a light chain constant region amino acid sequence comprising SEQ ID NO: 91.

[0198] In some instances, the binding molecule comprises a heavy chain constant region of SEQ ID NO: 119, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto and a light chain constant region of SEQ ID NO: 91, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, and further comprises a VH region comprising a HCDR1, a HCDR2 and a HCDR3, and a VL region comprises a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have amino acid sequences as set out for the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of a binding molecule in Table 1. In some instances, the binding molecule comprises a heavy chain constant region of SEQ ID NO: 119, and a light chain constant region of SEQ ID NO: 91, and further comprises a VHregion comprising a HCDR1, a HCDR2 and a HCDR3, and a VL region comprises a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have amino acid sequences as set out for the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of a binding molecule in Table 1.

[0199] In some instances, the binding molecule comprises a heavy chain constant region of SEQ ID NO: 119, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto and a light chain constant region of SEQ ID NO: 91, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, and further comprises a VH region and a VL region as disclosed herein, e.g., the VH and VL sequences of any of the binding molecules of Table 2. In some instances, the binding molecule comprises a heavy chain constant region of SEQ ID NO: 119 and a light chain constant region of SEQ ID NO: 91, and further comprises a VH region and a VL region as disclosed herein, e.g. the VH and VL sequences of any of the binding molecules of Table 2.

[0200] Table 3 provides the amino acid sequences of heavy chain (HC) and light chain (LC) for exemplary binding molecules of the disclosure. In some instances, the binding molecule comprises HC and LC amino acid sequences of any binding molecule illustrated in Table 3. A binding molecule of the disclosure may comprise the heavy chain (HC) and the light chain (LC) of any binding molecule of Table 3.

[0201] T able 3 : Amino acid sequences of heavy chain (HC) and light chain (LC) of exemplary binding molecules

[0202] Binding HC / LC amino acid sequence SEQ ID molecule

[0203] AB1 HC QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMYWVRQAPGKGLEWMGGFDP 76 E D GE T L YVQKF QGRVTMT E D T S T D T AYMD L S S L RS E D T AVY YC AT GGAVAL F D YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK LC DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAAST 77 LQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLYSFPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC AB2 HC QVQLVQSGAEVRKPGASVKVSCKVSGYTLTELSMYWVRQAPGKGLEWMGGFDP 78

[0204]

[0205] E D GE T L YVQKF QGRVTMT E D T S T D T AYMD L S S L RS E D T AVY YC AT GGAVAL F DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK LC DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAAST 79 LQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLYSFPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC AB3 HC EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISS 80

[0206] SSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGVLFFDY WGQGT LVTVS S AS TKGP S VF PL AP S S KS T S GGTAALGCL VKD YF PE PVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLS PGK LC DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWFQQKPGKAPKLLIYAASS 81 LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSITPRTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC AB10 HC EVQWE S GGDL VQ PGGS LRL S CAAS GFT F S S FAMS WVRQAPGKGLEWVS AT S V 82

[0207] SGDITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNGDVAMVY FDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKE YKC KVS NKAL PAS I E KT I S KAKGQ PRE P QVYT L P P S RE EMT KNQVS L T C L VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VF S C S VMHE AL HNHYTQKSLSLSPGK LC DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKVLIYVASS 83 LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC AB8 HC QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWTWIRQPPGKGLEWIGYI 84

[0208] YYSGSTNYNPSLKSRVTISVDTSRNQFSLKLSSVTAADTAVYYCAKYSSSWVF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG KEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLS PGK LC DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLL 85 IYWASTRESGVPDRFSGSGSGTEFTLTISSLQAEDVAVYYCQQYYSTPLTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVT KSFNRGEC AB5 HC QVQLQESGPGLVKPSETLSLTCTVSGASITPYYWSWIRQPPGKGLEWIGYIYH 86

[0209] SGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTRGPEPFDNWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR

[0210]

[0211] VEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNH YT QKS L S L S PGK LC DIVMTQSPDSLTVSLGERATINCKSSQSVLYRSNNKNYLAWYQQKPGQAPKLL 87 IYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVEVYYCQQYYSTPYTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVT KSFNRGEC AB16 HC EVQLVE S GGGL VQ PGGS LRL S CAAS GFT F S RYAMS GVRQAPGKGLEWVS VI S G 88 SGGYTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKFRGYSYGS FDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKE YKC KVS NKAL PAS I E KT I S KAKGQ PRE P QVYT L P P S RE EMT KNQVS L T C L VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VF S C S VMHE AL HNH YT QKS L S L S P GK LC DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKVPKLLIYDASN 89 LETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNFPLTFGGGTKVET KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC AB34 TM HC QVQL VQS GAE VKK PGAS VKVS CKVSGYHLEEL EMYWVRQAP GKGL E WMGGFD P 116 E D GE T L YVQKF QGRVTMT E D T S T D T AYMD L S S L RS E D T AVY YC AT GGAVAL F D YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK E YKC KVS NKAL PAS I E KT I S KAKGQ P RE P QVYT L P P S RE EMT KNQVS L T C L VK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK LC DIQLTQSPSFLSASVGDRVTITCRASQGIYQYLAWYQQKPGKAPKLLIYAASA 117 LQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLYSFPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC AB34 HC QVQL VQS GAE VKK PGAS VKVS CKVSGYHLEEL EMYWVRQAP GKGL E WMGGFD P 118 EDGETLYVQKFQGRVTMTEDTSTDTAYMDLSSLRSEDTAVYYCATGGAVALFD TM-YTE YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLYITREPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK LC DIQLTQSPSFLSASVGDRVTITCRASQGIYQYLAWYQQKPGKAPKLLIYAASA 117 LQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLYSFPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

[0212]

[0213] EC

[0214] In some instances, the binding molecule comprises:

[0215] (a) a HC amino acid sequence comprising SEQ ID NO: 118 and a LC amino acid sequence comprising SEQ ID NO: 117;1

[0216] (b) a HC amino acid sequence comprising SEQ ID NO: 116 and a LC amino acid sequence comprising SEQ ID NO: 117;

[0217] (c) a HC amino acid sequence comprising SEQ ID NO: 76 and a LC amino acid sequence comprising SEQ ID NO: 77;

[0218] (d) a HC amino acid sequence comprising SEQ ID NO: 78 and a LC amino acid sequence comprising SEQ ID NO: 79;

[0219] (e) a HC amino acid sequence comprising SEQ ID NO: 80 and a LC amino acid sequence comprising SEQ ID NO: 81;

[0220] (f) a HC amino acid sequence comprising SEQ ID NO: 82 and a LC amino acid sequence comprising SEQ ID NO: 83;

[0221] (g) a HC amino acid sequence comprising SEQ ID NO: 84 and a LC amino acid sequence comprising SEQ ID NO: 85;

[0222] (h) a HC amino acid sequence comprising SEQ ID NO: 86 and a LC amino acid sequence comprising SEQ ID NO: 87; or

[0223] (i) a HC amino acid sequence comprising SEQ ID NO: 88 and a LC amino acid sequence comprising SEQ ID NO: 89.

[0224] In preferred instances, the binding molecule comprises a HC amino acid sequence comprising SEQ ID NO: 76 and a LC amino acid sequence comprising SEQ ID NO: 77. In preferred instances, the binding molecule comprises a HC amino acid sequence comprising SEQ ID NO: 118 and a LC amino acid sequence comprising SEQ ID NO: 117.

[0225] In some instances, the binding molecule comprises:

[0226] (a) a HC amino acid sequence comprising SEQ ID NO: 118, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0227] a LC amino acid sequence comprising SEQ ID NO: 117, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto

[0228] (b) a HC amino acid sequence comprising SEQ ID NO: 116, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0229] a LC amino acid sequence comprising SEQ ID NO: 117, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; or

[0230] (c) a HC amino acid sequence comprising SEQ ID NO: 76, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; anda LC amino acid sequence comprising SEQ ID NO: 77, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0231] (d) a HC amino acid sequence comprising SEQ ID NO: 78, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0232] a LC amino acid sequence comprising SEQ ID NO: 79, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0233] (e) a HC amino acid sequence comprising SEQ ID NO: 80, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0234] a LC amino acid sequence comprising SEQ ID NO: 81, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0235] (f) a HC amino acid sequence comprising SEQ ID NO: 82, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0236] a LC amino acid sequence comprising SEQ ID NO: 83, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0237] (g) a HC amino acid sequence comprising SEQ ID NO: 84, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0238] a LC amino acid sequence comprising SEQ ID NO: 85, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;

[0239] (h) a HC amino acid sequence comprising SEQ ID NO: 86, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0240] a LC amino acid sequence comprising SEQ ID NO: 87, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; or

[0241] (i) a HC amino acid sequence comprising SEQ ID NO: 88, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and

[0242] a LC amino acid sequence comprising SEQ ID NO: 89, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[0243] In preferred instances, the binding molecule comprises a HC amino acid sequence comprising SEQ ID NO: 76, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and a LC amino acid sequence comprising SEQ ID NO: 77, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. In preferred instances, the binding molecule comprises a HC amino acid sequence comprising SEQ ID NO: 118, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and a LC amino acid sequencecomprising SEQ ID NO: 117, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[0244] In some instances, where the binding molecule comprises a HC amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a reference HC amino acid sequence; and a LC amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a reference LC amino acid sequence, the CDRs of such a binding molecule are identical to the CDRs of the reference HC and LC amino acid sequences, and the differences all he outside those CDRs.

[0245] For example, in some instances, the binding molecule comprises a HC and a LC, wherein the HC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a HC amino acid sequence of a reference binding molecule as set out in Table 3, and the LC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the LC amino acid sequence of the same binding molecule as set out in Table 3, and the VH region comprises a HCDR1, a HCDR2 and a HCDR3, and the VL region comprises a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have amino acid sequences as set out for the same binding molecule in Table 1.

[0246] In some instances, the binding molecule comprises a HC and a LC, wherein the HC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 76, and the VH region comprises a HCDR1 of SEQ ID NO: 1, a HCDR2 of SEQ ID NO: 2 and a HCDR3 of SEQ ID NO: 3; and wherein the LC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 77, and the VL region comprises a LCDR1 of SEQ ID NO: 4, a LCDR2 of SEQ ID NO: 5 and a LCDR3 of SEQ ID NO: 6. In some instances, the binding molecule comprises a HC and a LC, wherein the HC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 76, and the VH region comprises a HCDR1 of SEQ ID NO: 121, a HCDR2 of SEQ ID NO: 2 and a HCDR3 of SEQ ID NO: 3; and wherein the LC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 77, and the VL region comprises a LCDR1 of SEQ ID NO: 4, a LCDR2 of SEQ ID NO: 5 and a LCDR3 of SEQ ID NO: 6.In some instances, the binding molecule comprises a HC and a LC, wherein the HC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 118, and the VH region comprises a HCDR1 of SEQ ID NO: 108, a HCDR2 of SEQ ID NO: 109 and a HCDR3 of SEQ ID NO: 110; and wherein the LC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 117, and the VL region comprises a LCDR1 of SEQ ID NO: 111, a LCDR2 of SEQ ID NO: 112 and a LCDR3 of SEQ ID NO: 113. In some instances, the binding molecule comprises a HC and a LC, wherein the HC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 118, and the VH region comprises a HCDR1 of SEQ ID NO: 122, a HCDR2 of SEQ ID NO: 109 and a HCDR3 of SEQ ID NO: 110; and wherein the LC amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 117, and the VL region comprises a LCDR1 of SEQ ID NO: 111, a LCDR2 of SEQ IDNO: 112 and a LCDR3 of SEQ ID NO: 113.

[0247] In some instances, when the binding molecule comprises a HC amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a reference HC region amino acid sequence; and a LC amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a reference LC amino acid sequence, the binding molecule is a binding molecule as described herein that shows sequence variation at one or more CDRs when compared to the corresponding CDR sequences of a reference binding molecule, such as a reference binding molecule as illustrated in Table 1.

[0248] In some instances, the binding molecule is an isolated binding molecule.

[0249] Functional properties

[0250] Binding specificity

[0251] In one aspect, there is provided a binding molecule that binds to PAPPA. In some instances, the binding molecule has the CDRs of a binding molecule as described herein that binds to PAPPA. In some instances, the binding molecule has the VH / VL region of a binding molecule as described herein that binds to PAPPA. In some instances, the binding molecule has an Fc region as described herein. In some instances, the binding molecule has a heavy chain and / or a light chain as described herein.In some instances, the binding molecule binds to human PAPPA. In some instances, the human PAPPA comprises the amino acid sequence of SEQ ID NO: 57, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71, as shown in Table 4 below. In some instances, the binding molecule binds to human PAPPA, wherein the human PAPPA comprises the amino acid sequence of SEQ ID NO: 71. In some instances, the binding molecule binds to human PAPPA, wherein the human PAPPA consists of the amino acid sequence SEQ ID NO: 71. In some instances, the binding molecule binds to a protein having the amino acid sequence of SEQ ID NO: 71. In some instances, the binding molecule binds to an epitope within SEQ ID NO: 71.

[0252] In some instances, the binding molecule binds to cynomolgus PAPPA. In some instances, the binding molecule binds to cynomolgus PAPPA, wherein the cynomolgus PAPPA comprises the amino acid sequence of SEQ ID NO: 72. In some instances, the binding molecule binds to cynomolgus PAPPA, wherein the cynomolgus PAPPA consists of the amino acid sequence of SEQ ID NO: 72. In some instances, the binding molecule binds to a protein having the amino acid sequence of SEQ ID NO: 72. In some instances, the binding molecule binds to an epitope within SEQ ID NO: 72.

[0253] In some instances, the binding molecule binds to mouse PAPPA. In some instances, the binding molecule binds to mouse PAPPA, wherein the mouse PAPPA comprises the amino acid sequence of SEQ ID NO: 73. In some instances, the binding molecule binds to mouse PAPPA, wherein the mouse PAPPA consists of the amino acid sequence of SEQ ID NO: 73. In some instances, the binding molecule binds to a protein having the amino acid sequence of SEQ ID NO: 73. In some instances, the binding molecule binds to an epitope within SEQ ID NO: 73.

[0254] In some instances, the binding molecule binds to human PAPPA, cynomolgus PAPPA, and mouse PAPPA. In some instances, the binding molecule binds to human PAPPA, wherein the human PAPPA comprises the amino acid sequence of SEQ ID NO: 71, cynomolgus PAPPA, wherein the cynomolgus PAPPA comprises the amino acid sequence of SEQ ID NO: 72, and mouse PAPPA, wherein the mouse PAPPA comprises the amino acid sequence of SEQ ID NO: 73. In some instances, the binding molecule binds to human PAPPA, wherein the human PAPPA consists of the amino acid sequence of SEQ ID NO: 71, cynomolgus PAPPA, wherein the cynomolgus PAPPA consists of the amino acid sequence of SEQ ID NO: 72, and mouse PAPPA, wherein the mouse PAPPA consists of the amino acid sequence of SEQ ID NO: 73. In some instances, the bindingmolecule binds to an epitope within SEQ ID NO: 71, an epitope within SEQ ID NO: 72, and an epitope within SEQ ID NO: 73.

[0255] In some instances, the binding molecule does not bind to PAPPA-2. In some instances, the binding molecule does not bind to human PAPPA-2. In some instances, the binding molecule does not bind to human PAPPA-2, wherein the human PAPPA-2 comprises the amino acid sequence of SEQ ID NO: 75. In some instances, the binding molecule does not bind to human PAPPA-2, wherein the human PAPPA-2 consists of the amino acid sequence of SEQ ID NO: 75. In some instances, the binding molecule does not bind to a protein having the amino acid sequence of SEQ ID NO: 75. In some instances, the binding molecule does not bind to an epitope within SEQ ID NO: 75.

[0256] References herein to a binding molecule that binds to a particular target relate to the ability of the binding molecule to specifically bind to that target. For example, in some instances, the binding molecule specifically binds to PAPPA. By specific binding, it will be understood that the binding molecule binds to the target, with no significant cross-reactivity to another protein, such as an unrelated protein. For example, a binding molecule that specifically binds to PAPPA may have no significant cross-reactivity to any other pappalysin protein. For example, a binding molecule that specifically binds to PAPPA may have no significant cross-reactivity to PAPPA-2. A binding molecule that is specific for a target protein, may bind to multiple forms of that target protein, while having no significant cross-reactivity to other, different, proteins. For example, a binding molecule that is specific for PAPPA may bind to human PAPPA, cynomolgus PAPPA, and mouse PAPPA, and not bind to human PAPPA-2. In some instances, the binding molecule binds to human PAPPA, wherein the human PAPPA comprises the amino acid sequence of SEQ ID NO: 71, cynomolgus PAPPA, wherein the cynomolgus PAPPA comprises the amino acid sequence of SEQ ID NO: 72, and mouse PAPPA, wherein the mouse PAPPA comprises the amino acid sequence of SEQ ID NO: 73, and does not bind to human PAPPA-2, wherein the human PAPPA-2 comprises the amino acid sequence of SEQ ID NO: 74. In some instances, the binding molecule binds to human PAPPA, wherein the human PAPPA consists of the amino acid sequence of SEQ ID NO: 71, cynomolgus PAPPA, wherein the cynomolgus PAPPA consists of the amino acid sequence of SEQ ID NO: 72, and mouse PAPPA, wherein the mouse PAPPA consists of the amino acid sequence of SEQ ID NO: 73 and does not bind to human PAPPA-2, wherein the human PAPPA-2 consists of the amino acid sequence of SEQ ID NO: 74. In some instances, the binding molecule binds to an epitope within SEQ ID NO: 71, an epitope within SEQ ID NO: 72, and an epitope within SEQ ID NO: 73, and does not bind to an epitope within SEQ ID NO: 74.In some instances, the binding molecule does not bind to HEK293 cells. In some instances, the binding molecule does not show non-specific binding to HEK293 cells. Binding to HEK293 cells can be measured by any suitable means. In some instances, binding to HEK293 cells is assessed using a method described herein. In some instances, binding to HEK293 cells is assessed using a Mirrorball assay, such as the Mirrorball assay described in Example 7.

[0257] Binding of the binding molecule to PAPPA and / or PAPPA-2 can be assessed by any suitable means. In some instances, binding of the binding molecule to PAPPA and / or PAPPA-2 is assessed using a method described herein. In some instances, binding of the binding molecule to PAPPA and / or PAPPA-2 is assessed using a homogenous time-resolved fluorescence (HTRF) assay, such as an HTRF assay described in Example 4.

[0258] Table 4: PAPPA sequences

[0259] PAPPA Amino Acid Sequence

[0260] sequence

[0261] Human PAPPA MRLWS WVL HL GL L S AAL GC GL AE RP RRARRD P RAGR P P RPAAG PAT CAT RAARGRRAS P P P P P P PGGAWE AVRVP RRRQQRE ARGAT E E P S P P S RAL Y F S GRGE QL RL RAD L E L PR

[0262] (with DAFTLQVWLRAEGGQRSPAVITGLYDKCSYISRDRGWWGIHTISDQDNKDPRYFFSL KTDRARQVTTINAHRSYLPGQWVYLAATYDGQFMKLYVNGAQVATSGEQVGGIFSPLT

[0263] methionine, QKCKVLMLGGSALNHNYRGYIEHFSLWKVARTQREILSDMETHGAHTALPQLLLQENW signal and proDNVKHAWS PMKDGSSPKVEFSNAHGFLLDTSLEPPLCGQTLCDNTEVIASYNQLSSFR QPKVVRYRWNLYEDDHKNPTVTREQVDFQHHQLAEAFKQYNISWELDVLEVSNSSLR

[0264] peptide; SEQ ID RRLILANCDISKIGDENCDPECNHTLTGHDGGDCRHLRHPAFVKKQHNGVCDMDCNYE RFNFDGGECCDPEITNVTQTCFDPDSPHRAYLDVNELKNILKLDGSTHLNIFFAKSSE NO: 57) EELAGVATWPWDKEALMHLGGIVLNPSFYGMPGHTHTMIHEIGHSLGLYHVFRGISEI QSCSDPCMETEPSFETGDLCNDTNPAPKHKSCGDPGPGNDTCGFHSFFNTPYNNFMSY ADDDCTDSFTPNQVARMHCYLDLVYQGWQPSRKPAPVALAPQVLGHTTDSVTLEWFPP IDGHFFERELGSACHLCLEGRILVQYASNASSPMPCSPSGHWSPREAEGHPDVEQPCK SSVRTWSPNSAVNPHTVPPACPEPQGCYLELEFLYPLVPESLTIWVTFVSTDWDSSGA VNDIKLLAVSGKNISLGPQNVFCDVPLTIRLWDVGEEVYGIQIYTLDEHLEIDAAMLT STADTPLCLQCKPLKYKWRDPPLQMDVASILHLNRKFVDMDLNLGSVYQYWVITISG TEESEPSPAVTYIHGSGYCGDGIIQKDQGEQCDDMNKINGDGCSLFCRQEVSFNCIDE PSRCYFHDGDGVCEEFEQKTSIKDCGVYTPQGFLDQWASNASVSHQDQQCPGWVIIGQ PAASQVCRTKVIDLSEGISQHAWYPCTISYPYSQLAQTTFWLRAYFSQPMVAAAVIVH LVTDGTYYGDQKQETISVQLLDTKDQSHDLGLHVLSCRNNPLIIPWHDLSQPFYHSQ AVRVSFSSPLVAISGVALRSFDNFDPVTLSSCQRGETYSPAEQSCVHFACEKTDCPEL AVENASLNCSSSDRYHGAQCTVSCRTGYVLQIRRDDELIKSQTGPSVTVTCTEGKWNK QVACEPVDCSIPDHHQVYAASFSCPEGTTFGSQCSFQCRHPAQLKGNNSLLTCMEDGL WSFPEALCELMCLAPPPVPNADLQTARCRENKHKVGSFCKYKCKPGYHVPGSSRKSKK RAFKTQCTQDGSWQEGACVPVTCDPPPPKFHGLYQCTNGFQFNSECRIKCEDSDASQG LGSNVIHCRKDGTWNGSFHVCQEMQGQCSVPNELNSNLKLQCPDGYAIGSECATSCLD HN S E S 11 L PMNVT VRD I PHWL N P T RVE RWC T AGL KWY PH PAL I HC VKGC E P FMGDN Y CDAINNRAFCNYDGGDCCTSTVKTKKVTPFPMSCDLQGDCACRDPQAQEHSRKDLRGY

[0265]

[0266] SHGHuman PAPPA RLWSWVLHLGLLSAALGCGLAERPRRARRDPRAGRPPRPAAGPATCATRAARGRRASP PPPPPPGGAWEAVRVPRRRQQREARGATEEPSPPSRALYFSGRGEQLRLRADLELPRD

[0267] (without AFTLQVWLRAEGGQRSPAVITGLYDKCSYISRDRGWWGIHTISDQDNKDPRYFFSLK TDRARQVTTINAHRSYLPGQWVYLAATYDGQFMKLYVNGAQVATSGEQVGGIFSPLTQ

[0268] methionine, with KC KVLML GGSALNHNYRGYIEHFS L WKVART QRE ILS DME THGAHT AL P QL L L QE NWD signal and with NVKHAWSPMKDGSSPKVEFSNAHGFLLDTSLEPPLCGQTLCDNTEVIASYNQLSSFRQ PKWRYRWNLYEDDHKNPTVTREQVDFQHHQLAEAFKQYNISWELDVLEVSNSSLRR

[0269] pro-peptide; SEQ RLILANCDISKIGDENCDPECNHTLTGHDGGDCRHLRHPAFVKKQHNGVCDMDCNYER FNFDGGECCDPEITNVTQTCFDPDSPHRAYLDVNELKNILKLDGSTHLNIFFAKSSEE ID NO: 69) ELAGVATWPWDKEALMHLGGIVLNPSFYGMPGHTHTMIHEIGHSLGLYHVFRGISEIQ SCSDPCMETEPSFETGDLCNDTNPAPKHKSCGDPGPGNDTCGFHSFFNTPYNNFMSYA DDDCTDSFTPNQVARMHCYLDLVYQGWQPSRKPAPVALAPQVLGHTTDSVTLEWFPPI DGHFFERELGSACHLCLEGRILVQYASNASSPMPCSPSGHWSPREAEGHPDVEQPCKS SVRTWS PNSAVNPHTVPPACPEPQGCYLELEFLYPLVPESLTIWVTFVSTDWDSSGAV NDIKLLAVSGKNISLGPQNVFCDVPLTIRLWDVGEEVYGIQIYTLDEHLEIDAAMLTS TADTPLCLQCKPLKYKWRDPPLQMDVASILHLNRKFVDMDLNLGSVYQYWVITISGT EESEPSPAVTYIHGSGYCGDGIIQKDQGEQCDDMNKINGDGCSLFCRQEVSFNCIDEP SRCYFHDGDGVCEEFEQKTSIKDCGVYTPQGFLDQWASNASVSHQDQQCPGWVIIGQP AASQVCRTKVIDLSEGISQHAWYPCTISYPYSQLAQTTFWLRAYFSQPMVAAAVIVHL VTDGTYYGDQKQETISVQLLDTKDQSHDLGLHVLSCRNNPLIIPWHDLSQPFYHSQA VRVSFSSPLVAISGVALRSFDNFDPVTLSSCQRGETYSPAEQSCVHFACEKTDCPELA VENASLNCSSSDRYHGAQCTVSCRTGYVLQIRRDDELIKSQTGPSVTVTCTEGKWNKQ VACEPVDCSIPDHHQVYAASFSCPEGTTFGSQCSFQCRHPAQLKGNNSLLTCMEDGLW SFPEALCELMCLAPPPVPNADLQTARCRENKHKVGSFCKYKCKPGYHVPGSSRKSKKR AFKTQCTQDGSWQEGACVPVTCDPPPPKFHGLYQCTNGFQFNSECRIKCEDSDASQGL GSNVIHCRKDGTWNGSFHVCQEMQGQCSVPNELNSNLKLQCPDGYAIGSECATSCLDH NSES IILPMNVTVRDIPHWLNPTRVERWCTAGLKWYPHPALIHCVKGCEPFMGDNYC DAINNRAFCNYDGGDCCTSTVKTKKVTPFPMSCDLQGDCACRDPQAQEHSRKDLRGYS HG

[0270] Human PAPPA E R PRRARRD P RAGR P PR PAAG PAT CAT RAARGRRAS P P P P P P P GGAWE AVRVP RRRQQ REARGATEEPSPPSRALYFSGRGEQLRLRADLELPRDAFTLQVWLRAEGGQRSPAVIT

[0271] (without GLYDKCSYISRDRGWWGIHTISDQDNKDPRYFFSLKTDRARQVTTINAHRSYLPGQW VYLAATYDGQFMKLYVNGAQVATSGEQVGGIFSPLTQKCKVLMLGGSALNHNYRGYIE

[0272] methionine and HFSLWKVARTQREILSDMETHGAHTALPQLLLQENWDNVKHAWS PMKDGSS PKVEFSN signal, with proAHGFLLDTSLEPPLCGQTLCDNTEVIASYNQLSSFRQPKWRYRWNLYEDDHKNPTV TREQVDFQHHQLAEAFKQYNISWELDVLEVSNSSLRRRLILANCDISKIGDENCDPEC

[0273] peptide; SEQ ID NHTLTGHDGGDCRHLRHPAFVKKQHNGVCDMDCNYERFNFDGGECCDPEITNVTQTCF DPDSPHRAYLDVNELKNILKLDGSTHLNIFFAKSSEEELAGVATWPWDKEALMHLGGI NO: 70) VLNPSFYGMPGHTHTMIHEIGHSLGLYHVFRGISEIQSCSDPCMETEPSFETGDLCND TNPAPKHKSCGDPGPGNDTCGFHSFFNTPYNNFMSYADDDCTDSFTPNQVARMHCYLD LVYQGWQPSRKPAPVALAPQVLGHTTDSVTLEWFPPIDGHFFERELGSACHLCLEGRI LVQYASNASS PMPCS PSGHWS PREAEGHPDVEQPCKSSVRTWS PNSAVNPHTVPPACP EPQGCYLELEFLYPLVPESLTIWVTFVSTDWDSSGAVNDIKLLAVSGKNISLGPQNVF CDVPLTIRLWDVGEEVYGIQIYTLDEHLEIDAAMLTSTADTPLCLQCKPLKYKWRDP PLQMDVAS ILHLNRKFVDMDLNLGSVYQYWVITISGTEESEPS PAVT YIHGSGYCGDG IIQKDQGEQCDDMNKINGDGCSLFCRQEVSFNCIDEPSRCYFHDGDGVCEEFEQKTSI KDCGVYTPQGFLDQWASNASVSHQDQQCPGWVIIGQPAASQVCRTKVIDLSEGISQHA WYPCTISYPYSQLAQTTFWLRAYFSQPMVAAAVIVHLVTDGTYYGDQKQETISVQLLD TKDQSHDLGLHVLSCRNNPLIIPWHDLSQPFYHSQAVRVSFSSPLVAISGVALRSFD NFDPVTLSSCQRGETYSPAEQSCVHFACEKTDCPELAVENASLNCSSSDRYHGAQCTV SCRTGYVLQIRRDDELIKSQTGPSVTVTCTEGKWNKQVACEPVDCSIPDHHQVYAASF SCPEGTTFGSQCSFQCRHPAQLKGNNSLLTCMEDGLWSFPEALCELMCLAPPPVPNAD LQTARC RENKHKVGS FC KYKC KPGYHVPGS S RKS KKRAFKTQC T QDGSWQE GACVPVT CDPPPPKFHGLYQCTNGFQFNSECRIKCEDSDASQGLGSNVIHCRKDGTWNGSFHVCQ EMQGQCSVPNELNSNLKLQCPDGYAIGSECATSCLDHNSESIILPMNVTVRDIPHWLN PTRVERWCTAGLKWYPHPALIHCVKGCEPFMGDNYCDAINNRAFCNYDGGDCCTSTV

[0274]

[0275] KT KKVT PFPMSCDLQGD C AC RD P QAQE HS RKD L RGY S HGMature human EARGATEE PS PPSRALYFSGRGEQLRL RADLELPRDAFTLQVWL RAE GGQRS PAVITG LYDKCSYISRDRGWWGIHTISDQDNKDPRYFFSLKTDRARQVTTINAHRSYLPGQWV PAPPA (without YLAATYDGQFMKLYVNGAQVATSGEQVGGIFS PLTQKCKVLMLGGSALNHNYRGYIEH FSLWKVARTQREILSDMETHGAHTALPQLLLQENWDNVKHAWS PMKDGS S PKVEFSNA

[0276] methionine, HGFLLDTSLEPPLCGQTLCDNTEVIASYNQLSSFRQPKWRYRWNLYEDDHKNPTVT signal, and proREQVDFQHHQLAEAFKQYNISWELDVLEVSNSSLRRRLILANCDISKIGDENCDPECN HTLTGHDGGDCRHLRHPAFVKKQHNGVCDMDCNYERFNFDGGECCDPEITNVTQTCFD

[0277] peptide; SEQ ID PDSPHRAYLDVNELKNILKLDGSTHLNIFFAKSSEEELAGVATWPWDKEALMHLGGIV LNPSFYGMPGHTHTMIHEIGHSLGLYHVFRGISEIQSCSDPCMETEPSFETGDLCNDT NO: 71) NPAPKHKSCGDPGPGNDTCGFHSFFNTPYNNFMSYADDDCTDSFTPNQVARMHCYLDL VYQGWQPSRKPAPVALAPQVLGHTTDSVTLEWFPPIDGHFFERELGSACHLCLEGRIL VQYASNAS S PMPCS PSGHWS PREAEGHPDVEQPCKS SVRTWS PNSAVNPHTVPPACPE PQGCYLELEFLYPLVPESLTIWVTFVSTDWDSSGAVNDIKLLAVSGKNISLGPQNVFC DVPLTIRLWDVGEEVYGIQIYTLDEHLEIDAAMLTSTADTPLCLQCKPLKYKWRDPP LQMDVASILHLNRKFVDMDLNLGSVYQYWVITISGTEESEPSPAVTYIHGSGYCGDGI IQKDQGEQCDDMNKINGDGCSLFCRQEVSFNCIDEPSRCYFHDGDGVCEEFEQKTSIK DCGVYTPQGFLDQWASNASVSHQDQQCPGWVIIGQPAASQVCRTKVIDLSEGISQHAW YPCTISYPYSQLAQTTFWLRAYFSQPMVAAAVIVHLVTDGTYYGDQKQETISVQLLDT KDQSHDLGLHVLSCRNNPLIIPWHDLSQPFYHSQAVRVSFSSPLVAISGVALRSFDN FDPVTLSSCQRGETYSPAEQSCVHFACEKTDCPELAVENASLNCSSSDRYHGAQCTVS CRTGYVLQIRRDDELIKSQTGPSVTVTCTEGKWNKQVACEPVDCSIPDHHQVYAASFS CPEGTTFGSQCSFQCRHPAQLKGNNSLLTCMEDGLWSFPEALCELMCLAPPPVPNADL QTARCRENKHKVGSFCKYKCKPGYHVPGSSRKSKKRAFKTQCTQDGSWQEGACVPVTC DPPPPKFHGLYQCTNGFQFNSECRIKCEDSDASQGLGSNVIHCRKDGTWNGSFHVCQE MQGQCSVPNELNSNLKLQCPDGYAIGSECATSCLDHNSESIILPMNVTVRDIPHWLNP TRVERWCTAGLKWYPHPALIHCVKGCEPFMGDNYCDAINNRAFCNYDGGDCCTSTVK TKKVTPFPMSCDLQGDCACRDPQAQEHSRKDLRGYSHG

[0278] Mature GAAE E P S P P S RAL Y F S GRGE QL RL RAD L E L PRDAFT L QVWL RAE GGQRS PAVI T GL YD KCSYTSRDRGWWGIHTISDQGNRDPRYFFSLKTDRARQVTTINAHRSYLPGQWVYLA

[0279] cynomolgus ATYDGRFMKLYVNGAQVATSGEQVGGIFS PLTQKCKVLMLGGSALNHNYRGYIEHFSL WKVARTQREILSDMETHGTHTALPQLLLQENWDNVKHAWS PMKDGSS PKVEFSNAHGF PAPPA LLDTSLEPPLCGQTLCDNTEVIASYNQLPSFRQPKWRYRWNLYEDDHKNPMVTREQ

[0280] (SEQ ID NO: VDFQHQQLAEAFKHYNISWELKVLEVSNSSLRRRLILANCDISKIGDENCDPECNHTL TGHDGGDCRHLRHPAFMKKQQNGVCDMDCNYERFNFDGGECCDPKITDVTQTCFDPDS

[0281] 72) PHRAYL DVNE LKNILRLDGSTHLNIF FAN S S E E E L AGVAT WPWD KE ALMHL GG I VLN P SFYGMPGHTHTMIHEIGHSLGLYHVFRGISEIQSCSDPCMETEPSFETGDLCNDTNPA PKHKSCGDPGPGNDTCGFHSFFNTPYNNFMSYADDDCTDSFTPNQVARMHCYLDLVYQ SWQPSRKPAPVALAPQWGHTTDSVTLEWFPPIDGHFFERELGSACDLCLEGRILVQY AFNASS PMPCS PSGHWS PREAEGHPDVEQPCKS SVRTWS PNSAVNPHTVPPAC PE PQG CYLELEFLYPLVPESLTIWVTFVSTDWDSSGAVNDIKLLTVSGKNISLGPQNVFCDIP LTIRLQDVGEEVYGIQIYTLDEHLEIDAAMLTSTADTPLCLQCKPLQYKWRDPPLQV DVASILHLNRKFIDMDLNRSNVYQYWVITISGTEESEPSPAVTYIHGSGYCGDGIIQK DQGEECDDLNKINGDGCSLFCRQEVSFNCIDEPSRCYFHDGDGVCEEFEQKTSIKDCG VYTPQGFLDQWASNASVSHQDQQCPGWVIIGQPAASQVCRTKVIDLSEGISQHAWYPC TISYPYSQLAQTTFWLRAYFSQPMVAAAVIVHLVTDGTYYGDQKQETISVQLLDTKDQ SHDLGLHVLSCRNNPLIIPWHDLSQPFYHSQAVRVSFSSPLVAISGVALRSFDNFDP VTLSSCQRGETYSPAEQSCVHFACENTDCPELAVENASLNCSSSDRYHGAQCTVSCRT GYVL Q I RRDD E L I KS QMGP S VT VT C T E GKWNKQVAC E P VD C S I P DHHHVYAAS F S C PN GTTFGSQCSFQCRHPAQLKGNNSLLTCMEDGLWSFPEALCELMCLAPPPVPNADLQTP RCRENKHKVGSFCKYKCKPGYHVPGSSRKSKKRAFKTQCTQDGSWQEGACVPVTCDPP PSKFHGLYQCTNGFQFNSECRIKCEDSDASQGRGSNIINCRKDGTWSGSFHVCQEMQG QCSAPNQLNSNLKLQCPDGYAIGSECATSCLDHNSESIILPMNVTVRDIPHWLNPTRV ERWCTAGLKWYPHPALIHCVKGCEPFMGDNYCDAINNRAFCNYDGGDCCTSTVKTKK VTPFPMSCDLQGDCACRDPQAQEHSRKDLRGYSHG

[0282] Mature murine RGAEEPSPPSRALYFSGRGEQLRLRADLELPRDAFTLQVWLRAEGGQKSPAVITGLYD KCSYTSRDRGWVMGIHTTSDQGNRDPRYFFSLKTDRARKVTTIDAHRSYLPGQWVHLA PAPPA ATYDGRLMKLYMNGAQVATSAEQVGGIFS PLTQKCKVLMLGGSALNHNFRGHIEHFSL WKVARTQREIVSDMETRGLHTPLPQLLLQENWDNVKRTWS PMKDGNS PQVEFSNAHGF

[0283]

[0284] LLDTNLEPPLCGQTLCDNTEVISSYNQLPSFRQPKWRYRWNIYDDHHENPTVSWQQ(SEQ IDNO: IDFQHQQLAEAFQHYNISWELEVLNINSSSLRHRLILANCDISKIGDEKCDPECNHTL TGHDGGDCRQLRYPAFMKKQQNGVCDMDCNYERFNFDGGECCDPDITDVTKTCFDPDS

[0285] 73) PHRAYL DVNE LKNILRLDGSTHLNIF FAN S S E E E L AGVAT WPWD KE ALMHL GG I VLN P SFYGIPGHTHTMIHEIGHSLGLYHIFRGISEIQSCSDPCMETEPSFETGDLCNDTNPA PKHKFCGDPGPGNDTCGFHGFFNTPYNNFMSYADDDCTDSFTPNQVSRMHCYLDLVYQ SWQPSRKPAPVALAPQWGHTMDSVMLEWFPPIDGHFFERELGSACDLCLEGRILVQY AFNASS PMPCGPSGHWS PREAEGHPDVEQPCKSSVRTWS PNSAVNPHTVPPAC PE PQG CYLELEFRYPLVPESLTIWVTFVSSDWDSSGAVNDIKLLTISGKNISLGPQNVFCDIP LTIRLRDVGEEVYGIQIYTLDEHLEIDAAMLTSTVDSPLCLQCKPLQYKVLRDPPLLE DVASLLHLNRRFMDTDLKLGSVYQYRIITISGNEESEPSPAAIYTHGSGYCGDGVIQK DQGEECDDMNKVNGDGCSLFCKQEVSFNCIDEPSRCYFHDGDGMCEEFEQKTSIKDCG VYTPQGFLDQWASNASVSHQDQQCPGWWIGQPAASQVCRTKVIDLSEGISQHAWYPC TITYPYYHLPQTTFWLQTYFSQPMVAAAVIIHLVTDGTYYGDQKQETISVQLLDTKDQ SHDLGLHVLSCRNNPLIIPWHDLSQPFYHSQAVHVSFSSPLVAISGVALRSFDNFDP VTLSSCQRGETYSPAEQSCVHFACQAADCPELAVGNASLNCSSNHHYHGAQCTVSCQT GYVLQIQRDDELIKSQVGPSITVTCTEGKWNKQVACEPVDCGIPDHHHVYAASFSCPE GTTFGRRCSFQCRHPAQLKGNNSFLTCMEDGLWSFPEALCELMCLAPPPVPNADLQTA RCRENKHKVGSFCKYKCKPGYHVPGSSRKSKKRAFKTQCTQDGSWQEGTCVPVTCDPP PPKFHGLYQCTNGFQFNSECRIKCEDSDASQGRGSNIIHCRKDGTWSGSFHVCREMQG QCSAPNQLNSNLKLQCPDGYAIGSECAISCLDHNSESIILPVNLTVRDIPHWMNPTRV QRIVCTAGLQWYPHPALIHCVKGCEPFMGDNYCDAINNRAFCNYDGGDCCTSTVKTKK VTPFPMSCDLQNDCACRDPEAQEHNRKDLRGYSHG

[0286] Mature human PPEESNQNGGEGSYREAETFNSQVGLPILYFSGRRERLLLRPEVLAEIPREAFTVEAW VKPEGGQNNPAIIAGVFDNCSHTVSDKGWALGIRSGKDKGKRDARFFFSLCTDRVKKA PAPPA-2 TILISHSRYQPGTWTHVAATYDGRHMALYVDGTQVASSLDQSGPLNS PFMASCRSLLL GGDSSEDGHYFRGHLGTLVFWSTALPQSHFQHSSQHSSGEEEATDLVLTASFEPVNTE

[0287] (SEQ IDNO: WVPFRDEKYPRLEVLQGFEPEPEILSPLQPPLCGQTVCDNVELISQYNGYWPLRGEKV 74) IRYQWNICDDEGLNPIVSEEQIRLQHEALNEAFSRYNISWQLSVHQVHNSTLRHRW LVNCEPSKIGNDHCDPECEHPLTGYDGGDCRLQGRCYSWNRRDGLCHVECNNMLNDFD DGDCCDPQVADVRKTCFDPDS PKRAYMSVKELKEALQLNSTHFLNIYFASSVREDLAG AATWPWDKDAVTHLGGIVLSPAYYGMPGHTDTMIHEVGHVLGLYHVFKGVSERESCND PCKETVPSMETGDLCADTAPTPKSELCREPEPTSDTCGFTRFPGAPFTNYMSYTDDNC TDNFTPNQVARMHCYLDLVYQQWTESRKPTPIPIPPMVIGQTNKSLTIHWLPPISGW YDRASGSLCGACTEDGTFRQYVHTASSRRVCDSSGYWTPEEAVGPPDVDQPCEPSLQA WSPEVHLYHMNMTVPCPTEGCSLELLFQHPVQADTLTLWVTSFFMESSQVLFDTEILL ENKESVHLGPLDTFCDIPLTIKLHVDGKVSGVKVYTFDERIEIDAALLTSQPHSPLCS GC RPVRYQVL RD P P FAS GL PVWTHSHRKFTDVE VT PGQMYQYQVLAEAGGEL GE AS P PLNHIHGAPYCGDGKVSERLGEECDDGDLVSGDGCSKVCELEEGFNCVGEPSLCYMYE GDGICEPFERKTSIVDCGIYTPKGYLDQWATRAYSSHEDKKKCPVSLVTGEPHSLICT SYHPDLPNHRPLTGWFPCVASENETQDDRSEQPEGSLKKEDEVWLKVCFNRPGEARAI FIFLTTDGLVPGEHQQPTVTLYLTDVRGSNHSLGTYGLSCQHNPLIINVTHHQNVLFH HTTSVLLNFSSPRVGISAVALRTSSRIGLSAPSNCISEDEGQNHQGQSCIHRPCGKQD SCPSLLLDHADWNCTSIGPGLMKCAITCQRGFALQASSGQYIRPMQKEILLTCSSGH WDQNVSCLPVDCGVPDPSLVNYANFSCSEGTKFLKRCSISCVPPAKLQGLSPWLTCLE DGLWSLPEVYCKLECDAPPIILNANLLLPHCLQDNHDVGTICKYECKPGYYVAESAEG KVRNKLLKIQCLEGGIWEQGSCIPWCEPPPPVFEGMYECTNGFSLDSQCVLNCNQER EKLPILCTKEGLWTQEFKLCENLQGECPPPPSELNSVEYKCEQGYGIGAVCSPLCVIP P S D P VML P EN I T AD T L E HWME PVKVQS I VC T GRRQWH P D P VL VHC I Q S C E P FQAD GWC DTINNRAYCHYDGGDCCSSTLSSKKVIPFAADCDLDECTCRDPKAEENQ

[0288] Mature human E ARGAT E E P S P P S RAL Y F S GRGE QL RL RADL E L P RD AF T L QVWL RAE GGQRS P AVI T G LYDKCSYISRDRGWWGIHTISDQDNKDPRYFFSLKTDRARQVTTINAHRSYLPGQWV PAPPA [82-394] YLAATYDGQFMKLYVNGAQVATSGEQVGGIFS PLTQKCKVLMLGGSALNHNYRGYIEH FSLWKVARTQREILSDMETHGAHTALPQLLLQENWDNVKHAWS PMKDGS S PKVEFSNA

[0289] (SEQ IDNO: HGFLLDTSLEP

[0290] 102)

[0291]

[0292] Mature human EARGATEE PS PPSRALYFSGRGEQLRL RADLELPRDAFTLQVWL RAE GGQRS PAVITG LYDKCSYTSRDRGWWGIHTISDQDNKDPRYFFSLKTDRARQVTTINAHRSYLPGQWV PAPPA [82- YLAATYDGQFMKLYVNGAQVATSGEQVGGIFS PLTQKCKVLMLGGSALNHNYRGYIEH FSLWKVARTQREILSDMETHGAHTALPQLLLQENWDNVKHAWS PMKDGS S PKVEFSNA

[0293] 394][I147T] HGFLLDTSLEP

[0294] (SEQ ID NO:

[0295] 103)

[0296] PAPPA (82- E ARGAT E E P S P P S RAL Y F S GRGE QL RL RADL E L P RD AF T L QVWL RAE GGQRS P AVI T G LYDKCSYISRDRGWWGIHTISDQDNKDPRYFFSLKTDRARQVTTINAHRSYLPGQWV

[0297] 1183) (SEQ ID YLAATYDGQFMKLYVNGAQVATSGEQVGGIFS PLTQKCKVLMLGGSALNHNYRGYIEH FSLWKVARTQREILSDMETHGAHTALPQLLLQENWDNVKHAWS PMKDGS S PKVEFSNA NO: 104) HGFLLDTSLEPPLCGQTLCDNTEVIASYNQLSSFRQPKWRYRWNLYEDDHKNPTVT REQVDFQHHQLAEAFKQYNISWELDVLEVSNSSLRRRLILANCDISKIGDENCDPECN HTLTGHDGGDCRHLRHPAFVKKQHNGVCDMDCNYERFNFDGGECCDPEITNVTQTCFD PDSPHRAYLDVNELKNILKLDGSTHLNIFFAKSSEEELAGVATWPWDKEALMHLGGIV LNPSFYGMPGHTHTMIHEIGHSLGLYHVFRGISEIQSCSDPCMETEPSFETGDLCNDT NPAPKHKSCGDPGPGNDTCGFHSFFNTPYNNFMSYADDDCTDSFTPNQVARMHCYLDL VYQGWQPSRKPAPVALAPQVLGHTTDSVTLEWFPPIDGHFFERELGSACHLCLEGRIL VQYASNAS S PMPCS PSGHWS PREAEGHPDVEQPCKS SVRTWS PNSAVNPHTVPPACPE PQGCYLELEFLYPLVPESLTIWVTFVSTDWDSSGAVNDIKLLAVSGKNISLGPQNVFC DVPLTIRLWDVGEEVYGIQIYTLDEHLEIDAAMLTSTADTPLCLQCKPLKYKWRDPP LQMDVASILHLNRKFVDMDLNLGSVYQYWVITISGTEESEPSPAVTYIHGSGYCGDGI IQKDQGEQCDDMNKINGDGCSLFCRQEVSFNCIDEPSRCYFHDGDGVCEEFEQKTSIK DCGVYTPQGFLDQWASNASVSHQDQQCPGWVIIGQPAASQVCRTKVIDLSEGISQHAW YPCTISYPYSQLAQTTFWLRAYFSQPMVAAAVIVHLVTDGTYYGDQKQETISVQLLDT

[0298]

[0299] KDQSHDLGLHVLSCRNNPLIIPWHDLSQPFYHSQAVRVSFSSPLVAISGVALRSFDN

[0300] PAPPA domains

[0301] Human PAPPA comprises multiple distinct sub-domains. Human PAPPA comprises a laminin-G like (“LG”) domain, a proteolytic domain (“PD”), a scarf domain (“SD”), a beta-barrel domain (“BBD”), five complement control protein (“CCP”) domains, and a C-terminal domain (“CTD”). The PD of human PAPPA comprises two lin-12 / Notch repeats (“LNR1” and “LNR2”), and the CTD of human PAPPA comprises a third LNR domain (“LNR3”).

[0302] In some instances, the binding molecule binds to the LG domain of PAPPA. In some instances, the binding molecule binds to the LG domain of human PAPPA. In some instances, the binding molecule binds to an epitope in the LG domain of PAPPA. In some instances, the binding molecule binds to an epitope in the LG domain of human PAPPA. In some instances, the binding molecule binds to an epitope in PAPPA that comprises one or more amino acids of the LG domain of PAPPA. In some instances, the binding molecule binds to an epitope in human PAPPA that comprises one or more amino acids of the LG domain of human PAPPA.

[0303] The LG domain of human PAPPA is contained within amino acid residues 1-242 of mature human PAPPA, e.g., within amino acid residues 1-242 of SEQ ID NO: 71. In some instances, the bindingmolecule binds to an epitope of human PAPPA within amino acid residues 1-242 of SEQ ID NO: 71. In some instances, the binding molecule binds to an epitope in human PAPPA that comprises one or more amino acids of amino acid residues 1-242 of SEQ ID NO: 71. In some instances, the binding molecule binds to an epitope of human PAPPA within amino acid residues 65-69 of SEQ ID NO: 71. As used herein, reference to binding to an epitope “within” a range of amino acid residues encompasses binding of the binding molecule to the first and last amino acid residue within that range. Thus, a binding molecule which binds to an epitope within amino acid residues 65-69 of SEQ ID NO: 71 encompasses a binding molecule which binds to amino acid residue 65 and amino acid residue 69 of SEQ ID NO: 71. In some instances, the binding molecule binds to an epitope in human PAPPA that comprises one or more amino acids of amino acid residues 65-69 of SEQ ID NO: 71. In some instances, the binding molecule binds to amino acid residues 65-69 of SEQ ID NO: 71. In some instances, the binding molecule binds to amino acid residues 65-69 within SEQ ID NO: 71.

[0304] The LG domain of human PAPPA is contained within amino acid residues 82-324 of full length PAPPA, e.g., within amino acid residues 82-324 of SEQ ID NO: 57. In some instances, the binding molecule binds to an epitope of human PAPPA within amino acid residues 82-324 of SEQ ID NO: 57. In some instances, the binding molecule binds to an epitope in human PAPPA that comprises one or more amino acids of amino acid residues 82-324 of SEQ ID NO: 57.

[0305] In some instances, the binding molecule binds to an epitope of human PAPPA within amino acid residues 131-150 of SEQ ID NO: 57. In some instances, the binding molecule binds to an epitope in human PAPPA that comprises one or more amino acids of amino acid residues 131-150 of SEQ ID NO: 57. In some instances, the binding molecule binds to amino acid residues 131-150 of SEQ ID NO: 57. In some instances, the binding molecule binds to amino acid residues 131-150 within SEQ ID NO: 57.

[0306] In some instances, the binding molecule binds to an epitope of human PAPPA within amino acid residues 146-150 of SEQ ID NO: 57. In some instances, the binding molecule binds to an epitope in human PAPPA that comprises one or more amino acids of amino acid residues 146-150 of SEQ ID NO: 57. In some instances, the binding molecule binds to amino acid residues 146-150 of SEQ ID NO: 57. In some instances, the binding molecule binds to amino acid residues 146-150 within SEQ ID NO: 57.Amino acid residues 65-69 of SEQ ID NO: 71, or amino acid residues 146-150 of SEQ ID NO: 57, are present in the LG domain of PAPPA and consist of the amino acid residues Y-I-S-R-D (SEQ ID NO: 75). Thus, in some instances, the binding molecule binds to the amino acid sequence of SEQ ID NO: 75. In some instances, the binding molecule binds to the amino acid sequence of SEQ ID NO: 75 within SEQ ID NO: 71. In some instances, the binding molecule binds to an epitope comprising the amino acid sequence of SEQ ID NO: 75. In some instances, the binding molecule binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 75.

[0307] In some instances, the binding molecule binds to an epitope of human PAPPA within amino acid residues 227-250 of SEQ ID NO: 57. In some instances, the binding molecule binds to an epitope in human PAPPA that comprises one or more amino acids of amino acid residues 227-250 of SEQ ID NO: 57. In some instances, the binding molecule binds to amino acid residues 227-250 of SEQ ID NO: 57. In some instances, the binding molecule binds to amino acid residues 227-250 within SEQ ID NO: 57.

[0308] In some instances, the binding molecule binds to an epitope of human PAPPA within amino acid residues 131-150 and 227-250 of SEQ ID NO: 57. In some instances, the binding molecule binds to an epitope in human PAPPA that comprises one or more amino acids of amino acid residues 131-150 and one or more amino acids of amino acid residues 227-250 of SEQ ID NO: 57. In some instances, the binding molecule binds to amino acid residues 131-150 and 227-250 of SEQ ID NO: 57. In some instances, the binding molecule binds to amino acid residues 131-150 and 227-250 within SEQ ID NO: 57.

[0309] Binding of a binding molecule to the LG domain of PAPPA, such as within amino acid residues of 1-242 of SEQ ID NO: 71, or within amino acid residues 82-324 of SEQ ID NO: 57 can be determined by any suitable means. In some instances, binding of a binding molecule to the LG domain of PAPPA is determined using a method described herein. In some instances, binding of a binding molecule to the LG domain of PAPPA is determined using an HTRF assay. In some instances, binding of a binding molecule to the LG domain of PAPPA is determined using bio-layer interferometry (BLI), e.g., using BLI as described in Example 14.

[0310] Binding of a binding molecule within amino acid residues 131-150, 227-250 and / or 65-69 of SEQ ID NO: 71 can be determined using any suitable means. In some instances, binding of a binding molecule within amino acid residues 131-150, 227-250 and / or 65-69 of SEQ ID NO: 71 isdetermined using a method described herein. In some instances, binding of a binding molecule within amino acid residues 131-150, 227-250 and / or 65-69 of SEQ ID NO: 71 is determined using cryogenic electron microscopy (cryo-EM), e.g., using cryo-EM as described in Example 15.

[0311] Advantageously, binding of the binding molecule to the LG domain of human PAPPA, such as within amino acid residues 1-242 of SEQ ID NO: 71, or within amino acid residues 65-69 of SEQ ID NO: 71, may allow native PAPPA inhibitors to bind to PAPPA. Stanniocalcin-1 (STC1) and stanniocalcin-2 (STC2) are native inhibitors of PAPPA. STC1 and STC2 bind to the LNR3 domain of PAPPA and inhibit PAPPA-induced cleavage of IGFBPs. Binding of the binding molecule to the LG domain of human PAPPA, such as within amino acid residues 1-242 of SEQ ID NO: 71, or within amino acid residues 65-69 of SEQ ID NO: 71, may allow STC1 and / or STC2 to bind to the LNR3 domain of PAPPA. Thus, in some instances, the binding molecule does not compete with STC1 and / or STC2 for binding to PAPPA, e.g., human PAPPA. In some instances, the binding molecule does not compete with STC1 and / or STC2 for binding to PAPPA, e.g., human PAPPA, as determined using an epitope competition assay, such as an epitope competition assay described in Example 13. In some instances, the binding molecule does not compete with STC1 for binding to PAPPA, e.g., human PAPPA. In some instances, the binding molecule does not compete with STC 1 for biding to PAPPA, e.g., human PAPPA, as determined using an epitope competition assay, such as an epitope competition assay described in Example 13.

[0312] PAPPA epitope competition

[0313] In some instances, the binding molecule competes for binding to PAPPA with another binding molecule of the invention, such as competes for binding to PAPPA with a binding molecule as described above. In some instances, the binding molecule competes for binding to PAPPA with a binding molecule comprising a heavy chain variable region (VH) which comprises HCDR1, HCDR2, HCDR3 as indicated for any one of the binding molecules illustrated in Table 1, and a light chain variable region (VL) which comprises LCDR1, LCDR2 and LCDR3 as indicated for the same binding molecule illustrated in T able 1. In preferred instances, the binding molecule competes for binding to PAPPA with a binding molecule comprising a heavy chain variable region (VH) which comprises HCDR1, HCDR2, HCDR3 as indicated for AB1 illustrated in Table 1, and a light chain variable region (VL) which comprises LCDR1, LCDR2 and LCDR3 as indicated for AB1 illustrated in Table 1. Thus, in some instances, the binding molecule competes for binding to PAPPA with a reference binding molecule comprising a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set ofCDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence of the reference binding molecule comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and wherein the VL region amino acid sequence of the reference binding molecule comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6. In some instances, the binding molecule competes for binding to PAPPA with a reference binding molecule comprising a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence of the reference binding molecule comprises HCDR1 of SEQ ID NO : 121 , HCDR2 of SEQ ID NO : 2 and HCDR3 of SEQ ID NO: 3 , and wherein the VL region amino acid sequence of the reference binding molecule comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6.

[0314] In preferred instances, the binding molecule competes for binding to PAPPA with a binding molecule comprising a heavy chain variable region (VH) which comprises HCDR1, HCDR2, HCDR3 as indicated for AB34 illustrated in Table 1, and a light chain variable region (VL) which comprises LCDR1, LCDR2 and LCDR3 as indicated for AB34 illustrated in Table 1. In some instances, the binding molecule competes for binding to PAPPA with a reference binding molecule comprising a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence of the reference binding molecule comprises HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 110, and wherein the VL region amino acid sequence of the reference binding molecule comprises LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113. In some instances, the binding molecule competes for binding to PAPPA with a reference binding molecule comprising a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence of the reference binding molecule comprises HCDR1 of SEQ ID NO: 122, HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 110, and wherein the VL region amino acid sequence of the reference binding molecule comprises LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113.

[0315] In some instances, the binding molecule competes for binding to PAPPA with a binding molecule comprising a VH and VL region amino acid sequence of any binding molecule illustrated in Table 2. In preferred instances, the binding molecule competes for binding to PAPPA with a bindingmolecule comprising a VH and VL region amino acid sequence of AB 1 illustrated in T able 2. Thus, in some instances, the binding molecule competes for binding to PAPPA with a reference binding molecule comprising a VH region amino acid sequence comprising SEQ ID NO: 7 and a VL region amino acid sequence comprising SEQ ID NO: 8. In preferred instances, the binding molecule competes for binding to PAPPA with a binding molecule comprising a VH and VL region amino acid sequence of AB34 illustrated in Table 2. Thus, in some instances, the binding molecule competes for binding to PAPPA with a reference binding molecule comprising a VH region amino acid sequence comprising SEQ ID NO: 114 and a VL region amino acid sequence comprising SEQ ID NO: 115.

[0316] In some instances, the binding molecule competes for binding to PAPPA with a binding molecule comprising a HC and LC amino acid sequence of any binding molecule illustrated in Table 3. In preferred instances, the binding molecule competes for binding to PAPPA with a binding molecule comprising a HC and LC amino acid sequence of AB1 illustrated in Table 3. Thus, in some instances, the binding molecule competes for binding to PAPPA with a reference binding molecule comprising a HC amino acid sequence comprising SEQ ID NO: 76 and a LC amino acid sequence comprising SEQ ID NO: 77. In preferred instances, the binding molecule competes for binding to PAPPA with a binding molecule comprising a HC and LC amino acid sequence of AB34 illustrated in Table 3. Thus, in some instances, the binding molecule competes for binding to PAPPA with a reference binding molecule comprising a HC amino acid sequence comprising SEQ ID NO: 118 and a LC amino acid sequence comprising SEQ ID NO: 117. In some instances, the binding molecule competes for binding to PAPPA with a reference binding molecule comprising a HC amino acid sequence comprising SEQ ID NO: 116 and a LC amino acid sequence comprising SEQ ID NO: 117.

[0317] In some instances, the binding molecule does not compete for binding to PAPPA with another binding molecule of the invention, such as does not compete for binding to PAPPA with a binding molecule as described above. In some instances, the binding molecule does not compete for binding to PAPPA with a binding molecule comprising a heavy chain variable region (VH) which comprises HCDR1, HCDR2, HCDR3 as indicated for any one of the binding molecules illustrated in Table 1, and a light chain variable region (VL) which comprises LCDR1, LCDR2 and LCDR3 as indicated for the same binding molecule illustrated in Table 1. In some instances, the binding molecule does not compete for binding to PAPPA with a binding molecule comprising a heavy chain variable region (VH) which comprises HCDR1, HCDR2, HCDR3 as indicated for AB1 illustrated in Table1, and a light chain variable region (VL) which comprises LCDR1, LCDR2 and LCDR3 as indicated for AB1 illustrated in Table 1. Thus, in some instances, the binding molecule does not compete for binding to PAPPA with a reference binding molecule comprising a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence of the reference binding molecule comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and wherein the VL region amino acid sequence of the reference binding molecule comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6. In some instances, the binding molecule does not compete for binding to PAPPA with a reference binding molecule comprising a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence of the reference binding molecule comprises HCDR1 of SEQ ID NO: 121, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and wherein the VL region amino acid sequence of the reference binding molecule comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6.

[0318] In some instances, the binding molecule does not compete for binding to PAPPA with a binding molecule comprising a heavy chain variable region (VH) which comprises HCDR1, HCDR2, HCDR3 as indicated for AB1 illustrated in Table 1, and a light chain variable region (VL) which comprises LCDR1, LCDR2 and LCDR3 as indicated for AB34 illustrated in Table 1. Thus, in some instances, the binding molecule does not compete for binding to PAPPA with a reference binding molecule comprising a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence of the reference binding molecule comprises HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 110, and wherein the VL region amino acid sequence of the reference binding molecule comprises LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113. In some instances, the binding molecule does not compete for binding to PAPPA with a reference binding molecule comprising a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein the VH region amino acid sequence of the reference binding molecule comprises HCDR1 of SEQ ID NO: 122, HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 110, and wherein the VL region amino acid sequence of the reference binding molecule comprises LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113.In some instances, the binding molecule does not compete for binding to PAPP A with a binding molecule comprising a VH and VL region amino acid sequence of any binding molecule illustrated in Table 2. In some instances, the binding molecule does not compete for binding to PAPPA with a binding molecule comprising a VH and VL region amino acid sequence of AB 1 illustrated in Table 2. Thus, in some instances, the binding molecule does not compete for binding to PAPPA with a reference binding molecule comprising a VH region amino acid sequence comprising SEQ ID NO: 7 and a VL region amino acid sequence comprising SEQ ID NO: 8. In some instances, the binding molecule does not compete for binding to PAPPA with a binding molecule comprising a VH and VL region amino acid sequence of AB34 illustrated in Table 2. Thus, in some instances, the binding molecule does not compete for binding to PAPPA with a reference binding molecule comprising a VH region amino acid sequence comprising SEQ ID NO: 114 and a VL region amino acid sequence comprising SEQ ID NO: 115.

[0319] In some instances, the binding molecule does not compete for binding to PAPPA with a binding molecule comprising a HC and LC amino acid sequence of any binding molecule illustrated in Table 3. In some instances, the binding molecule does not compete for binding to PAPPA with a binding molecule comprising a HC and LC amino acid sequence of AB1 illustrated in Table 3. Thus, in some instances, the binding molecule does not compete for binding to PAPPA with a reference binding molecule comprising a HC amino acid sequence comprising SEQ ID NO: 76 and a LC amino acid sequence comprising SEQ ID NO: 77. In some instances, the binding molecule does not compete for binding to PAPPA with a binding molecule comprising a HC and LC amino acid sequence of AB34 illustrated in Table 3. Thus, in some instances, the binding molecule does not compete for binding to PAPPA with a reference binding molecule comprising a HC amino acid sequence comprising SEQ ID NO: 118 and a LC amino acid sequence comprising SEQ ID NO: 117. In some instances, the binding molecule does not compete for binding to PAPPA with a reference binding molecule comprising a HC amino acid sequence comprising SEQ ID NO: 116 and a LC amino acid sequence comprising SEQ ID NO: 117.

[0320] The ability of a binding molecule to compete for binding to PAPPA with another binding molecule can be measured by any suitable means. In some instances, the ability of a binding molecule to compete for binding to PAPPA with another binding molecule is measured using a method described herein. In some instances, the ability of a binding molecule to compete for binding to PAPPA with another binding molecule is assessed using an epitope binning assay, such as anepitope binning assay described in Example 12. In some instances, a binding molecule competes for binding to PAPPA with another binding molecule when no signal is observed in an epitope binning assay, such as an epitope binning assay described in Example 12. In some instances, a binding molecule does not compete for binding to PAPPA with another binding molecule when a signal is observed in an epitope binning assay, such as an epitope binning assay described in Example 12.

[0321] Inhibitory activity against PAPPA

[0322] In some instances, the binding molecule is a PAPPA antagonist. In some instances, the binding molecule inhibits PAPPA protease activity. In some instances, the binding molecule inhibits IGFBP4 cleavage mediated by PAPPA protease activity. In some instances, the binding molecule inhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% of IGFBP4 cleavage mediated by PAPPA protease activity. In preferred instances, the binding molecule inhibits at least 90% of IGFBP4 cleavage mediated by PAPPA protease activity.

[0323] Inhibition of IGFBP4 cleavage mediated by PAPPA protease activity can be assessed by any suitable means. In some instances, inhibition of IGFBP4 cleavage mediated by PAPPA protease activity is assessed using a method described herein. In some instances, inhibition of IGFBP4 cleavage mediated by PAPPA protease activity is assessed using a homogeneous time-resolved fluorescence (HTRF) assay, such as an HTRF assay described in Example 5. In some instances, inhibition of IGFBP4 cleavage mediated by PAPPA protease activity is assessed using an assay which measures inhibition of native PAPPA, such as an assay which measures inhibition of native PAPPA described in Example 16.

[0324] In some instances, the binding molecule inhibits formation of N-terminal fragments of IGFBP4 mediated by PAPPA protease activity. In some instances, inhibition of formation of N-terminal fragments of IGFBP4 mediated by PAPPA protease activity is assessed using methods described herein. In some instances, inhibition of formation of N-terminal fragments of IGFBP4 mediated by PAPPA protease activity is assessed using an assay which measures inhibition of native PAPPA, such as an assay which measures inhibition of native PAPPA described in Example 16.

[0325] Binding of the binding molecule to the LG domain of human PAPPA, such as within amino acid residues 65-69 of SEQ ID NO: 71, may induce a conformational change in human PAPPA. In some instances, binding of the binding molecule to human PAPPA induces a conformational change inhuman PAPPA. In some instances, binding of the binding molecule to the LG domain of human PAPPA, such as within amino acid residues 65-69 of SEQ ID NO: 71, induces a conformational change in human PAPPA.

[0326] Binding of the binding molecule to the LG domain of human PAPPA, such as within amino acid residues 65-69 of SEQ ID NO: 71, may induce a conformational change in human PAPPA that prevents the correct formation of the substrate binding pocket, Thus, in some instances, binding of the binding molecule to PAPPA (e.g., the LG domain of PAPPA) prevents substrate binding to PAPPA. In some instances, binding of the binding molecule to PAPPA prevents IGFBP4 binding to PAPPA. In some instances, binding of the binding molecule to PAPPA (e.g., the LG domain of PAPPA) reduces substrate binding to PAPPA. In some instances, binding of the binding molecule to PAPPA reduces IGFBP4 binding to PAPPA. In some instances, binding of the binding molecule to PAPPA reduces substrate binding (e.g., IGFBP4 binding) to PAPPA by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100%. In preferred instances, binding of the binding molecule to PAPPA reduces substrate binding (e.g., IGFBP4 binding) to PAPPA by at least 90% .

[0327] Binding affinity

[0328] In some instances, the binding molecule binds to PAPPA with a KD of less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM or less than 0.5 nM. In some instances, the binding molecule binds to PAPPA with a KD of less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, less than 0.15 nM, or less than 0.1 nM. In some instances, the binding molecule binds to PAPPA with a KD of 0.055 nM. In some instances, the binding molecule binds to PAPPA with a KD of 0.238 nM. In some instances, the binding molecule binds to PAPPA with a KD of 25.4 nM.

[0329] In some instances, the binding molecule binds to human PAPPA, such as human PAPPA having the amino acid sequence of SEQ ID NO: 71, with a KD of less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, less than 0.15 nM, less than 0.1 nM, less than 0.05 nM, or less than 0.01 nM. In some instances, the binding molecule binds to human PAPPA, such as human PAPPA having the amino acid sequence of SEQ ID NO: 71, with a KD of less than 100 nM and more than 0.01 nM, less than lOnM and more than 0.01 nM, less than lOnM and more than 0.05 nM, less than 10 nM and more than 0.1 nM, less than1 nM and more than 0.2 nM, or less than 0.5 nM and more than 0.2 nM. In some instances, the binding molecule binds to human PAPPA, such as human PAPPA having the amino acid sequence of SEQ ID NO: 71, with a KD of less than 0.5 nM and more than 0.01 nM, less than 0.25 nM and more than 0.05 nM. In some instances, the binding molecule binds to human PAPPA, such as human PAPPA having the amino acid sequence of SEQ ID NO: 71, with a KD of 0.238 nM. In some instances, the binding molecule binds to human PAPPA, such as human PAPPA having the amino acid sequence of SEQ ID NO: 71, with a KD of 0.055 nM.

[0330] In some instances, the binding molecule binds to cynomolgus PAPPA, such as cynomolgus PAPPA having the amino acid sequence of SEQ ID NO: 72, with a KD of less than 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM or less than 0.5 nM. In some instances, the binding molecule binds to cynomolgus PAPPA, such as cynomolgus PAPPA having the amino acid sequence of SEQ ID NO: 72, with a KD of less than 500 nM and more than 0.5 nM, less than 250 nM and more than 1 nM, less than 100 nM and more than 5 nM, less than 50 nM and more than 20 nM, or less than 30 nM and more than 20 nM. In some instances, the binding molecule binds to cynomolgus PAPPA, such as cynomolgus PAPPA having the amino acid sequence of SEQ ID NO: 72, with a KD of 25.4nM.

[0331] In some instances, the binding molecule binds to human PAPPA, such as human PAPPA having the amino acid sequence of SEQ ID NO: 71, with a KD of less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, less than 0.15 nM, less than 0.1 nM, less than 0.05 nM, or less than 0.01 nM and binds to cynomolgus PAPPA, such as cynomolgus PAPPA having the amino acid sequence of SEQ ID NO: 72, with a KD of less than 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM or less than 0.5 nM. In some instances, the binding molecule binds to human PAPPA, such as human PAPPA having the amino acid sequence of SEQ ID NO: 71, with a KD of less than 100 nM and more than 0.01 nM, less than 10 nM and more than 0.1 nM, less than 1 nM and more than 0.2 nM, or less than 0.5 nM and more than 0.2 nM and binds to cynomolgus PAPPA, such as cynomolgus PAPPA having the amino acid sequence of SEQ ID NO: 72, with a KD of less than 500 nM and more than 0.5 nM, less than 250 nM and more than 1 nM, less than 100 nM and more than 5 nM, less than 50 nM and more than 20 nM, or less than 30 nM and more than 20 nM. In someinstances, the binding molecule binds to human PAPPA, such as human PAPPA having the amino acid sequence of SEQ ID NO: 71, with a KD of less than 5 nM, and binds to cynomolgus PAPPA, such as cynomolgus PAPPA having the amino acid sequence of SEQ ID NO: 72, with a KD of less than 30 nM. In some instances, the binding molecule binds to human PAPPA, such as human PAPPA having the amino acid sequence of SEQ ID NO: 71, with a KD of less than 1 nM, and binds to cynomolgus PAPPA, such as cynomolgus PAPPA having the amino acid sequence of SEQ ID NO: 72, with a KD of less than 30 nM. In some instances, the binding molecule binds to human PAPPA with a KD of 0.238 nM, and binds to cynomolgus PAPPA with a KD of 25.4 nM.

[0332] In some instances, the binding molecule binds to the LG domain of human PAPPA (e.g., the amino acid sequence of SEQ ID NO: 102) with a KD of less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM or less than 1 nM. In some instances, the binding molecule binds to the LG domain of human PAPPA (e.g., the amino acid sequence of SEQ ID NO: 102) with a KD of less than 10 nM and more than 0.01 nM, less than 5 nM and more than 0.05 nM, less than 4 nM and more than 0.06 nM, less than 3 nM and more than 0.07 nM, less than 2 nM and more than 0.08 nM, or less than 1 nM and more than 0.09 nM. In some instances, the binding molecule binds to the LG domain of human PAPPA (e.g., the amino acid sequence of SEQ ID NO: 102) with a KD of 0.095 nM.

[0333] In some instances, the binding molecule binds to the amino acid sequence of SEQ ID NO: 103 with a KD of less than 20 nM, less than 10 nM, less than 8nM, less than 6 nM, or less than 4nM. In some instances, the binding molecule binds to the amino acid sequence of SEQ ID NO: 103 with a KD of less than 20 nM and more than 0.2 nM, less than 10 nM and more than 0.5 nM, less than 8 nM and more than InM, less than 6 nM and more than 2 nM, or less than 4nM and more than 3 nM. In some instances, the binding molecule binds to the amino acid sequence of SEQ ID NO: 103 with a KD of 3.93 nM.

[0334] The binding affinity of the binding molecule to PAPPA (e.g., human PAPPA and / or cynomolgus PAPPA) can be measured by any suitable means. In some instances, the binding affinity of the binding molecule to PAPPA is measured using a method described herein, such as the method described in Example 6. In some instances, the binding affinity of the binding molecule to PAPPA is measured using Biacore. In some instances, the binding affinity of the binding molecule to PAPPA is measured using Biacore at 25°C. In some instances, the binding affinity of the binding molecule to PAPPA is measured using Biacore 8K+ at 25°C.Non-specific binding

[0335] Non-specific binding or polyreactivity of monoclonal antibodies (mAbs) using HEK (Human Embryonic Kidney) helps identify antibodies that bind to off-target antigens, affecting the specificity, dose and safety profile of therapeutic antibodies. Thus, in some instances, the binding molecule does not have a propensity for non-specific binding. In some instances, the binding molecule has a low propensity for non-specific binding. In some instances, the binding molecule does not bind non-specifically. Propensity for non-specific binding can be measured by any suitable means. In some instances, propensity for non-specific binding is measured using a method described herein. In some instances, propensity for non-specific binding is assessed using a Mirrorball assay, such as the Mirrorball assay described in Example 7. In some instances, a binding molecule does not have a propensity for non-specific binding, or a has a low propensity to bind non-specifically, when the binding molecule has an FI value less than 5x the FI value of its isotype control in a Mirrorball assay, such as the Mirrorball assay described in Example 7.

[0336] Self-association propensity

[0337] Self-associated binding molecules may appear foreign to a patient’s immune system and therefore elicit an immune response that reduces the therapeutic activity of the binding molecule. It is therefore preferable for binding molecules of the present disclosure to have a low propensity for self-association. In some instances, the binding molecule does not have a propensity for selfassociation. In some instances, the binding molecule has a low propensity to self-associate. In some instances, the binding molecule does not self- associate. Self-association can be measured by any suitable means. In some instances, self-association is measured using a method described herein. In some instances, self-association is assessed using an affinity capture self-interaction nanoparticle spectrometry (AC-SINS) assay, such as the AC-SINS assay described in Example 8. In some instances, a binding molecule does not have a risk of self-association, or has a low propensity for self-aggregation, when the binding molecule exhibits a redshift of less than or equal to 10 nM in an AC-SINS assay, e.g., an AC-SINS assay described in Example 8. In some instances, the binding molecule exhibits a redshift of less than or equal to 10 nM. In some instances, the binding molecule exhibits a redshift of less than or equal to 10 nM in an AC-SINS assay, e.g., an AC-SINS assay described in Example 8.

[0338] In vivo clearanceIn vivo clearance refers to the pharmacokinetic parameter that describes the efficiency with which a drug is removed from the body. Clearance is a crucial determinant of a drug's half-life within the body. Removal of a binding molecule from the circulation of a subject may limit the therapeutic utility of the binding molecule. A binding molecule which exhibits fast in vivo clearance may therefore need to be administered more frequently compared to a binding molecule which exhibits slow in vivo clearance. Thus, in some instances, the binding molecule does not show fast in vivo clearance. Fast in vivo clearance of a binding molecule can be measured by any suitable means. In some instances, fast in vivo clearance of a binding molecule is measured using a method described herein. In some instances, fast in vivo clearance is assessed using a baculovirus (BV) particle ELISA, such as the BV particle ELISA described in Example 9. In some instances, a binding molecule does not show fast in vivo clearance when the binding molecule exhibits a BV score of less than or equal to 5 in a BV particle ELISA, e.g., a BV particle ELISA as described in Example 9. In some instances, the binding molecule exhibits a BV score of less than or equal to 5. In some instances, the binding molecule exhibits a BV score of less than or equal to 5 in a BV particle ELISA, e.g., a BV particle ELISA as described in Example 9.

[0339] Antibody or antigen-binding fragment thereof

[0340] In some instances, the binding molecule is an antibody, or an antigen-binding fragment thereof. In some instances, the binding molecule is an immunoglobulin molecule. In some instances, the binding molecule is an antibody that is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, e.g., disulfide bonds.

[0341] In some instances, the antibody or antigen-binding fragment thereof, or the immunoglobulin molecule has the CDRs of a binding molecule that binds to PAPPA as described herein. Thus, in some instances, the antibody or antigen-binding fragment thereof comprises a set of six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) described herein. In some instances, the antibody, or antigen-binding fragment thereof, or the immunoglobulin molecule has the VH and / or VL region of a binding molecule that binds to PAPPA as described herein. Thus, in some instances, the antibody or antigen-binding fragment thereof comprises a VH region amino acid sequence described herein. In some instances, the antibody or antigen-binding fragment thereof comprises a VL region amino acid sequence described herein. In some instances, the antibody or antigen-binding fragment thereof comprises VH and VL region amino acid sequences described herein. In some instances, the antibody or antigen-binding fragment thereof, or the immunoglobulinmolecule, comprises an Fc region described herein. In some instances, the antibody or antigenbinding fragment thereof comprises a heavy chain described herein. In some instances, the antibody or antigen-binding fragment thereof comprises a light chain described herein. In some instances, the antibody or antigen-binding fragment thereof comprises a heavy chain described herein, and a light chain described herein. In some instances, the antibody, or antigen-binding fragment thereof, or the immunoglobulin molecule has one or more of the functional properties as described herein. Thus, in some instances, the antibody or antigen-binding fragment thereof, or the immunoglobulin molecule, has a binding specificity as described herein. In some instances, the antibody or antigenbinding fragment thereof, or the immunoglobulin molecule, binds to one or more PAPPA sequences or domains as disclosed herein. In some instances, the antibody or antigen-binding fragment thereof, or the immunoglobulin molecule, binds to the LG domain of PAPPA as described herein. In some instances, the antibody or antigen-binding fragment thereof, or the immunoglobulin molecule has inhibitory activity against PAPPA as described herein. In some instances, the antibody or antigenbinding fragment thereof, or the immunoglobulin molecule has binding affinity to PAPPA as described herein. In some instances, the antibody or antigen-binding fragment thereof, or the immunoglobulin molecule has the non-specific binding propensity described herein. In some instances, the antibody or antigen-binding fragment thereof, or the immunoglobulin molecule has the self-association propensity described herein. In some instances, the antibody or antigen-binding fragment thereof, or the immunoglobulin molecule has the in vivo clearance properties described herein.

[0342] The term "antibody" includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda. The antibodies or antigen-binding fragments thereof of the disclosure may have any antibody format. An antibody is a protein including at least one or two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one or two light (L) chain variable regions (abbreviated herein as VL). 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, termed "framework regions" (FW). The extent of the framework region and CDRs has been precisely defined (see, Rabat, E.A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, C. et al, J. Mol. Biol. 196:901-917, 1987). Preferably, each VH and VL region is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy -terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. In some instances,the binding molecule is, or comprises, a full-length antibody, or an antigen-binding fragment thereof. In some instances, the antibody has the “conventional” format described above. In some instances, the antibody or antigen-binding fragment is a Fab', an Fv, an scFv, an Fd, a V NAR domain, an IgNAR, an infrabody, an IgG CH2, a minibody, a single-domain antibody, an Fcab, an scFv-Fc, F(ab')2, a di-scFv, a bi-specific T-cell engager (BiTE®), a F(ab')3, a tetrabody, a triabody, a diabody, a DVD-Ig, an (scFv)2, or a mAb2.

[0343] The term “antigen binding fragment”, as used herein, refers to a portion of an antibody that binds to PAPPA, e.g., a molecule in which one or more immunoglobulin chains is not full length, but which binds to PAPPA. In some instances, the antigen-binding fragment is one or more selected from a Fv fragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv fragment, an sc(Fv)2 fragment, a dAb fragment, a single chain antibody, or a combination thereof. For example, in some instances, the antigen-binding fragment is a Fab fragment. In some instances, the binding molecule comprises a Fab domain.

[0344] Various techniques are known for the production of antibody fragments. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies, as described, for example, by Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993) and Brennan et al., Science 229:81 (1985). In some instances, anti-PAPPA antibody fragments are produced recombinantly. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus allowing the production of large amounts of these fragments. Such anti-PAPPA antibody fragments can also be isolated from the antibody phage libraries discussed above. The anti-PAPPA antibody fragments can also be linear antibodies as described in U.S. Patent No.

[0345] 5,641,870. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0346] According to the present disclosure, techniques can be adapted for the production of single-chain antibodies specific to PAPPA. See, e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for PAPPA, or derivatives, fragments, analogs or homologs thereof. See, e.g., Huse et al., Science 246:1275-1281 (1989). Antibody fragments can be produced by techniques known in the art including, but not limited to: F(ab')2 fragment produced by pepsin digestion of an antibody molecule; Fab fragment generated byreducing the disulfide bridges of an F(ab')2 fragment; Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent; or Fv fragments.

[0347] In some instances, the antibody or antigen-binding fragment thereof is monoclonal. In some instances, the antibody or antigen-binding fragment thereof is a monoclonal antibody (mAb). In some instances, the antibody or antigen-binding fragment thereof is polyclonal. In some instances, the antibody or antigen-binding fragment thereof is a polyclonal antibody.

[0348] A “monoclonal antibody” (mAb) refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0349] The antibodies of the disclosure and antigen-binding fragments thereof may be derived from any species by recombinant means. For example, the antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. For use in administration to humans, non-human derived antibodies or antigenbinding fragments may be genetically or structurally altered to be less antigenic upon administration to the human patient.

[0350] In some instances, the antibody or antigen binding fragment is one or more selected from a murine antibody, a humanised antibody, a chimeric antibody, a fully human antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a multispecific antibody, a combination of any thereof, or an antigen binding fragment of any thereof. Especially preferred are human or humanised antibodies or antigen-binding fragments thereof, especially as recombinant human or humanised antibodies or antigen-binding fragments thereof.

[0351] In some instances, the antibody or antigen-binding fragment thereof is a humanised antibody or antigen-binding fragment thereof. In some instances, the antibody or antigen-binding fragmentthereof is a recombinant antibody or antigen-binding fragment thereof. The term “humanised antibody” refers to an antibody derived from a non-human (e.g., murine) immunoglobulin, which has been engineered to contain minimal non-human (e.g., murine) sequences. Typically, humanised antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability.

[0352] Humanised antibodies can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or capability. In general, humanised antibodies will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Humanised antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanised antibodies are described in U.S. Pat. Nos. 5,225,539 or 5,639,641.

[0353] Methods for engineering, humanising or resurfacing non-human or human antibodies or antigenbinding fragments thereof can also be used and are well known in the art. A humanised, resurfaced or similarly engineered antibody can have one or more amino acid residues from a source that is non-human, e.g. , but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are replaced by residues that are often referred to as “import” residues, which are typically taken from an “import” variable, constant or other domain of a known human sequence. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. Suitably, the CDR residues may be directly and most substantially involved in influencing PAPPA binding. Accordingly, part or all of the non-human or human CDR sequences are preferably maintained while the non-human sequences of the variable and constant regions can be replaced with human or other amino acids.Antibodies or antigen-binding fragments thereof can also optionally be humanised, resurfaced, engineered with retention of high affinity for the antigen PAP PA and other favourable biological properties. To achieve this goal, humanised (or human) or engineered anti-PAPPA antibodies and resurfaced antibodies or antigen-binding fragments thereof can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanised and engineered products using three-dimensional models of the parental, engineered, and humanised sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen, such as PAPPA. In this way, FW residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

[0354] Humanisation, resurfacing or engineering of anti-PAPPA antibodies or antigen-binding fragments thereof of the present disclosure can be performed using any known method, such as but not limited to those describedin, Jones et al., Nature 321:522 (1986); Riechmannet al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988); Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993); U.S. Pat. Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567, 7,557,189; 7,538,195; and 7,342,110; International Application Nos. PCT / US98 / 16280; PCT / US96 / 18978; PCT / US91 / 09630; PCT / US91 / 05939; PCT / US94 / 01234; PCT / GB89 / 01334; PCT / GB91 / 01134; PCT / GB92 / 01755; International Patent Application Publication Nos. WO90 / 14443; WO90 / 14424; W090 / 14430; and European Patent Publication No. EP 229246; each of which is entirely incorporated herein by reference, including the references cited therein.

[0355] Anti-PAPPA humanised antibodies and antigen-binding fragments thereof can also be made in transgenic mice containing human immunoglobulin loci that are capable upon immunization of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.In some instances, the antibody or antigen-binding fragment thereof is a human antibody or antigenbinding fragment thereof. The term “human antibody” means an antibody produced in a human or an antibody having an amino acid sequence corresponding to an antibody produced in a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated. See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147 (l):86-95 (1991); U.S. Patent 5,750,373.

[0356] In some instances, an antibody or antigen-binding fragment thereof of the disclosure is a chimeric antibody or antigen-binding fragment thereof. The term “chimeric antibody” refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

[0357] Antibodies or antigen-binding fragments thereof of the present disclosure can be raised in various animals including mice, rats, rabbits, goats, sheep, monkeys or horses. Antibodies may be raised following immunisation with individual capsular polysaccharides, or with a plurality of capsular polysaccharides. Blood isolated from these animals contains polyclonal antibodies - multiple antibodies that bind to the same antigen. Antigens may also be injected into chickens for generation of polyclonal antibodies in egg yolk. To obtain a monoclonal antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from an animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies. Methods for producing monoclonal antibodies are conventional techniques known to those skilled in the art (see e.g. Making and Using Antibodies: A Practical Handbook. GC Howard.CRC Books. 2006. ISBN 0849335280). Polyclonal and monoclonal antibodies are often purified using Protein A / G or antigen-affinity chromatography.

[0358] The antibody or antigen binding fragment thereof of the disclosure may be prepared as a monoclonal anti-PAPPA antibody, which can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature 256:495 (1975). Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting, or an in vitro binding assay, e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), can then be propagated either in in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid using known methods.

[0359] Alternatively, the antibody or antigen-binding fragment thereof can also be made using recombinant DNA methods as described in U.S. Patent No. 4,816,567. The polynucleotides encoding an antibody or antigen-binding fragment thereof may be isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody or antigen-binding fragment thereof, and their sequence is determined using conventional procedures. As disclosed herein, further aspects relate to polynucleotides that encode an antibody or antigen-binding fragment as described herein, and methods of using those polynucleotides to produce a binding molecule of the disclosure. The isolated polynucleotides encoding the heavy and light chains may be cloned into suitable expression vectors. Those vectors may be transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, and monoclonal antibodies generated by the host cells. Also, recombinant monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described in McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991).The polynucleotide(s) encoding an antibody or antigen-binding fragment thereof of the disclosure can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies or antigen-binding fragments thereof. In some instances, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted (1) for those regions of, for example, a human antibody to generate a chimeric antibody or (2) for a non-immunoglobulin polypeptide to generate a fusion antibody. In some instances, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

[0360] In some instances, the antibody or antigen-binding fragment thereof can be selected from a phage library, where that phage library expresses human antibodies, as described, for example, in Vaughan et al., Nat. Biotech. 14:309-314 (1996); Sheets et al., Proc. Natl. Acad. Sci. USA, 95:6157-6162 (1998); Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); and Marks et al., J. Mol. Biol.

[0361] 222:581 (1991). Techniques for the generation and use of antibody phage libraries are also described in U.S. Patent Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., J. Molec. Biol. 376: 1182-1200 (2008), each of which is incorporated by reference in its entirety.

[0362] Affinity maturation strategies and chain shuffling strategies are known in the art and can be employed to generate high affinity human antibodies or antigen-binding fragments thereof. See Marks et al., BioTechnology 10:779-783 (1992), incorporated by reference in its entirety.

[0363] In some instances, the variable domains in the heavy or light chains of an antibody or antigenbinding fragment thereof are altered by at least partial replacement of one or more CDRs and / or by partial framework region replacement and sequence changing. In some instances, the variable domains in both the heavy and light chains of an antibody or antigen-binding fragment thereof are altered by at least partial replacement of one or more CDRs and / or by partial framework region replacement and sequence changing. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and in certain instances from an antibody from a different species. It is not necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen-binding capacity of onevariable domain to another. Rather, it is only necessary to transfer those residues that are necessary to maintain the activity of the antigen-binding site. Given the explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within the competence of those skilled in the art to carry out routine experimentation to obtain a functional antibody with reduced immunogenicity .

[0364] Alterations to the variable region notwithstanding, those skilled in the art will appreciate that an antibody or antigen-binding fragment thereof of this disclosure can comprise an antibody or antigen-binding fragment thereof in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as decreased effector function including reduced ability to induce ADCC when compared with an antibody or antigen-binding fragment thereof of approximately the same immunogenicity comprising a native or unaltered constant region.

[0365] The “Rabat numbering system” is generally used when referring to a residue in the variable region (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Rabat et al., Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0366] The amino acid position numbering as in Rabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FW or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Rabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Rabat) after heavy chain FW residue 82.

[0367] The Rabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Rabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop, when numbered using the Rabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because theKabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The table below lists the positions of the amino acids comprising the variable regions of the antibodies in each system.

[0368] Region Kabat AbM Chothia

[0369] LCDR1 L24-L34 L24-L34 L24-L34

[0370] LCDR2 L50-L56 L50-L56 L50-L56

[0371] LCDR3 L89-L97 L89-L97 L89-L97

[0372] HCDR11H31-H35B H26-H35B H26-H32..34

[0373] HCDR12H31-H35 H26-H35 H26-H32

[0374] HCDR2 H50-H65 H50-H58 H52-H56

[0375] HCDR3 H95-H102 H95-H102 H95-H102

[0376]

[0377] 'Kabat Numbering

[0378] 2Chothia Numbering

[0379] ImMunoGeneTics (IMGT) also provides a numbering system for the immunoglobulin variable regions, including the CDRs. See, e.g., Lefranc, M.P. et al., Dev. Comp. Immunol. l 55-77(2003). The IMGT numbering system is based on an alignment of more than 5,000 sequences, structural data, and characterization of hypervariable loops and allows for easy comparison of the variable and CDR regions for all species. According to the IMGT numbering schema, HCDR1 is at positions 26 to 35, HCDR2 is at positions 51 to 57, HCDR3 is at positions 93 to 102, LCDR1 is at positions 1 to 32, LCDR2 is at positions 50 to 52, and LCDR3 is at positions 89 to 97.

[0380] In some instances, the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3 present in the VH region and VL region of any binding molecule of Table 2, defined using the Kabat numbering system. In some instances, the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3 present in the VH region and VL region of any binding molecule of Table 2, defined using the Chothia numbering system. In some instances, the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3 present in the VH region and VL region of anybinding molecule of Table 2, defined using the AbM numbering system. In some instances, the binding molecule comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3 present in the VH region and VL region of any binding molecule of Table 2, defined using the IMGT numbering system.

[0381] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol.

[0382] 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

[0383] Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

[0384] The present disclosure further embraces variants and equivalents that are substantially homologous to a binding molecule of the disclosure (e.g. an antibody, such as a murine, chimeric, humanised or human antibody, or antigen-binding fragments thereof). These can contain, for example, conservative substitution mutations, i.e., the substitution of one or more amino acids by similar amino acids. For example, conservative substitution refers to the substitution of an amino acid with another within the same general class such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art.

[0385] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0386] CONSERVATIVE AMINO ACID SUBSTITUTIONS

[0387] Basic: arginine, lysine, histidine

[0388] Acidic: glutamic acid, aspartic acid,

[0389] Polar: glutamine, asparagine

[0390] Hydrophobic: leucine, isoleucine, valine

[0391] Aromatic: phenylalanine, tryptophan, tyrosine

[0392] Small: glycine, alanine, serine, threonine, methionine1

[0393] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2- aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of the binding molecules of the present disclsoure. A limited number of nonconservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The binding molecules of the present disclosure can also comprise non-naturally occurring amino acid residues.

[0394] Non-naturally occurring amino acids include, without limitation, trans-3 -methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271: 19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3 -azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0395] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of binding molecules of the present disclosure.Essential amino acids in the binding molecules of the present disclosure can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffmity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaveret al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the binding molecules of the present disclosure.

[0396] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988).

[0397] Related aspects

[0398] Another aspect relates to nucleic acid molecules and polynucleotides encoding the binding molecules of the disclosure. The term “nucleic acid molecules” is used interchangeably with the term “polynucleotides” to designate a series of nucleic acid residues that are covalently bonded in a chain. In some instances, provided is an isolated nucleic acid molecule or isolated polynucleotide encoding a binding molecule as described herein. In some instances, provided are one or more isolated nucleic acid molecules or isolated polynucleotides encoding a binding molecule as described herein. For example, where the binding molecule comprises multiple polypeptide chains, those chains may be encoded by the same polynucleotide or by more than one polynucleotide, e.g., each polypeptide chain may be encoded by a separate polynucleotide. One of skill in the art could readily obtain nucleic acid molecules encoding the disclosed binding molecules, such as one ormore nucleic acid molecules encoding the polypeptide chains that are comprised within a binding molecule as described herein.

[0399] The polynucleotide may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence or nucleic acid sequence encompasses a DNA molecule with the specified sequence, and encompasses an RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

[0400] The present disclosure also provides constructs such as plasmids, vectors, phagemids, transcription cassettes or expression cassettes which comprise nucleic acid sequence(s) encoding a binding molecule of the disclosure. In some instances, the construct comprises one or more polynucleotides as disclosed herein. In some instances, the construct is an expression construct, such as an expression vector or plasmid. In some instances, the construct is capable of expressing the binding molecule of the disclosure.

[0401] Suitable vectors can be chosen or constructed, so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. In some instances, the vector comprises suitable regulatory sequences to allow for expression of the binding molecule of the disclosure. Vectors may be plasmids or viral, e.g., phage, or phagemid, as appropriate. For further details see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for manipulation of nucleic acid, for example, in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, 2nd Edition, eds. Ausubel et al., John Wiley & Sons, 1992.

[0402] A further aspect relates to a host cell comprising a polynucleotide as disclosed herein, and / or a construct as disclosed herein, such as a vector as disclosed herein. In some instances, the host cell is capable of expressing a binding molecule as disclosed herein from the polynucleotide, construct or vector.

[0403] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known in the art. For cells suitable for producing antibodies, see Gene Expression Systems,Academic Press, eds. Fernandez et al., 1999. Briefly, suitable host cells include bacteria, plant cells, mammalian cells, and yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse myeloma cells, and many others. A common bacterial host is E. coli. Any protein expression system compatible with the invention may be used to produce the disclosed binding molecules. Suitable expression systems include transgenic animals described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

[0404] Introducing a nucleic acid into a host cell may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

[0405] The introduction of the nucleic acid into the cells may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene. Accordingly, in one aspect is provided a method of producing a binding molecule as disclosed herein, comprising expressing a polynucleotide of the disclosure, or a vector of the disclosure in a host cell. In some instances this method is performed in vitro or ex vivo.

[0406] Following production by expression, a binding molecule, or its constituent polypeptides, may be isolated and / or purified using any suitable technique, then used as appropriate. Binding molecules, polypeptides and encoding nucleic acid molecules and vectors may be isolated and / or purified from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function.

[0407] Compositions and Kits

[0408] A further aspect relates to a composition comprising a binding molecule as disclosed herein, a polynucleotide (or one or more polynucleotides) as disclosed herein, a construct (such as a vector) as disclosed herein, and / or a host cell as disclosed herein. In some instances, the composition further comprises a carrier.In some instances, the composition is a pharmaceutical composition. Accordingly, in one aspect is provided a pharmaceutical composition comprising a binding molecule as disclosed herein, a polynucleotide (or one or more polynucleotides) as disclosed herein, a construct (such as a vector) as disclosed herein, and / or a host cell as disclosed herein, wherein the composition further comprises a pharmaceutically acceptable carrier or diluent.

[0409] The phrase "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.

[0410] A pharmaceutical composition as disclosed herein is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. The administration may, for example, be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous or transdermal. The dosage ranges for administration of the binding molecule, polynucleotide(s), vector and / or cell are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the binding molecule or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician.

[0411] The pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration. In one aspect, there is provided a kit comprising a binding molecule as disclosed herein, a polynucleotide (or one or more polynucleotides) as disclosed herein, a construct (such as a vector) as disclosed herein, a host cell as disclosed herein, and / or a composition as disclosed herein. In some instances, the kit comprises one or more containers. In some instances, the kit comprises all of the components necessary and / or sufficient to perform a method of treatment as disclosed herein. In some instances, the kit comprises instructions for performing a method of treatment as disclosed herein, or instructions for using the components of the kit, such as a binding molecule or composition as disclosed herein, in a method of treatment as disclosed herein.Methods of Treatment

[0412] Also disclosed herein are methods for the prevention or treatment of PAPPA-mediated diseases or disorders. Accordingly, provided is a method for preventing or treating a PAPPA-mediated disease, disorder or condition in a subject, comprising administering to a subject in need thereof an effective amount of a binding molecule as disclosed herein, polynucleotide(s) as disclosed herein, construct or vector as disclosed herein, cell as disclosed herein, and / or pharmaceutical composition as disclosed herein. Also provided is a binding molecule as disclosed herein, polynucleotide(s) as disclosed herein, construct or vector as disclosed herein, cell as disclosed herein, and / or pharmaceutical composition as disclosed herein, for use in medicine or for use as a medicament. Also provided is a binding molecule as disclosed herein, polynucleotide (s) as disclosed herein, construct or vector as disclosed herein, cell as disclosed herein, and / or pharmaceutical composition as disclosed herein, for use in the prevention or treatment of a PAPPA-mediated disease, disorder or condition in a subject. Also provided is the use of a binding molecule as disclosed herein, polynucleotide(s) as disclosed herein, construct or vector as disclosed herein, cell as disclosed herein, and / or pharmaceutical composition as disclosed herein, in the manufacture of a medicament for use in the prevention or treatment of a PAPPA-mediated disease, disorder or condition in a subject.

[0413] In some instances, the PAPPA-mediated disease, disorder or condition is a disease, disorder or condition of the kidneys. In some instances, the PAPPA-mediated disease, disorder or condition is kidney disease. In some instances, the PAPPA-mediated disease, disorder or condition is polycystic kidney disease. In some instances, the PAPPA-mediated disease, disorder or condition is autosomal dominant polycystic kidney disease (ADPKD).

[0414] In some instances, the subject has any of these PAPPA-mediated diseases, disorders or conditions. In some instances, the method is for the prevention or treatment of any of these PAPPA-mediated diseases, disorders or conditions. In some instances, the method is for the treatment of ADPKD. In some instances, the PAPPA-mediated disorder treated by the method disclosed herein is ADPKD.

[0415] In some instances, the subject is a mammal. In some instances, the subject is a human.

[0416] Binding molecule key:

[0417] Binding molecule Component SEQ ID NO

[0418] AB1 H-CDR1 1 or 121

[0419]

[0420] H-CDR2 2H-CDR3 3 L-CDR1 4 L-CDR2 5 L-CDR3 6 VH 7 VL 8 HC 76 LC 77 AB2 H-CDR1 9 or 121

[0421] H-CDR2 10 H-CDR3 11 L-CDR1 12 L-CDR2 13 L-CDR3 14 VH 15 VL 16 HC 78 LC 79 AB3 H-CDR1 17 H-CDR2 18 H-CDR3 19 L-CDR1 20 L-CDR2 21 L-CDR3 22 VH 23 VL 24 HC 80 LC 81 AB 10 H-CDR1 25 H-CDR2 26 H-CDR3 27 L-CDR1 28 L-CDR2 29 L-CDR3 30 VH 31 VL 32 HC 82 LC 83 AB8 H-CDR1 33 H-CDR2 34 H-CDR3 35 L-CDR1 36 L-CDR2 37 L-CDR3 38 VH 39 VL 40 HC 84 LC 85

[0422]

[0423] AB5 H-CDR1 41H-CDR2 42

[0424] H-CDR3 43

[0425] L-CDR1 44

[0426] L-CDR2 45

[0427] L-CDR3 46

[0428] VH 47

[0429] VL 48

[0430] HC 86

[0431] LC 87

[0432] AB 16 H-CDR1 49

[0433] H-CDR2 50

[0434] H-CDR3 51

[0435] L-CDR1 52

[0436] L-CDR2 53

[0437] L-CDR3 54

[0438] VH 55

[0439] VL 56

[0440] HC 88

[0441] LC 89

[0442] AB34 H-CDR1 108 or 122

[0443] H-CDR2 109

[0444] H-CDR3 110

[0445] L-CDR1 111

[0446] L-CDR2 112

[0447] L-CDR3 113

[0448] VH 114

[0449] VL 115

[0450] HC TM 116

[0451] LC TM 117

[0452] HC TM-YTE 118

[0453]

[0454] LC TM-YTE 117

[0455] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of instances of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0456] The headings provided herein are not limitations of the various aspects or instances of this disclosure.EXAMPLES

[0457] Example 1: PAPPA inhibition is beneficial in the treatment of autosomal dominant polycystic kidney disease (ADPKD)

[0458] Materials & Methods

[0459] Animals

[0460] Ksp-TamCre x PkdlLox animals were bred at InnoSer Belgie NV, Diepenbeek, Belgium. Animals were transferred to the rodent unit where they were housed in individually ventilated cages (IV C) from Innovive with a filter top, sterilized corncob bedding (irradiated, dust-free supplied by Innovive, USA) and a shelter-like device (if feasible; PLEXX, The Netherlands) to stimulate natural patterns of behaviour. Animals were maintained at 21 ± 2 °C and a relative humidity between 40 to 70%, on a 12 / 12 light / dark cycle with air renewed 8-20 times per hour. During the in-life period, the animals had free access to standard pelleted food (Diet No. V1534-703, supplied by SNIFF, Germany). The animals had free access to water from Aquavive drinking bottles (Innovive), which were refreshed when necessary.

[0461] Ethical approval

[0462] This study was approved by the Animal Experimentation Committee (EC MxCL 2018-109). All procedures and methods of euthanasia that were used during this study were considered to avoid or minimize discomfort, distress, and pain to the animals. Animals experiencing severe or chronic distress or pain during this study, that could not be relieved, were euthanized after consultation of the animal welfare officer. However, animals that were moribund or in state of impending death were humanely killed immediately as stated in the study plan.

[0463] Treatment regimen

[0464] Animals were administered with one oral injection of Tamoxifen (150 mg / kg body weight; volume of 10 ml / kg) on post-natal day (PND) 18, 19 and 20. The compounds isotype control R347 hulgGl (dose 3 mg / kg and 30mg / kg) and a tool PAPPA antibody (doses 0.1, 0.3, 1, 3, 10 and 30mg / kg) were dosed per subcutaneous (SC) injection. In the first week of treatment, the animals were dosed twice: on PND42 and PND45. Thereafter, the animals were dosed once weekly starting from PND49 until PND 107. Tolvaptan was dosed in the food (0.1%) and dosed daily via oral gavage (75 mg / kg). The CDR, VH / VL region and HC / LC amino acid sequences of the tool PAPPA antibody are provided below.Tool PAPPA mAb CDR amino acid sequences:

[0465] Tool HCDR1 GYTFTNYWLG 92 LCDR1 RSSKSLLHSNGITYLY 95 PAPPA

[0466] mAb HCDR2 GDLYPGSGYTNYNEKFKG 93 LCDR2 QMSNLAS 96 (derived

[0467] from

[0468] mAb-PA HCDR3 ARNWFAY 94 LCDR3 AQNLELPWT 97 1 / 41)

[0469]

[0470] Tool PAPPA mAb VH and VL region amino acid sequences:

[0471] Tool VH QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDLYP 98 PAPPA GSGYTNYNEKF KGKAT L TAD T S S S T AYMQL S S L T S E D S AVY F FVY Y ARNWFAY

[0472] mAb WGQGTLVTVSS

[0473] (derived

[0474] from VL DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQVLI 99 mAb-PA YQMS NL AS GVPDRFSSSGSGTD FAL RTS RVE AE DVGVY YC AQNL E L PWT F GGG 1 / 41) TKLEIK

[0475]

[0476] Tool PAPPA mAb heavy chain (HC) and light chain (LC) amino acid sequences

[0477] Tool HC QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDLYP 100 GSGYTNYNEKF KGKAT L TAD T S S S T AYMQL S S L T S E D S AVY F FVY Y ARNWFAY PAPPA WGQGT LVTVS S AS TKGP S VF PL AP S S KS T S GGTAALGCL VKD YF PE PVTVSWN

[0478] mAb SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWD

[0479] (derived VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE

[0480] from YKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

[0481] mAB-PA CSVMHEALHNHYTQKSLSLS PGK

[0482] 1 / 41) LC DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQVLI 101 YQMS NL AS GVPDRFSSSGSGTD FAL R I S RVE AE DVGVY YC AQNL E L PWT F GGG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK

[0483]

[0484] SFNRGEC

[0485] Body weight

[0486] Body weight was determined daily from PND18 to PND20 and weekly from PND20 to PND110. The initial 24 hours after dosing and at least daily during the study period, the animals were observed for any behavioral and physical abnormalities.Ultrasound analysis

[0487] Kidney volume was determined at three time points during the study: at PND40, PND75, and PND96. Mice were anesthetized via isoflurane anaesthesia. As the right kidney volume was determined, the right side of the abdomen was shaved, and all remaining hairs were removed with hair removal creme. The mouse was kept under anaesthesia while being placed on a heating path under the 3D motor with echo probe connected to the Vevo3100 ultrasound. The right kidney was imaged from the ventral aspect of the abdomen creating the transverse sagittal plane. The right kidney was centralized, and a complete 3D scan was taken whereafter the mouse was removed from isoflurane anaesthesia and placed on a heating path until completely awake.

[0488] Urine sampling and osmolality

[0489] Urine was collected the day before sacrifice in the termination week by placing the animals individually in an Innovive cage from which the com cob bedding was replaced by hydrophobic Labsand (Kit4Cat). Animals were housed in these cages for 8 hours to obtain approximately 0.5 mL of urine. Subsequently, 100 pl of fresh urine from each animal was utilized for urine osmolality determination, which was performed using the freezing point depression osmometry method with a Micro-Osmometer (Model 3300, Advanced Instruments).

[0490] Termination and sample collection

[0491] Starting from PND99, analgesia was administered to the animals before heart puncture and perfusion were performed. Buprenorphine (Vetergesic ® Multidosis 0.3 mg / ml) was subcutaneously administered approximately one hour before thoracotomy at a dose of 0.1 mg / kg. All animals were sacrificed humanely by exsanguination from the heart under deep isoflurane anesthesia (ISL-04-15), using a 1 mL syringe and needle. Thereafter, all animals were perfused with saline via the heart to remove residual blood before tissue collection (notes were added for those mice whose kidneys were not sufficiently perfused). Termination was reassured by cervical dislocation. Plasma and kidneys were collected from all animals. In selected groups, urine, blood, femurs, and sternum were collected.

[0492] Kidney collection and processing

[0493] From all animals, both kidneys were collected and weighed before processing. The kidneys were transversely cut into several pieces. For histology, one piece was immediately immersed in a 4% formaldehyde solution. After 48 hours of fixation, the tissues were dehydrated, paraffin-embeddedin FFPE blocks, and then cut into 2 pm-thin sections. These sections were deparaffinized, rehydrated, and stained for Periodic acid-Schiff (PAS) and hematoxylin and eosin (H&E) using an autostainer (Leica ST5010). Histopathological assessment of the kidneys was performed by pathologists. The remaining kidney pieces (20-150mg) were snap-frozen directly in 2 ml Precellys tubes with pre-filled beads (432-0142, VWR).

[0494] Blood urea nitrogen

[0495] Plasma was used for urea level determination via the Urea Nitrogen (BUN) colorimetric detection kit (EIABUN; Invitrogen, Thermo Fisher Scientific). 2.5 pl plasma was added to a 96-well microplate, together with two test reagents and sterile water. Furthermore, a standard dilution was added to the 96-well microplate to analyse the data. Results were obtained via a 96-well microplate reader (Tecan, Infinite M200) with optical absorption at 450 nm.

[0496] Histopathology

[0497] The fixated kidney was processed for paraffin embedding and further pathological assessment after hematoxylin-eosin staining. This was done by a board-certified pathologist. For each kidney one section was evaluated. The cystic areas (any tubules that have lumen diameter >9 microns, which is the normal tubular diameter) were identified by a colour thresholding method using an image analysis system (HALO-Indica Labs-Albuquerque, NM, USA). The cystic areas of a kidney were calculated as the sum of the areas (calculated in millimetres) of the cystic lesions found in all the section analysed, in which dilated tubules were included, while dilated veins and pelvic cavity were excluded by manual tissue classifier option: (cyst area / total area)xl00% iv.

[0498] Kidney tissue lysis for proteomics and western blot analysis

[0499] Snap-frozen kidney tissues were lysed in 0.5 -1 mL RIPA buffer (Thermo Fisher Scientific) supplemented with Protease inhibitors (Complete Mini EDTA, Roche) and Phosphatase inhibitors (PhosStop, Roche) using Percellys®24 tissue homogeniser, 2 x 20 s, 5500 rpm. Homogenates were then centrifuged at 20,000 ref for 5 min at 4°C, and the supernatants were transferred to new tubes and stored at -80°C. Total protein concentrations were determined using the Pierce BCA Assay kit (ThermoFisher)

[0500] Kidney tissue proteomics

[0501] Protein lysates were subjected to reduction and alkylation, and subsequent protease treatment using Trypsin / LysC (Thermo Fisher Scientific) using the SP3 sample preparation method. Prior to LC-MS analysis peptide samples were loaded onto Evosep tips (Evosep, Odense, Denmark) according to the manufacturer's instructions. LC-MS / MS analysis was conducted on a timsTOF PRO 2 mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled with an Evosep LC-system (Evosep). Samples were run using a default SPD30 LC gradient and separated on an analytical C18-RP column (Endurance, 15 cm x 150 pm, 1.9 pm, Evosep). The MS was operated in data-independent acquisition-parallel accumulation-serial fragmentation (DIA-PASEF) mode, using a default DIA-PASEF method with a TIMS window of 0.6 1 / K0 - 1.6 1 / KO.

[0502] DIA-PASEF raw data were processed by DIA-NN software suite. Protein intensities coming from DIA-NN analysis were analysed using R (v. 4.2.3), tidyverse and DEP package (v.1.20.0). Briefly, proteins with values obtained for at least 50% of the samples in each condition. The data was normalized using VSN (v. 3.66.0), and missing values were imputed using missForest function (v.1.5). Finally, differential analysis was done using linear models and empirical Bayes statistics using limma package (v. 3.54.2). PCA was calculated using the normalized imputed values. Enrichment analysis was performed using clusterProfiler package (v.4.6.2) with KEGG, mSigdb and Reactome databases. Most graphs were prepared using ggplot2 (v. 3.5.1)

[0503] Western Blot

[0504] Total tissue or cell protein lysates were subjected to electrophoresis on either Novex 4-20% Trisglycine mini gel (pS6K) or BioRad Mini Protein TGX 4-15% stain- free gel (pAKT, AKT), and subsequently transferred onto PVDF membrane. The membranes were then incubated with primary antibodies: pAkt (Ser473) #4058, pAkt (Thr308) #4056, total Akt #2920, Phospho-p70 S6 Kinase (Thr389) #9234, all from Cell Signaling Technology, and GAPDH (MA5- 15738 , Thermo Fisher Scientific), followed by incubation with HRP secondary antibodies (Dako). Chemiluminescent signal were detected using Clarity Western ECL substrate (BioRad). The resulting images were captured by BioRad ChemiDoc Imaging System and analysed with the Image Lab 5.2 software.

[0505] Automated capillary Western blot

[0506] PhosphoAkt (pAkt) / Akt and PhosphoErk (pErk) levels were determined using the Jess system and Protein Normalization Assay Module (ProteinSimple), following the manufacturer’s instructions. Total kidney protein lysates were analysed at a concentration of 1 mg / mL. The following antibodies were employed: pAkt (Ser473) (193H12) Rabbit mAb (Cell Signaling Technology) and Goat antiRabbit Secondary HRP Antibody to detect pAkt / Akt proteins. For pErk proteins, pErkl / 2 (Thr202 / Tyr204) Rabbit mAb #4370 (Cell Signaling Technology) and 20x Anti-Rabbit SecondaryNIR Antibody (Bio-Techne) were utilised. Data were analysed using the integrated application Compass for SW.

[0507] ELISA

[0508] To measure IGF1 and PAPPA levels in mouse plasma, Mouse & Rat IGF-I / IGF-1 ELISA Kit (MG100, R&D Systems) and PAPP-A (Mouse) ELISA (AL-158, AnshLabs) were utilised following the manufacturer’s protocol.

[0509] Gene expression analysis

[0510] Snap-frozen kidney tissues were lysed in 0.6 mL RLT buffer (Thermo Fisher Scientific) supplemented with 40 mM DTT and homogenised using Percellys®24 tissue homogeniser for 2 x 20 s at 5500rpm. The resulting homogenates were then centrifuged at 20,000 ref for 5 min at 4°C, and the supernatants were transferred to new tubes. Subsequently, total RNA was extracted using RNeasy Mini kit and automated processing in QIAcube (Qiagen). Following this, cDNA was synthesized using High-capacity cDNA reverse transcription kit (Applied Biosystems). RT-qPCR was conducted using TaqMan™ Fast Advanced Master Mix on QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems) following the manufacturer’s instructions. TaqMan™ Gene Expression Assays (murine PAPPA Mm01259244_ml, Ccl2: Mm00441242_ml, Tnf: Mm00443258_ml, Tgfbl: Mm01178820_ml, Collal: Mm00801666_gl, Havcrl: Mm00506686_ml and Tbp: Mm01277042_ml) were purchased from Applied Biosystem. Quantification cycle (Cq) values were determined by the software using the Auto Baseline and Auto Threshold options, which were then used to calculate relative gene expression (2A-dCq) normalized against the reference gene Tbp.

[0511] PCNA Immunofluorescence in kidney FFPE sections

[0512] Immunofluorescence was conducted on the automated Ventana Discovery Ultra platform following the manufacturers’ protocol. All reagents were purchased from Roche (Ventana Medical systems, Roche, Basel, Switzerland). FFPE tissue blocks were sectioned to 2 pm-thin tissue sections, deparaffmized, and rehydrated in Ventana discovery Ultra instrument. Following antigen retrieval (CC1, 40 min at 95C), anti-PCNA antibody (1:4000, #13110S, Cell Signaling) was incubated for 1 hour at 37°C, followed by OmmiMap anti -rabbit- HRP (12 min), visualized with Cy5 (5min), and counterstained with DAPI. The slides were then scanned using a whole slide imaging (WSI) scanner at 40 x magnification (Olympus).Digital files were analysed using Visiopharm image analyses software (Visiopharm Inc., Hoersholm, Denmark). PCNA quantification was carried out by initially identifying nuclei using a commercially available application protocol for Nuclei Detection Al immunofluorescence (Visiopharm), followed by the detection of PCNA Cy5 positive nuclei using a customized trained Al DeepLabv3 algorithm. The percentage of PCNA positive nuclei was calculated by dividing the number of PCNA positive nuclei by the total number of nuclei counted in the tissue.

[0513] RNAscope detection of Pappa mRNA

[0514] Prior to RNAscope, FFPE blocks were sectioned to 4 pm-thin tissue sections. RNAscope was carried out on the automated Ventana Discovery Ultra platform in accordance with the manufacturer's protocol. All reagents were purchased from Roche (Ventana Medical systems, Roche, Basel, Switzerland). Briefly, following target retrieval (24 min at 97°C for tissues) and protease treatment (16 min at 37°C), sections were hybridized with MmPappa target probe (443929, ACD), control probe MmPPIP (313919,ACD), and negative control probe dapB (312039) for 2 h at 43°C, followed by RNAscope amplification and DAB chromogenic detection using VS detection reagents (323200, ACD). The slides were then scanned using a whole slide imaging (WSI) scanner at 40 x magnification (3D Histech). The localisation of Pappa mRNA was evaluated by a histologist and a pathologist.

[0515] Digital files were analysed using Visiopharm image analyses software (Visiopharm Inc., Hoesholm, Denmark). Pappa mRNA quantification was performed by nuclei detection using a commercially available application protocol, Nuclei Detection Al Brightfield (Visiopharm), followed by the detection and quantification of Pappa mRNA using a customized trained Al DeepLabv3 algorithm. The number of Pappa mRNA dots per cell was calculated by dividing the number of Pappa mRNA dots by the total number of nuclei in the tissue.

[0516] Cell culture and lysis

[0517] MCF7 (ACC 317, DSMZ), a human breast carcinoma cell line, was cultured in RPMI160 (GibcoTM, Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (GibcoTM, Thermo Fisher Scientific) in a 37°C and 5% CO2 incubator. For the experiment, MCF7 cells were seeded at 24,000 cells / cm2in a 24 well Costar plate (Coming). After 24 h, the media was changed to serum-free media, and the cells were starved overnight. On the day of the experiment, IGF1 (291 -Gl-200, R&D systems) + / - IGFBP4 (804-GB-025, R&D systems) and PAPPA (2487-ZNF-020, R&D systems) + / - PAPPA mAb (Tool or AB1) were pre-incubated in two separate tubes at 37°Cfor 90 min, then pooled together and further incubated for 90 min before being added to the cells. The final concentrations in the wells were as follows: IGF1 (6.4 nM), IGFBP4 (25 nM), PAPPA (5 nM), PAPPA mAb (200 nM). The cells were incubated for 30 min before being harvested in RIPA buffer (Thermo Fisher Scientific) supplemented with Protease inhibitors (Complete Mini EDTA, Roche) and Phosphatase inhibitors (PhosStop, Roche). Lysates were stored at -80°C until Western blot analysis.

[0518] Measurement of PAPPA mAb concentrations in mouse plasma

[0519] The plasma concentration of the PAPPA mAb was measured on Gyrolab using either an in-house developed ligand-binding universal PK assay or commercially available Gyrolab Generic PK kit for human IgG. For the in-house assay, the mAb was captured using a mouse anti-human IgG monoclonal antibody and detected using Alexa-647 labelled sheep anti-human IgG.

[0520] Cleaved IGFBP4 measurements in mouse plasma

[0521] Cleaved IGFBP4 levels in mouse plasma were measured by in-house developed ligand-binding MSD immunoassays. The cleaved NT-fragments were captured to the bottom of MSD-plates by antibodies specific for the neo-epitopes that are exposed after cleavage of IGFBP4. Detection antibodies recognizing the NT-fragments and ECL reagents were used to generate a signal. In house generated recombinant NT IGFBP4 proteins (mouse) were used as calibration curves. The mouse assay was qualified with regards to specificity, precision, recovery and parallelism according to internal standards.

[0522] Total PAPPA levels in mouse plasma

[0523] Total levels of PAPPA (bound and unbound to compound) were used as a target engagement biomarker in mouse plasma. PAPPA levels were measured using a commercial kit (AL- 158 AnshLabs) that were qualified in house. The PAPPA antibodies tested did not interfere with the analysis.

[0524] Statistics

[0525] For group comparison, One-way ANOVA and Dunnett’s multiple comparisons test, or Brown-Forsythe and Welch ANOVA if not equal variance were used. For dose dependent response MCP mod analysis has been used. For pair-wise comparison unpaired parametric two-tailed t test, or unpaired t test with Welch’s correction if not equal variance were used. *P<0.05, **P<0.01, ****P<0.001, ****P<0.0001PAPPA is upregulated and selectively expressed in epithelial cells lining cysts or dilated tubules in a mouse model of ADPKD

[0526] To determine whether ADPKD is associated with abnormal PAPPA expression, kidneys from ADPKD mice were compared with kidneys from control mice. ADPKD mice displayed a significant increase in PAPPA mRNA expression in kidney tissues, as measured by qPCR, in comparison to healthy control mice (FIG. 1A). Furthermore, using RNAscope, localised expression of PAPPA mRNA was identified in kidney cysts or dilated tubules in ADPKD mice (FIG. IB). The number of Pappa mRNA-positive cells also exhibited a significant increase in ADPKD mouse kidneys (FIG. 1C). Concurrently, ADPKD mice demonstrated a significant elevation in the plasma level of cleaved Igfbp4 compared to healthy control mice (FIG. ID).

[0527] These results demonstrate that increased PAPPA expression and increased plasma levels of cleaved IGFBP4 is present in the kidneys of ADPKD mice compared to healthy controls. PAPPA may therefore be a therapeutic target for ADPKD.

[0528] PAPPA mAb ameliorates ADPKD progression at all doses in a proof-of-concept study in a mouse model of ADPKD

[0529] A study was initiated to investigate whether a PAPPA monoclonal antibody (tool PAPPA mAb) is able to improve ADPKD progression. The study timeline is illustrated in FIG. 2A. During the treatment period, ADPKD mice were subjected to weekly subcutaneous injections of either an isotype control antibody or tool PAPPA mAb at various doses: 1, 3, 10, and 30 mg / kg. Healthy control mice were treated with PBS in a similar manner. Kidney volume was measured by ultrasound at postnatal day 96, revealing a significant reduction in the tool PAPPA mAb treatment group compared to the isotype control (FIG. 2B). At postnatal day 109-112, blood urea levels were assayed, demonstrating a significant decrease following tool PAPPA mAb treatment (FIG. 2C). Following termination, kidney sections were stained with H&E and the kidney index was analysed, revealing a significant decrease in the tool PAPPA mAb treatment group (FIG. 2D). H&E sections were examined by a board-certified pathologist, confirming that animals treated with tool PAPPA mAb demonstrated a reduction in the severity of dilated tubules and cysts, mononuclear infiltrate, and the presence of hyaline casts. Reduced cyst size is shown in FIG. 2E.

[0530] These results demonstrate that a PAPPA mAb improves ADPKD progression in a mouse model of ADPKD.PAPPA mAb dose-dependently treats ADPKD without causing polyuria in a further proof-of-concept study in a mouse model of ADPKD

[0531] To determine whether lower doses of the tool PAPPA mAb could improve ADPKD progression, ADPKD mice received weekly subcutaneous injections of either an isotype control antibody or tool PAPPA mAb at various doses: 0.1, 0.3, and 3 mg / kg. Healthy control mice were treated with PBS in a similar manner. Additionally, ADPKD mice were treated with tolvaptan by daily gavage at 75 mg / kg in combination with 0.1% tolvaptan in food, and a diet control group of ADPKD mice were handled in a similar manner without tolvaptan. Kidney volume was measured by ultrasound on postnatal day 98, revealing a significant dose-dependent reduction in the tool PAPPA mAb treatment group (FIG. 3A). At postnatal day 109-112, blood urea levels were assayed, demonstrating a significant dose -dependent decrease following tool PAPPA mAb treatment (FIG.

[0532] 3B). Following termination, the kidney weight-to-body-weight ratio was measured, revealing a significant dose -dependent decrease in the tool PAPPA mAb treatment group (FIG. 3C). Tool PAPPA mAb at 3 mg / kg exhibited a treatment effect similar to that of tolvaptan in reducing kidney volume, blood urea levels, and kidney-to-body-weight ratio. Urine was collected from mice one day before termination, and urine osmolality was assayed, demonstrating that tool PAPPA mAb did not change urine osmolality, while tolvaptan significantly reduced urine osmolality (FIG. 3D).

[0533] These results demonstrate that lower doses of the tool PAPPA mAb could improve ADPKD progression in a mouse model of ADPKD.

[0534] PAPPA mAb dose-dependently blocks Igfbp4 cleavage, with no observable effect on systemic Igfl levels in a mouse model of ADPKD

[0535] To determine whether the tool PAPPA mAb could inhibit IGFBP4 cleavage, ADPKD mice were treated with weekly subcutaneous injections of either an isotype control antibody or tool PAPPA mAb at various doses: 0.1, 0.3, and 3 mg / kg. ADPKD mice treated with tool PAPPA mAb demonstrated a dose-dependent rise in the plasma levels of endogenous murine PAPPA protein compared to the isotype control (FIG. 4A). However, the administration of tool PAPPA mAb at 0.1 mg / kg did not yield a significant effect, nor did the tolvaptan treatment. In parallel, tool PAPPA mAb led to a dose-dependent reduction in the plasma levels of cleaved endogenous Igfbp4 (FIG.

[0536] 4B). Interestingly, the treatment with tool PAPPA mAb did not regulate the plasma levels of IGF 1 (FIG. 4C).These results demonstrate that the tool PAPPA mAh dose-dependently inhibits IGFBP4 cleavage, and has no observable effect on systemic levels of IGF 1 in a mouse model of ADPKD.

[0537] PAPPA mAb inhibits IGF signalling and cyst proliferation in a mouse model of ADPKD To determine whether the tool PAPPA mAb could inhibit IGF signalling and cyst proliferation, ADPKD mice were treated with weekly subcutaneous injection of either an isotype control antibody or tool PAPPA mAb at 3 mg / kg. ADPKD mice treated with tool PAPPA mAb at 3 mg / kg exhibited a significant decrease in the phosphorylation of Erk, Akt, and S6k in kidney tissues compared to the isotype control (FIG. 5A). Furthermore, tool PAPPA mAb at 3 mg / kg also led to a significant reduction in the presence of proliferation marker Pcna-positive cells in kidney tissues (FIG. 5B).

[0538] These results demonstrate that the tool PAPPA mAb inhibits IGF signalling and cyst proliferation in a mouse model of ADPKD.

[0539] PAPPA mAb dose-dependently downregulated mRNA expression of inflammation, fibrosis, and injury markers in ADPKD mouse kidneys

[0540] To determine whether the tool PAPPA mAb could improve markers of inflammation, fibrosis and injury, ADPKD mice were treated with either a single concentration of an isotype control antibody or the tool PAPPA mAb at various doses (0.1, 0.3 and 3 mg / kg). ADPKD mice under isotype control and diet control conditions showed increased mRNA expression of inflammatory markers Ccl2 (FIG. 6A) and Tnf (FIG. 6B), fibrosis markers Tgfbl (FIG. 6C) and Collal (FIG. 6D), as well as the kidney injury marker Haver 1 (FIG. 6E). Upon treatment with tool PAPPA mAb at various doses (0.1, 0.3, and 3 mg / kg), a dose-dependent reduction in these markers was observed, with a significant decrease in the expression of all markers at 3 mg / kg compared to the isotype control. Tolvaptan treatment also resulted in a reduction in the expression of these markers compared to diet control mice, although it did not reach statistical significance.

[0541] These results demonstrate that the PAPPA mAb provided a greater improvement in markers of inflammation, fibrosis and injury compared to tolvaptan in a mouse model of ADPKD.

[0542] PAPPA mAb reversed the aberrantly regulated proteins in a mouse model of ADPKD To determine whether the tool PAPPA mAb could improve the levels of aberrantly regulated proteins caused by ADPKD, ADPKD mice were treated with either isotype control or the tool PAPPA mAb at 3mg / kg. Kidney tissues from healthy control mice and ADPKD mice, treated witheither isotype control or tool PAPPA mAb at 3 mg / kg, underwent proteomic analysis. In comparison with healthy control mice, the isotype control group displayed 1091 significantly upregulated proteins and 1070 downregulated proteins (FIG. 7A). The significantly altered genes induced by ADPKD were compared with those altered by tool PAPPA mAb treatment at 3 mg / kg based on their fold change, as shown in FIG. 7B. A number of genes were significantly altered under both conditions (indicated by dark circles), but notably in opposite directions. Specifically, genes upregulated in ADPKD were downregulated following tool PAPPA mAb treatment, and vice versa. Additionally, genes significantly altered in one condition (either ADPKD or tool PAPPA mAb treatment) demonstrated a clear opposite direction of alteration in the other condition. This negative correlation between the effects of the disease and the treatment clearly demonstrates that tool PAPPA mAb treatment effectively counteracts the aberrant changes observed in the disease (FIG.

[0543] 7B). These results demonstrate that the tool PAPPA mAb improved the aberrant protein changes observed in the isotype control group.

[0544] PAPPA mAb shows improvement in blood urea and reduction of cleaved IGFBP4 in plasma and increase in total PAPPA in plasma

[0545] Treatment of ADPKD mice with the tool PAPPA mAb across a dose range of 0.1-30 mg / kg resulted in a dose-dependent improvement in kidney function as demonstrated by the blood urea reduction from baseline upon mAb treatment (FIG. 8A). Target engagement was confirmed by a dosedependent reduction in cleaved IGFBP4 formation (FIG. 8B) and a dose-dependent increase in total murine PAPPA (FIG. 8C).

[0546] Example 2: Generation of PAPPA proteins

[0547] The protein sequences used in the design of constructs for expression of human, cynomolgus and mouse mature PAPPA proteins were derived from UniProt entries Q13219 (human), A0A2K5WE98 (cynomolgus (cyno)) and Q8R4K8 (mouse).

[0548] The protein sequences used in the design of constructs for expression of human, cynomolgus and mouse mature PAPPA-2 proteins were derived from UniProt entries Q9BXP8 (human), A0A7N9CY00 (cynomolgus (cyno)) and E9PZ87 (mouse).

[0549] The amino acid sequences used in the design of constructs are shown in SEQ ID NOs: 63-68 and 105-107.Human PAPPA: SEQ ID No 63; signal peptide amino acids 1-22 underlined, pro-peptide amino acids 23-81 in bold MRLWSWVLHLGLLSAALGCGLAERPRRARRDPRAGRPPRPAAGPATCATRAARGRR ASPPPPPPPGGAWEAVRVPRRRQQREARGATEEPSPPSRALYFSGRGEQLRLRADLELP RDAFTLQVWLRAEGGQRSPAVITGLYDKCSYISRDRGWVVGIHTISDQDNKDPRYFFSLK TDRARQVTTINAHRSYLPGQWVYLAATYDGQFMKLYVNGAQVATSGEQVGGIFSPLTQ KCKVLMLGGSALNHNYRGYIEHFSLWKVARTQREILSDMETHGAHTALPQLLLQENWD NVKHAWSPMKDGSSPKVEFSNAHGFLLDTSLEPPLCGQTLCDNTEVIASYNQLSSFRQPK VVRYRVVNLYEDDHKNPTVTREQVDFQHHQLAEAFKQYNISWELDVLEVSNSSLRRRLI LANCDISKIGDENCDPECNHTLTGHDGGDCRHLRHPAFVKKQHNGVCDMDCNYERFNF DGGECCDPEITNVTQTCFDPDSPHRAYLDVNELKNILKLDGSTHLNIFFAKSSEEELAGVA TWPWDKEALMHLGGIVLNPSFYGMPGHTHTMIHEIGHSLGLYHVFRGISEIQSCSDPCME TEPSFETGDLCNDTNPAPKHKSCGDPGPGNDTCGFHSFFNTPYNNFMSYADDDCTDSFTP NQVARMHCYLDLVYQGWQPSRKPAPVALAPQVLGHTTDSVTLEWFPPIDGHFFERELGS ACHLCLEGRILVQYASNASSPMPCSPSGHWSPREAEGHPDVEQPCKSSVRTWSPNSAVNP HTVPPACPEPQGCYLELEFLYPLVPESLTIWVTFVSTDWDSSGAVNDIKLLAVSGKNISLG PQNVFCDVPLTIRLWDVGEEVYGIQIYTLDEHLEIDAAMLTSTADTPLCLQCKPLKYKVV RDPPLQMDVASILHLNRKFVDMDLNLGSVYQYWVITISGTEESEPSPAVTYIHGSGYCGD GIIQKDQGEQCDDMNKINGDGCSLFCRQEVSFNCIDEPSRCYFHDGDGVCEEFEQKTSIK DCGVYTPQGFLDQWASNASVSHQDQQCPGWVIIGQPAASQVCRTKVIDLSEGISQHAWY PCTISYPYSQLAQTTFWLRAYFSQPMVAAAVIVHLVTDGTYYGDQKQETISVQLLDTKD QSHDLGLHVLSCRNNPLIIPVVHDLSQPFYHSQAVRVSFSSPLVAISGVALRSFDNFDPVTL SSCQRGETYSPAEQSCVHFACEKTDCPELAVENAYLNCSSSDRYHGAQCTVSCRTGYVL QIRRDDELIKSQTGPSVTVTCTEGKWNKQVACEPVDCSIPDHHQVYAASFSCPEGTTFGS QCSFQCRHPAQLKGNNSLLTCMEDGLWSFPEALCELMCLAPPPVPNADLQTARCRENKH KVGSFCKYKCKPGYHVPGSSRKSKKRAFKTQCTQDGSWQEGACVPVTCDPPPPKFHGLY QCTNGFQFNSECRIKCEDSDASQGLGSNVIHCRKDGTWNGSFHVCQEMQGQCSVPNELN SNLKLQCPDGYAIGSECATSCLDHNSESIILPMNVTVRDIPHWLNPTRVERVVCTAGLKW YPHPALIHCVKGCEPFMGDNYCDAINNRAFCNYDGGDCCTSTVKTKKVTPFPMSCDLQG DCACRDPQAQEHSRKDLRGYSHGGGGGSGLNDIFEAQKIEWHEGSHHHHHHHHHH

[0550] Cyno PAPPA: SEQ ID No 64; signal peptide amino acids 1-16 underlined MPLLLLLPLLWAGALAGAAEEPSPPSRALYFSGRGEOLRLRADLELPRDAFTLOVWLRA EGGQRSPAVITGLYDKCSYTSRDRGWVVGIHTISDQGNRDPRYFFSLKTDRARQVTTINAHRSYLPGQWVYLAATYDGRFMKLYVNGAQVATSGEQVGGIFSPLTQKCKVLMLGGSAL NHNYRGYIEHFSLWKVARTQREILSDMETHGTHTALPQLLLQENWDNVKHAWSPMKDG SSPKVEFSNAHGFLLDTSLEPPLCGQTLCDNTEVIASYNQLPSFRQPKVVRYRVVNLYED DHKNPMVTREQVDFQHQQLAEAFKHYNISWELKVLEVSNSSLRRRLILANCDISKIGDEN CDPECNHTLTGHDGGDCRHLRHPAFMKKQQNGVCDMDCNYERFNFDGGECCDPKITDV TQTCFDPDSPHRAYLDVNELKNILRLDGSTHLNIFFANSSEEELAGVATWPWDKEALMH LGGIVLNPSFYGMPGHTHTMIHEIGHSLGLYHVFRGISEIQSCSDPCMETEPSFETGDLCND TNPAPKHKSCGDPGPGNDTCGFHSFFNTPYNNFMSYADDDCTDSFTPNQVARMHCYLDL VYQSWQPSRKPAPVALAPQVVGHTTDSVTLEWFPPIDGHFFERELGSACDLCLEGRILVQ YAFNASSPMPCSPSGHWSPREAEGHPDVEQPCKSSVRTWSPNSAVNPHTVPPACPEPQGC YLELEFLYPLVPESLTIWVTFVSTDWDSSGAVNDIKLLTVSGKNISLGPQNVFCDIPLTIRL QDVGEEVYGIQIYTLDEHLEIDAAMLTSTADTPLCLQCKPLQYKVVRDPPLQVDVASILH LNRKFIDMDLNRSNVYQYWVITISGTEESEPSPAVTYIHGSGYCGDGIIQKDQGEECDDLN KINGDGCSLFCRQEVSFNCIDEPSRCYFHDGDGVCEEFEQKTSIKDCGVYTPQGFLDQWA SNASVSHQDQQCPGWVIIGQPAASQVCRTKVIDLSEGISQHAWYPCTISYPYSQLAQTTF WLRAYFSQPMVAAAVIVHLVTDGTYYGDQKQETISVQLLDTKDQSHDLGLHVLSCRNN PLIIPVVHDLSQPFYHSQAVRVSFSSPLVAISGVALRSFDNFDPVTLSSCQRGETYSPAEQS CVHFACENTDCPELAVENASLNCSSSDRYHGAQCTVSCRTGYVLQIRRDDELIKSQMGPS VTVTCTEGKWNKQVACEPVDCSIPDHHHVYAASFSCPNGTTFGSQCSFQCRHPAQLKGN NSLLTCMEDGLWSFPEALCELMCLAPPPVPNADLQTPRCRENKHKVGSFCKYKCKPGYH VPGSSRKSKKRAFKTQCTQDGSWQEGACVPVTCDPPPSKFHGLYQCTNGFQFNSECRIKC EDSDASQGRGSNIINCRKDGTWSGSFHVCQEMQGQCSAPNQLNSNLKLQCPDGYAIGSE CATSCLDHNSESIILPMNVTVRDIPHWLNPTRVERVVCTAGLKWYPHPALIHCVKGCEPF MGDNYCDAINNRAFCNYDGGDCCTSTVKTKKVTPFPMSCDLQGDCACRDPQAQEHSRK DLRGYSHGGSGLNDIFEAQKIEWHEGSHHHHHHHHHH

[0551] Murine PAPPA: SEQ ID No 65; signal peptide amino acids 1-16 underlined.

[0552] MPLLLLLPLLWAGALARGAEEPSPPSRALYFSGRGEOLRLRADLELPRDAFTLOVWLRAE GGQKSPAVITGLYDKCSYTSRDRGWVMGIHTTSDQGNRDPRYFFSLKTDRARKVTTIDA HRSYLPGQWVHLAATYDGRLMKLYMNGAQVATSAEQVGGIFSPLTQKCKVLMLGGSA LNHNFRGHIEHFSLWKVARTQREIVSDMETRGLHTPLPQLLLQENWDNVKRTWSPMKD GNSPQVEFSNAHGFLLDTNLEPPLCGQTLCDNTEVISSYNQLPSFRQPKVVRYRVVNIYD DHHENPTVSWQQIDFQHQQLAEAFQHYNISWELEVLNINSSSLRHRLILANCDISKIGDEK CDPECNHTLTGHDGGDCRQLRYPAFMKKQQNGVCDMDCNYERFNFDGGECCDPDITDVTKTCFDPDSPHRAYLDVNELKNILRLDGSTHLNIFFANSSEEELAGVATWPWDKEALMH LGGIVLNPSFYGIPGHTHTMIHEIGHSLGLYHIFRGISEIQSCSDPCMETEPSFETGDLCNDT NPAPKHKFCGDPGPGNDTCGFHGFFNTPYNNFMSYADDDCTDSFTPNQVSRMHCYLDL VYQSWQPSRKPAPVALAPQVVGHTMDSVMLEWFPPIDGHFFERELGSACDLCLEGRILV QYAFNASSPMPCGPSGHWSPREAEGHPDVEQPCKSSVRTWSPNSAVNPHTVPPACPEPQ GCYLELEFRYPLVPESLTIWVTFVSSDWDSSGAVNDIKLLTISGKNISLGPQNVFCDIPLTIR LRDVGEEVYGIQIYTLDEHLEIDAAMLTSTVDSPLCLQCKPLQYKVLRDPPLLEDVASLL HLNRRFMDTDLKLGSVYQYRIITISGNEESEPSPAAIYTHGSGYCGDGVIQKDQGEECDD MNKVNGDGCSLFCKQEVSFNCIDEPSRCYFHDGDGMCEEFEQKTSIKDCGVYTPQGFLD QWASNASVSHQDQQCPGWVVIGQPAASQVCRTKVIDLSEGISQHAWYPCTITYPYYHLP QTTFWLQTYFSQPMVAAAVIIHLVTDGTYYGDQKQETISVQLLDTKDQSHDLGLHVLSC RNNPLIIPVVHDLSQPFYHSQAVHVSFSSPLVAISGVALRSFDNFDPVTLSSCQRGETYSPA EQSCVHFACQAADCPELAVGNASLNCSSNHHYHGAQCTVSCQTGYVLQIQRDDELIKSQ VGPSITVTCTEGKWNKQVACEPVDCGIPDHHHVYAASFSCPEGTTFGRRCSFQCRHPAQL KGNNSFLTCMEDGLWSFPEALCELMCLAPPPVPNADLQTARCRENKHKVGSFCKYKCKP GYHVPGSSRKSKKRAFKTQCTQDGSWQEGTCVPVTCDPPPPKFHGLYQCTNGFQFNSEC RIKCEDSDASQGRGSNIIHCRKDGTWSGSFHVCREMQGQCSAPNQLNSNLKLQCPDGYAI GSECAISCLDHNSESIILPVNLTVRDIPHWMNPTRVQRIVCTAGLQWYPHPALIHCVKGCE PFMGDNYCDAINNRAFCNYDGGDCCTSTVKTKKVTPFPMSCDLQNDCACRDPEAQEHN RKDLRGYSHGGGGGSGLNDIFEAQKIEWHEGSHHHHHHHHHH

[0553] Human PAPPA-2: SEQ ID No 66; Human CD33 signal peptide amino acids 1-16 underlined MPLLLLLPLLWAGALAPPEESNONGGEGSYREAETFNSOVGLPILYFSGRRERLLLRPEVL AEIPREAFTVEAWVKPEGGQNNPAIIAGVFDNCSHTVSDKGWALGIRSGKDKGKRDARF FFSLCTDRVKKATILISHSRYQPGTWTHVAATYDGRHMALYVDGTQVASSLDQSGPLNS PFMASCRSLLLGGDSSEDGHYFRGHLGTLVFWSTALPQSHFQHSSQHSSGEEEATDLVLT ASFEPVNTEWVPFRDEKYPRLEVLQGFEPEPEILSPLQPPLCGQTVCDNVELISQYNGYWP LRGEKVIRYQVVNICDDEGLNPIVSEEQIRLQHEALNEAFSRYNISWQLSVHQVHNSTLRH RVVLVNCEPSKIGNDHCDPECEHPLTGYDGGDCRLQGRCYSWNRRDGLCHVECNNMLN DFDDGDCCDPQVADVRKTCFDPDSPKRAYMSVKELKEALQLNSTHFLNIYFASSVREDL AGAATWPWDKDAVTHLGGIVLSPAYYGMPGHTDTMIHEVGHVLGLYHVFKGVSERESC NDPCKETVPSMETGDLCADTAPTPKSELCREPEPTSDTCGFTRFPGAPFTNYMSYTDDNC TDNFTPNQVARMHCYLDLVYQQWTESRKPTPIPIPPMVIGQTNKSLTIHWLPPISGVVYD RASGSLCGACTEDGTFRQYVHTASSRRVCDSSGYWTPEEAVGPPDVDQPCEPSLQAWSPEVHLYHMNMTVPCPTEGCSLELLFQHPVQADTLTLWVTSFFMESSQVLFDTEILLENKES VHLGPLDTFCDIPLTIKLHVDGKVSGVKVYTFDERIEIDAALLTSQPHSPLCSGCRPVRYQ VLRDPPFASGLPWVTHSHRKFTDVEVTPGQMYQYQVLAEAGGELGEASPPLNHIHGAP YCGDGKVSERLGEECDDGDLVSGDGCSKVCELEEGFNCVGEPSLCYMYEGDGICEPFER KTSIVDCGIYTPKGYLDQWATRAYSSHEDKKKCPVSLVTGEPHSLICTSYHPDLPNHRPLT GWFPCVASENETQDDRSEQPEGSLKKEDEVWLKVCFNRPGEARAIFIFLTTDGLVPGEHQ QPTVTLYLTDVRGSNHSLGTYGLSCQHNPLIINVTHHQNVLFHHTTSVLLNFSSPRVGISA VALRTSSRIGLSAPSNCISEDEGQNHQGQSCIHRPCGKQDSCPSLLLDHADVVNCTSIGPG LMKCAITCQRGFALQASSGQYIRPMQKEILLTCSSGHWDQNVSCLPVDCGVPDPSLVNY ANFSCSEGTKFLKRCSISCVPPAKLQGLSPWLTCLEDGLWSLPEVYCKLECDAPPIILNAN LLLPHCLQDNHDVGTICKYECKPGYYVAESAEGKVRNKLLKIQCLEGGIWEQGSCIPVVC EPPPPVFEGMYECTNGFSLDSQCVLNCNQEREKLPILCTKEGLWTQEFKLCENLQGECPPP PSELNSVEYKCEQGYGIGAVCSPLCVIPPSDPVMLPENITADTLEHWMEPVKVQSIVCTGR RQWHPDPVLVHCIQSCEPFQADGWCDTINNRAYCHYDGGDCCSSTLSSKKVIPFAADCD LDECTCRDPKAEENQGGGGSGLNDIFEAQKIEWHEGSHHHHHHHHHH

[0554] Cyno PAPPA-2: SEQ ID No 67; signal peptide amino acids 1-22 underlined, pro-peptide amino acids 23-233 in bold MMCLNILRISLAILAGWALCSANSELGWTRKKSLVEREHLNOVLLEGERCWLGAKV RRPRASPQHHLFGVYPSRAGNYLRPYSVEEQEIRHTGRSRPDTEGNAVSLVPRDLTE NPAGLRGAVEHPPAPWVENSPTGQSELLGDDDTYLGNQGSKESLGEAGTQKGSAV AATTATIFTTPKEPKPETQRRGWTKSRQRRQVWKRRAEDVWGDSDISSHFQPWSK HSLKHRVRKSPPDKSNQNGGEDSYREAETFNSQVGLPILYFSGRRERLLLRPEVLAEIPRE AFTVEAWVKPEGGQNNPAIIAGVFDNCSHTVSDKGWALGIRSGKDKGKRDARFFFSLRT DRMKKATILISHSRYQPGTWTHVAATYDGRRMALYVHGTQVASSLDQSGPLNSPFMAS CRSLLLGGDSSEDGRYFRGHLGTLVFWSTALPQNHFQHSSQHSSGEEEATDLVLTASFEP LNTEWVPFRDEKYPRLEVLQGFEPEPEILSPLRPPLCGQTVCDNVELISRYNGYWPLRGEK VIRYQVVNICDDEGLNPIVSEEQIRLQHEALNEAFSRYNISWQLSVHQVHNSTLRHRVVL VNCEPSKIGNDHCDPECEHPLTGYDGGDCRLQGRCYSWNRRDGLCHAECNNMLNDFDD GDCCDPQVADVRKTCFDPDSPKRAYMSVKELKEALQLNSTHFLNIYFASSVREDLAGAA TWPWDKDAVTHLGGIVLSPAYYGMPGHMDTMIHEVGHVLGLYHVFKGVSERESCNDP CKETVPSMETGDLCADTAPTPKSELCREPEAMSDTCGFTRFPGAPFTNYMSYTDDNCTD NFTPNQVARMHCYLDLVYQQWTESRKPTPIPIPPMIIGQTNKSLTIHWLPPLSGVVYDRAS GSMCGACTEDGTFRQYVHTASSRRVCDSSGYWTPEEAVGPPDVDQPCEPSLQAWSPEVHLYHMNMTVPCPTEGCSLELLFQYPVQADTLTLWVTSFFLDSSQVLFDTEILLENKESVHL GPLDTFCDIPLTIKLHVDGKVSGVKVYTFDERIEIDAALLTSQPHSPLCSGCRPVRYQVLR DPPFASGLPVVVTHPHRKFTDVEVTPGQMYRYQVLAEAGGELGEASPPLNHIHGAPYCG DGKVSESLGEECDDGDLVSGDGCSKACELEEGFNCVGEPSLCYMYEGDGICEPFERKTSI VDCGIYTPKGYLDQWATQAYSSHEDKNKCPVSLVTGEPHSLICTSYHPDLPNHRPLTGW FPCVASENETQDDRSEQPESSLKKEDEVWLKVCFNRPGVARAIFIFLTTDGLDPGEHRQPT VTLYLTDVRGSNHSLGTYGLSCQHNPLIINVTHHQNILFHHTTSVLLNFSSPRVGISAVAL RTFSHIGLSAPSNCISEDEGQNHQGQSCVHQPCGKQDSCPSLLLDHADWNCTSIGPGLM KCAITCQRGFALQASGGQYIRPMQKEILLTCSSGHWDQNVSCLPVDCGVPDLSLVNYAN FSCSEGTNFLKRCSISCVPPAKLQGLSPWLTCLEDGLWSLPEVYCKLECDAPPVILNANLL LPHCLQDNHDVGTICKYECKPGYYVAESAEGKVRNKFLKIQCLEGGIWEQGSCIPVVCEP PPPVFEGMYECTNGFNLDSQCVLNCNQESEKLPILCTKEGLWTQEFKLCENLQGECPPPPS ELNSVEYKCEQGYGIGAVCSPLCVIPPSDPVMLPENITADTLEHWMEPVKVQSIVCTGRR QWHPDPVLVHCIQSCEPFQADGWCDTINNRAYCHYDGGDCCSSTLSSKKVIPFAADCDL DECTCRDPKAEENQGGGGSGLNDIFEAQKIEWHEGSHHHHHHHHHH

[0555] Murine PAPPA-2: SEQ ID No 68; Human CD33 signal peptide amino acids 1-16 underlined

[0556]

[0557] TAPQHHLFGVYPSRLENYPRSYPVGTQQQTLYAEWSQRSPERQAPSGIPRDLTENPAGAR RESQQPAAPWVGDGPIGQSQLLRNDDTYLGEGGSKEALGEPAIQGSSEIAVSTIATFAYQ KGPTAETQRKGQSKSRLPRQVQKRQAEDMTGDPQNTPQGFQLWPKEPLKHGDSDSPLE GTIQNGGGASLRRVETFNSQGGLPVLYFTGKRERLLLRPEVLAEIPREAFTVEAWVRPEG GQSNPAIIAGVFDNCSHTVNDKGWALGIRSGKDKGWRDARFFFSLRTDRMKKATIVTGH SRYQPGMWTHVAATYDGQHTALYVDGTRVASSRDQFGPLNSPFMASCRSLILGGDSSED GHYFRGYLGTLVIWSTALSQTHLQHSPLHPSAADELTTLILTATFDPLMEQWAPFRDDLY PRLEVLQDSESQPEILSPLQPPPCGQTACDNVELISQYNKHGPLRREKVIRYQVVNIYDDE GLHPIVSDHQIRRQHEALNQAFSRYNISWQLSVHRVHNSTLRHRVVLVNCEPSKIGNDHC DPECEHPLTGFDGGDCRLQGRCYSWNRRDGLCHVECNNMLNDFDDGDCCDPEVTDVR KTCFDPDSPKRAYMSVKELKEALQLNSTHFLNVYFASSVREDLAGSATWPWDKEALSHL GGVVLNPTYYGMLGHTNIMIHEVGHVLGLYHVFKGVSERESCDDPCRETVPSMETGDLC ADTAPTPKSKLCRDPEPANDTCGFTLFPGAPFNNYMSYTDDECTDNFTPNQVARMHCYL DLVYQQWSESQKPTPIPIPPMVIGQTHKSLTIHWLPPISGVVYDRAPDSMCSACTEDGTFR QYVYKASSGRVCDSSGYWTPEEAVGPPDVDQPCEPSLQAWSPELHLNHMNMTVPCPAE GCSLELLFQYPVQADTLTLWVTYLSMNSSKAFFDIEILLEHKKSMHLGPLNTFCDIPLTIKLHMDEKVMGAKVYTFDERMEIDAALLTSRPNSSLCSGCKPMSYQVLREPPFSSGLPMVV THPHRKFTDTEVIPGQMYQYQVQAEAVGELGEASPPLSHIHGGPYCGDGKVERSIGEMC DDGDLLNGDGCSRACELEEGFNCAGEPSLCYRYEGDGVCEDFEKESIITDCGLHTPEGYL DQWASQAYSYHEDKEKCPVSLVTGEPHSMICTSWHPDSSPYHSFPGWFPCVFSLKKDQD AGSEQAKDSLQKDNEIWLEVCFNRPGVAVAIYIFLASDGVTPGEHQQATVTLYLIDVSGS NHSLGTYGLSCQQNPLVINVSHHVNVYPQHTSSMLLNFSSPLVGISAVALRTSSQTSSSAP SNCIPEQNYEGKSCAQRPCGEQGRCAPLLLDHTDMVNCTSSSPGHMECAITCQRGYVLQ TSSGQYLRNMQKEILLTCSSGHWDKDVICKRLDCGVPDSSLVNYANFSCLEGTDFLKQCS ISCVPPAKLQGLNPWLTCLEDGLWSLPEVYCKLECEAPPVIPNANLLLPHFLEGNHDVGTI CKYECKPGYYVKETSGSQGKNKFLKIQCLDDGSWEQGSCVPVVCEPPPPVFEGMYECTD GFKLDSQCVLNCNQETEGIPILCTKEGLWTQEFKLCENLQGECPPPTSELNSVEYKCGQG YGIGAVCSPSCVIPPSDPVILPENVTTDTLEHWMEPVKVQSIVCTGRRQWHPDPSLVHCIQ SCEPFQADGWCDTINNRAYCNYDGGDCCSSTLSSKKVIPFAADCDLDECTCRDPKAEEN QGGGGSGLNDIFEAQKIEWHEGSHHHHHHHHHH

[0558] Laminine G domain of Human PAPPA: SEQ ID No 105; signal peptide amino acids 1-16 underlined.

[0559] MPLLLLLPLLWAGALAGLNDIFEAOKIEWHEGSHHHHHHHHHHGSEARGATEEPSPPSR ALYFSGRGEQLRLRADLELPRDAFTLQVWLRAEGGQRSPAVITGLYDKCSYISRDRGWV VGIHTISDQDNKDPRYFFSLKTDRARQVTTINAHRSYLPGQWVYLAATYDGQFMKLYVN GAQVATSGEQVGGIFSPLTQKCKVLMLGGSALNHNYRGYIEHFSLWKVARTQREILSDM ETHGAHTALPQLLLQENWDNVKHAWSPMKDGSSPKVEFSNAHGFLLDTSLEP

[0560] Laminine G domain of Human PAPPA mutated: SEQ ID NO 106; signal peptide amino acids 1-16 underlined, mutation in bold MPLLLLLPLLWAGALAGLNDIFEAOKIEWHEGSHHHHHHHHHHGSEARGATEEPSPPSR ALYFSGRGEQLRLRADLELPRDAFTLQVWLRAEGGQRSPAVITGLYDKCSYTSRDRGWV VGIHTISDQDNKDPRYFFSLKTDRARQVTTINAHRSYLPGQWVYLAATYDGQFMKLYVN GAQVATSGEQVGGIFSPLTQKCKVLMLGGSALNHNYRGYIEHFSLWKVARTQREILSDM ETHGAHTALPQLLLQENWDNVKHAWSPMKDGSSPKVEFSNAHGFLLDTSLEP

[0561] Laminin G domain ofcynoPAPPA: SEQIDNO 120; signal peptide amino acids 1-16 underlined.

[0562] MPLLLLLPLLWAGALAGLNDIFEAOKIEWHEGSHHHHHHHHHHGGAAEEPSPPSRALYF SGRGEQLRLRADLELPRDAFTLQVWLRAEGGQRSPAVITGLYDKCSYTSRDRGWVVGIHTISDQGNRDPRYFFSLKTDRARQVTTINAHRSYLPGQWVYLAATYDGRFMKLYVNGAQ VATSGEQVGGIFSPLTQKCKVLMLGGSALNHNYRGYIEHFSLWKVARTQREILSDMETH GTHTALPQLLLQENWDNVKHAWSPMKDGSSPKVEFSNAHGFLLDTSLEP

[0563] Human PAPPA [82-1183]: SEQ ID No 107; signal peptide amino acids 1-16 underlined MPLLLLLPLLWAGALAGHHHHHHHHHHGENLYFOSEARGATEEPSPPSRALYFSGRGEO LRLRADLELPRDAFTLQVWLRAEGGQRSPAVITGLYDKCSYISRDRGWVVGIHTISDQDN KDPRYFFSLKTDRARQVTTINAHRSYLPGQWVYLAATYDGQFMKLYVNGAQVATSGEQ VGGIFSPLTQKCKVLMLGGSALNHNYRGYIEHFSLWKVARTQREILSDMETHGAHTALP QLLLQENWDNVKHAWSPMKDGSSPKVEFSNAHGFLLDTSLEPPLCGQTLCDNTEVIASY NQLS SFRQPKVVRYRVVNLYEDDHKNPTVTREQVDFQHHQLAEAFKQ YNI SWELDVLE VSNSSLRRRLILANCDISKIGDENCDPECNHTLTGHDGGDCRHLRHPAFVKKQHNGVCD MDCNYERFNFDGGECCDPEITNVTQTCFDPDSPHRAYLDVNELKNILKLDGSTHLNIFFA KSSEEELAGVATWPWDKEALMHLGGIVLNPSFYGMPGHTHTMIHEIGHSLGLYHVFRGI SEIQSCSDPCMETEPSFETGDLCNDTNPAPKHKSCGDPGPGNDTCGFHSFFNTPYNNFMS YADDDCTDSFTPNQVARMHCYLDLVYQGWQPSRKPAPVALAPQVLGHTTDSVTLEWFP PIDGHFFERELGSACHLCLEGRILVQYASNASSPMPCSPSGHWSPREAEGHPDVEQPCKSS VRTWSPNSAVNPHTVPPACPEPQGCYLELEFLYPLVPESLTIWVTFVSTDWDSSGAVNDI KLLAVSGKNISLGPQNVFCDVPLTIRLWDVGEEVYGIQIYTLDEHLEIDAAMLTSTADTPL CLQCKPLKYKVVRDPPLQMDVASILHLNRKFVDMDLNLGSVYQYWVITISGTEESEPSPA VTYIHGSGYCGDGIIQKDQGEQCDDMNKINGDGCSLFCRQEVSFNCIDEPSRCYFHDGDG VCEEFEQKTSIKDCGVYTPQGFLDQWASNASVSHQDQQCPGWVIIGQPAASQVCRTKVI DLSEGISQHAWYPCTISYPYSQLAQTTFWLRAYFSQPMVAAAVIVHLVTDGTYYGDQKQ ETISVQLLDTKDQSHDLGLHVLSCRNNPLIIPVVHDLSQPFYHSQAVRVSFSSPLVAISGVA LRSFDN

[0564] Mammalian expressed Avi- and poly-His- tagged human, cynomolgus and mouse PAPPA and PAPPA-2 proteins

[0565] Constructs for human, cynomolgus and mouse PAPPA and PAPPA-2 proteins encoding full length mature proteins including C-terminal Avi- and poly -histidine (poly-His) tags (FIG. 9) were cloned into the pDEST12.2 OriP (Thermo Fisher) vector. The constructs were expressed in a Chinese Hamster Ovary (CHO) cells and purified from the conditioned media using standard immobilized metal affinity chromatography (IMAC) and size exclusion chromatography purification.Example 3: Generation of anti-human PAPPA antibodies

[0566] This study was designed to isolate fully human PAPPA-specific monoclonal antibodies (mAbs) by screening and characterizing the properties of these mAbs for binding to PAPPA, specificity and species cross-reactivity. Antibodies specifically binding to PAPPA were generated according to the following protocol using transgenic mice engineered to contain the full human repertoire of variable heavy and variable light (Kappa and Lambda) chain sequences.

[0567] Immunisations

[0568] Recombinant human PAPPA and PAPPA-DTA prepared using the protocol set out in Example 2 were used to immunise transgenic mice bearing the human variable heavy and variable light (Kappa and Lambda) chain regions. T helper epitopes (DTA tag) were incorporated into one of the PAPPA immunogens as described in Percival-Alwyn et al. (2015) to break tolerance and generate a robust immune response to the human PAPPA in mice.

[0569] The recombinant proteins were diluted in PBS, emulsified with equal volumes of complete Freund’s adjuvant, and injected into the mice at two sites. For the subsequent three injections, the proteins were emulsified in Freund’s incomplete adjuvant and injections were performed as above. The final boost was carried out on day 24 by injecting recombinant protein in PBS intraperitoneally.

[0570] Tail vein bleeds were obtained from mice before immunisation, on day 13 after the first immunisation, and on day 20 after second immunisation. The IgG titres to human PAPPA were determined by serum ELISA.

[0571] Assessment of mouse immune response to PAPPA

[0572] The serum IgG titres to human PAPPA and an irrelevant protein control were determined by ELISA in 96-well microtitre plates using standard techniques. Antibodies were detected using an HRP labelled polyclonal goat anti-mouse IgG specific secondary antibody (Jackson Immunolabs), and the assay was developed using TMB substrate (Sigma) followed by the addition of 0.5 M sulphuric acid to stop the reaction. The plates were then read using a PerkinElmer EnVision 2103 multilabel plate reader.

[0573] The serum titration curves for human PAPPA and the irrelevant protein were plotted and the respective area under the curves (AUG) were calculated.Monoclonal IgG isolation

[0574] Four days after the final boost, lymph nodes were aseptically harvested, and cells were isolated by mechanical disruption and then counted. These cells were mixed with SP2 / 0 myeloma cells and fused using an electrofusion apparatus. The resultant fusions were mixed with a methylcellulose -based semi-solid media and plated out into OmniTray plates. The semi-solid media comprised CloneMatrix and DMEM supplemented with 20% FCS, 10% BM Condimed Hl, 1 mM sodium pyruvate and OPI media supplement, 2% hypoxanthine azaserine, and FITC conjugated goat antimouse IgG. The cells in semi-solid media were cultured for 13 days at 37°C in a 7.5% CO2 incubator. During this incubation period, clonal colonies are formed from a single progenitor hybridoma cell. These colonies secrete IgG that is trapped in the vicinity of the colony by the FITC conjugated anti-IgG present in the semi-solid media. The resultant immune complex formation can be observed around the cell as a fluorescent ‘halo’ when visualised by ClonePix FL colony picker (Molecular Devices). These haloed colonies are then picked into 96 well microtitre plates.

[0575] After 3-5 days in culture, the supernatants of the picked colonies were harvested and screened for human PAPPA binding.

[0576] DNA sequencing and purification of IgGs

[0577] Messenger RNA (mRNA) was extracted from hybridoma cells using magnetic oligo (dT) particles and reverse transcribed into cDNA. PCR amplification was performed using poly-C and constant region VH or VL primers specific to all human IgG subclasses.

[0578] IgG purifications

[0579] Cells were propagated in 24-well plates and overgrown in serum free HL- 1 medium supplemented with HyberZero and glutamine. After 10 days, the supernatants were transferred to 96-well master blocks, and IgGs of all subclasses (IgGl, IgG2a, IgG2b and IgG3) were purified from overgrown cell culture supernatants on ProPlus resin (Phynexus) using Hamilton Liquid Handler. The captured mouse IgGs were eluted with 100 mM HEPES, 140 mM NaCl pH 3.0 and then neutralised with an equal volume of 200 mM HEPES pH 8.0. The purified IgGs were quantified using an absorbance reading at 280 nm in UV-Star 384 well plate.

[0580] Reformatting of IgGs

[0581] Chimeric hybridoma IgG clones were molecularly reformatted to generate constructs expressing human VH and VL domains and the relevant human IgG constant domains for each hybridomaessentially as described by Persic et al., 1997. The VH domain was cloned into the relevant vector containing the human heavy chain constant domains and regulatory elements to express whole IgG heavy chain in mammalian cells. Similarly, the VL domain was cloned into a vector for the expression of the appropriate human light chain (lambda or kappa) constant domains and regulatory elements to express whole IgG light chain in mammalian cells. To obtain IgGs, mammalian suspension CHO cells were transiently transfected with the heavy and light chain IgG vectors. IgGs were expressed and secreted into the medium. IgGs were purified from clarified supernatants using MabSelect SuRe chromatography columns (GE Healthcare Lifesciences Cat no: 11003493 for 1ml columns; 11003495 for 5ml columns) and the AktaXpress™ purification system from GE Healthcare Lifesciences. The eluted material was buffer exchanged into PBS using PD- 10 desalting columns (GE Healthcare Lifesciences; Cat no: 17085101). The concentration of IgG was determined spectrophotometrically using extinction coefficients based on the amino acid sequences of the IgGs (Pace et al., 1995), and the purified IgGs were analysed for purity using SDS-PAGE and HP-SEC analysis. Resultant antibodies were then screened for PAPPA selectivity using a high throughput homogeneous time resolved fluorescence (HTRF) assay.

[0582] Affinity maturation of AB 1

[0583] AB34 was identified from phage display selections against recombinant human PAPPA from libraries of variants of AB1 with randomization in HCDR1 and HCDR2, or LCDR1 and LCDR2. Beneficial mutations were recombined and antibodies reformatted to human IgGl containing the TM (L234F / L235E / P331S) mutations in the Fc (AB34 TM) or human IgGl containing TM and YTE (M252Y / S254T / T256E) mutations in the Fc (AB34 TM-YTE) (See reformatting section above).

[0584] Example 4: Profiling PAPPA binding antibodies

[0585] Screening hybridoma IgGs for specific binding to recombinant PAPPA proteins by homogeneous time-resolved fluorescence (HTRF)

[0586] Supernatants generated from the immunisations with human PAPPA and purified IgGs were screened to identify IgGs with specific binding to human and mouse PAPPA. PAPPA-reactive antibodies were then counter screened for human PAPPA-2 binding. Biotinylated recombinant protein encompassing the PAPPA protein (PAPPA and PAPPA-2) were generated in-house via standard recombinant protein expression techniques. Reactions consisting of test or control antibodies, lOnM of AF647-labelled anti-human fragment crystallisable (Fc)y (Jackson ImmunoResearch, 109-605-008) or AF647-labelled anti-mouse Fc (Jackson ImmunoResearch,115-605-164), InM streptavidin conjugated terbium cryptate (Revvity, 610SATLB) and 8nM of recombinant biotinylated PAPPA construct in assay buffer (lOOmM sodium-chloride, 50mM TRIS-hydrochloride and ImM calcium-chloride) were incubated for 4 hours at room temperature. Time resolved fluorescence at 620 nm and 665 nm emission wavelengths were determined using a PHERAstar FSX plate reader (BMG Labtech). The 665 / 620 nm sample ratio followed by the % deltaF values for each sample were calculated, as follows (NSB denotes values achieved by the non-specific binding controls where test antibody was not included):

[0587] acceptor 665nm

[0588] sample ratio = — - - * 10000

[0589] aonor 620nm

[0590] sample ratio — mean NSB ratio

[0591] %deltaF * 100

[0592]

[0593] mean NSB ratio

[0594] 3514 hybridoma clones were generated from the hybridoma process and screened for PAPPA -reactivity using a single point HTRF-based PAPPA binding assay. From this screen, 176 putative PAPPA reactive clones were identified - mAbs giving a Max %deltaF of >60 was used as a cut off. Antibodies from the supernatant of these clones was purified and binding to PAPPA re-confirmed using the same HTRF-based binding assay. 28 clones from this panel were chosen that gave a Max %deltaF value of >100 for human PAPPA binding and were reformatted to fully human IgGs (See reformatting section above). These antibodies were characterised in a multi-point screening assay to obtain full binding curves using the HTRF-based PAPPA binding assay.

[0595] The results are shown in Table 5 below.

[0596] Table 5: Max % deltaF values for mAbs binding to PAPPA or PAPPA-2 in HTRF assay Max % Human

[0597] Max % Mouse Max % Human deltaF PAPPA

[0598] Clone ID deltaF (huma PAPPA1 deltaF (mous PAPPA2 (human 1

[0599] n PAPPA1) Binding e PAPP Al) Binding PAPPA2 Binding

[0600] ) AB1 Yes 1525.08 Yes 735.41 No -0.89 AB2 Yes 456.00 Yes 413.00 No 4.00 AB3 Yes 535.78 Yes 133.45 No 12.34

[0601]

[0602] AB4 Yes 276.00 Yes 361.00 No 0.00 AB5 Yes 276.00 Yes 0.00 No 3.00 AB6 Yes 78.00 Yes 19.00 No 3.00 AB7 Yes 260.00 Yes 226.00 No 1.00 AB8 Yes 281.00 Yes 33.00 No 8.00 AB9 Yes 225.00 Yes -3.00 No 9.00 AB1O Yes 203.00 Yes -2.00 No 4.00 AB11 Yes 223.00 Yes 0.00 No 11.00 AB12 Yes 236.00 Yes 1.00 No 20.00 AB13 Yes 441.00 Yes 413.00 No 5.00 AB14 Yes 318.00 Yes 14.00 No 3.00 AB15 Yes 218.00 Yes 2.00 No 10.00 AB16 Yes 506.00 Yes 303.00 No 18.00 AB17 Yes 214.00 Yes 122.00 No 10.00 AB18 Yes 335.00 Yes 52.00 No 5.00 AB19 Yes 269.00 Yes 106.00 No 10.00 AB20 Yes 396.00 Yes 68.00 No 20.00 AB21 Yes 434.00 Yes 75.00 No 9.00 AB22 Yes 335.00 Yes 210.00 No 11.00 AB23 Yes 189.00 Yes 46.00 No 9.00 AB24 Yes 335.00 Yes 83.00 No 20.00 AB25 Yes 300.00 Yes 44.00 No 9.00 AB26 Yes 286.00 Yes 144.00 No 0.00 AB27 Yes 538.00 Yes 64.00 No 2.00 AB28 Yes 652.00 Yes 240.00 No 4.00 isotype

[0603] No -0.46 No 0.23 No 1.45 control

[0604] PAPPA2

[0605] positive

[0606] No n.a. No n.a. Yes 544.58 control

[0607] antibody

[0608]

[0609] AB34 was confirmed to bind to human PAPPA1 and was counter-screened for human PAPPA2 binding, essentially as described above, but with 2 nM PAPPA2 and overnight incubation. The Max % deltaF value for human PAPPA2 binding is shown in Table 6.

[0610] Table 6: Max % deltaF values for mAbs binding to PAPPA-2 in HTRF assay

[0611] Clone ID Human PAPPA2 Binding Max % deltaF (human PAPPA2) AB34 TM No 2.5

[0612] isotype control No 2.0

[0613] PAPPA2 positive control

[0614] Yes 843.25

[0615] antibody

[0616]

[0617] Example 5: Inhibitory activity of antibodies against PAPPA

[0618] Screening PAPPA-binding antibodies for inhibition of IGFBP4 cleavage mediated by PAPPA protease activity

[0619] To determine whether the substrate huIGFBP-4 undergoes cleavage by human PAPPA using an HTRF (Homogeneous Time-Resolved Fluorescence) assay, the N-terminal fragment of cleaved human IGFBP-4 was detected using a pair of antibodies (HyTest): IBP 144 binds to both full length huIGFBP-4 and the N-terminal fragment, IBP3cc binds only to the N-terminal fragment of cleaved huIGFBP-4. A FRET donor and receptor pair were obtained by labelling these detection antibodies with biotin and DyLight® 650 using Lightning-Link® fast conjugation kits (abeam). Streptavidin conjugated terbium cryptate (Revvity, 610SATLB) binds to the biotinylated detection antibody and served as a FRET donor fluorophore. The DyLight® 650 -labelled detection antibody acts as a FRET acceptor fluorophore, and thus FRET is detected when both antibodies bind to the huIGFBP- 4 N-terminal fragment following cleavage by PAPPA.

[0620] A decrease in PAPPA activity upon addition of inhibitory antibodies resulted in decreased formation of the cleaved huIGFBP-4 N-terminal fragment and consequently a lowered FRET signal.

[0621] Following overnight pre-incubation of huIGFBP-4 with a 4-fold excess of huIGF-1 (12.5nM and 50nM final concentrations, respectively), stock solutions of PAPPA (1.5nM final concentration) and combined detection antibodies (lOnM final concentration) / streptavidin conjugated terbium (InM final concentration), are prepared in assay buffer (lOOmM sodium-chloride, 50mM TRIS- hydrochloride and ImM calcium-chloride) and added to a 384-well microtitre plate, along with thesubstrate premix and sample antibody (final mab concentration in the range of 600-1300nM), and incubated for 5 hours at room temperature before transferring to 4°C overnight.

[0622] Time resolved fluorescence at 620 nm and 665 nm emission wavelengths were determined using a PHERAstar FSX plate reader (BMG Labtech). The 665 / 620 nm sample ratio followed by the % deltaF values for each sample were calculated, as follows (NSB denotes values achieved by the non-specific binding controls where substrate was omitted):

[0623] acceptor 665nm

[0624] sample ratio = — - - * 10000

[0625] aonor 620nm

[0626] sample ratio — mean NSB ratio

[0627] %deltaF = - - - * 100

[0628]

[0629] mean NSB ratio

[0630] Of the 28 PAPPA -binding antibodies identified from the HTRF binding screen, 20 antibodies demonstrated some degree of PAPPA protease activity inhibition. 7 antibodies exhibited a % inhibition of PAPPA-mediated IGFBP4 cleavage equal to or higher than 90%, and 6 antibodies exhibited complete inhibition of PAPPA-mediated IGFBP4 cleavage. Antibodies that gave a partial degree of inhibition at the concentration of PAPPA enzyme used in the assay protocol are shown in Table 7.

[0631] Table 7: % inhibition of IGFBP4 cleavage by PAPPA antibodies tested in a protease activity assay measuring the cleavage of IGFBP4 by PAPPA enzymes

[0632] Clone ID % inhibition [Final mAb] (nM)

[0633] AB 16 100 1279

[0634] AB 15 75.21 1136

[0635] AB 12 65.21 911

[0636] AB 13 30.32 1129

[0637] AB29 16.13 1184

[0638] AB 14 21.97 994

[0639] AB30 19.99 1098

[0640] AB9 52.37 1268

[0641] AB31 5.33 1158

[0642] AB5 90.33 772

[0643] AB 10 100.00 866

[0644] AB11 15.59 1091 AB32 8.98 1122

[0645] AB8 100.00 780

[0646]

[0647] AB4 65.30 668Clone ID % inhibition [Final mAb] (nM)

[0648] AB7 55.20 1090

[0649] AB 17 34.70 604

[0650] AB33 16.88 1050

[0651]

[0652] AB2 100.00 1000

[0653] Example 6: Affinity analysis

[0654] The affinities of antigen binding fragments (Fabs) of the human anti-PAPPA antibodies were measured using the Biacore 8K or Biacore 8K+ (Cytiva) at 25°C. The experiments were carried out using mature recombinant PAPPA protein or LG domain (LGD) fragments from the appropriate species (detailed in the tables below) with N or C-terminal Avi-His-tags. All species were either biotinylated using the Avi-tag or by chemical biotinylation using EZ link Sulfo-NHS-LC-Biotin (Thermo). Anti-PAPPA Fabs were obtained by either papain digestion or by using the FabALACTICA Fab Kit (Genovis).

[0655] Streptavidin (Thermo) was covalently immobilised to a C 1 chip (Cytiva) surface using standard amine coupling techniques at a concentration of 16 pg / ml in 10 mM Sodium acetate pH 5. Alternatively, EZ-Link Amine-PEG3-Biotin (Thermo) was coupled to a Cl chip at a concentration of 20 mg / ml in 10 mM acetate pH 4.0 using standard amine coupling. This enabled reversible immobilization of biotinylated PAPPA species using switchavidin (Tampere University / BioMediTech) (Laitinen et al., (2001), J. Biol. Chem. 276, 8219-8224; Pollheimer et al., (2013), Bioconjug. Chem. 24(10): 1656-68). Recombinant biotinylated PAPPA species were titrated onto the streptavidin or switchavidin chip surface in HBS-P+, ImM CaC12 or HBS-EP+ buffer to enable Fab binding.

[0656] The anti-PAPPA Fabs were serially diluted (0.123-10 nM or 1.23-100 nM) in HBS-P+, ImM CaC12 and injected in sequentially increasing concentrations (single cycle kinetics) over the chip at 30-60 ql / m incite. with 2 or 3 minute association steps and 10 or 30 minutes dissociation at the end of the complete binding cycle (Karlsson et al., (2006) Analytical Biochemistry 349:136-147). A buffer-only injection was made under the same conditions to allow for double reference subtraction of the final sensorgram sets. The reversible Switchavidin chip was regenerated using 2.5% citric acid, pH 2, 0.5% SDS. Fully corrected binding data were globally fitted to a 1:1 binding model using the Biacore Insight Evaluation Software (Cytiva).All tested antibodies showed high affinity binding to human PAPPA ranging from 10 nM to approximately 50 pM. Cross species affinity to cyno PAPPA was not maintained in all clones. AB1 has a high affinity against the LGD fragment of human PAPPA 1; however, this affinity is significantly reduced when mutation I147T is introduced into the LGD domain, demonstrating the crucial role of the residue on the binding. Biacore affinity results for select clones are provided in Table 8.

[0657] Table 8: Kinetics of anti-PAPPA Fabs binding to recombinant human and cynomolgus monkey

[0658] Fab Aa(M1s1) Ad (s1) AD (nM) PAPPA1 species N name Average SD Average SD Average SD Human 3.73E+06 8.00E+05 8.76E- 1.15E- 0.238 0.003 3

[0659] 04 04

[0660] AB1

[0661] Cyno 2.00E+06 4.84E+05 5.05E- 1.05E- 25.4 2.23 3

[0662] 02 02

[0663] Human 4.71E+05 1.44E+05 1.62E- 4.36E- 3.45 0.130 2

[0664] 03 04

[0665] AB3

[0666] Cyno 5.69E+05 3.12E+05 1.71E- 9.46E- 3.00 0.02 2

[0667] 03 04

[0668] Human 2.64E+05 5.99E+04 7.60E- 8.91E- 2.92 0.324 2

[0669] 04 05

[0670] AB10

[0671] Cyno 4.65E+05 1.02E+04 1.60E- 3.45E- 3.45 0.818 2

[0672] 03 04

[0673] Human 3.37E+04 3.56E- 10.6 1

[0674] 04

[0675] AB8

[0676] Cyno 1.61E+05 7.02E- 4.36 1

[0677] 04

[0678] Human 8.23E+05 8.76E+04 1.16E- 6.17E- 1.41 0.10 4

[0679] 03 05

[0680] AB16

[0681] Cyno 1.20E+06 1.77E+05 1.10E- 2.01E- 0.917 0.10 4

[0682] 03 04

[0683] Human 4.62E+05 5.75E+03 4.11E- 1.92E- 0.890 0.036 3 AB5

[0684] 04 05

[0685]

[0686] Cyno 4.08E+05 1.83E+04 3.62E- 3.48E- 0.892 0.123 3

[0687] 04 05

[0688] Human 3.69E+06 2.00E+05 7.62E- 6.00E- 0.207 0.02 6

[0689] 04 05

[0690] AB2

[0691] Cyno 1.71E+06 4.35E- 25.4 1

[0692] 02

[0693] LGD 6.67E+06 6.30E- 0.095 1

[0694] 04

[0695] AB1

[0696] Mutated LGD 1.19E+07 4.64E- 3.93 1

[0697] 02

[0698] AB34 Human 1.75E+06 5.55E+04 9.66E- 1.12E- 0.055 0.005 5

[0699] 05 05

[0700]

[0701] Biotinylated PAPPA was captured onto the chip surface and anti-PAPPA Fabs flowed over. SD = Standard Deviation

[0702] Affinity measurements of full-length IgGs for LGD fragments were performed on a Biacore 8K+ (Cytiva) at 25°C using a Series S Sensor Chip Cl (Cytiva, BR100535) surface prepared to support capture of oriented Fc capture of full-length anti-PAPPA IgG via Protein G' (PrG'). Running buffer was experiments was HBS-P+, ImM CaC12. Recombinant PAPPA LG domain (LGD) analytes from the appropriate species (as detailed in the accompanying tables) were diluted in the running buffer immediately prior to analysis.

[0703] Cl surfaces were prepared by using standard amine coupling techniques, covalently immobilizing PrG'. For capturing IgGs, PrG' (Sigma P4689) was diluted to 20 pg / ml in 10 mM sodium acetate, pH 4.0, to generate an oriented Fc capture surface suitable for reversible IgG capture and regeneration.

[0704] In this assay setup, full-length anti-PAPPA IgG was captured via Fc to the PrG' surface by injecting at 5 pl / min. Recombinant PAPPA LGD analyte was then injected at sequentially increasing concentrations (0.123-10 nM or 1.23-100 nM) in either multi-cycle kinetics or single-cycle kinetics at 40-60 pl / min, with association phases of 2-3 minutes and dissociation phases of 10-30 minutes. Between cycles, the PrG' surface was regenerated with two sequential injections of 6M guanidine -HC1. A buffer-only injection was made under the same conditions to allow for double reference subtraction of the final sensorgram sets. Fully corrected binding data were globally fitted to a 1:1 binding model using the Biacore Insight Evaluation Software (Cytiva).

[0705] All tested antibodies showed high affinity binding to LGD Human PAPPA1 ranging from 60 pM to 1.2 pM. Biacore affinity results for select clones are provided in Table 9.

[0706] Table 9: Kinetics of recombinant human and cynomolgus monkey LGD PAPPA binding to anti-PAPPA IgGs

[0707] IPAPPA ka (M-l s— 1) kd (s— 1) KD (M) Antibody name 1 n LGD Species Average SD Average SD Average SD Human 1.72E+07 3.42E+06 8.82E-04 1.10E-04 5.24E-11 8.90E-12 8 AB1

[0708] Cyno 2.01E+07 9.98E+06 7.76E-02 3.25E-02 4.12E-09 7.90E-10 4 Human 2.07E+07 3.73E+06 2.63E-05 6.73E-06 1.28E-12 2.52E-13 7 AB34

[0709] Cyno 1.76E+07 4.40E+06 7.59E-04 7.23E-05 4.68E-11 1.69E-11 8

[0710]

[0711] SD = Standard Deviation

[0712] Example 7 : Assessment of non-specific binding of antibodies to HEK293 cells

[0713] Non-specific binding of selected antibodies to HEK293 cells was assessed using a Mirrorball assay. Frozen HEK293 cells (ECACC) were rapidly thawed, added to 10 mL of assay medium containing 0.5% bovine serum albumin (PAA, K05-013) in Hanks Balanced Salt Solution (HBSS, Sigma, H8264), and collected by centrifugation (300g, 5 min, room temperature). The cells were resuspended in assay buffer to 2.5e5 cells / mL. An 8-point, 1 in 5 serial dilutions of test and control antibodies was performed in assay buffer, and 10 pL was added in duplicate to black 384well assay plates (Coming, 3655). 10 pL of a solution containing 16 nM AF647-labelled anti-human H+L (Invitrogen, A21445) was added to all assay plates. 20 pL of cells was added to one set of assay plates to assess non-specific cell binding.. The plates were sealed (Perkin Elmer, 6050185 Topseal) and incubated in the dark for 2 h at room temperature. The plates were read using a Mirrorball plate reader (TTP Labtech) in the FL3 channel (640 nm) at 4 pm resolution and 500 V. The signal was gated to remove low intensity background fluorescence based on the buffer only controls and high intensity fluorescence due to aggregation. As shown in Figure 10A and Figure 10C, the results show that none of the tested PAPPA mAbs show non-specific binding to HEK293 cells. Two control antibodies that are known to non-specifically bind to HEK cells are included as positive controls -Ctrl mAbl and Ctrl mAb2.Ill

[0714] Example 8: Assessment of propensity for self-association of antibodies

[0715] Propensity for self-association of selected antibodies was assessed using an affinity capture selfinteraction nanoparticle spectrometry (AC-SINS) assay. This method was adapted from the following sources: Sule et al, Mol. Pharmaceutics. 2013, 10, 1322-1331, and Liu et al, mAbs. 2014, 6(2), 483-492. 9mL gold nanoparticles (Sigma Aldrich, 753610) are coated with 400pg of a 1:4 mixture of whole goat IgG (Jackson ImmunoResearch, 005-000-003) and goat anti-human Fey (Jackson ImmunoResearch, 109- 005-098) in binding buffer (20 mM potassium acetate, pH 4.3) for 1-16 h at room temperature. The nanoparticles are blocked with 100 nM PEG (Sigma, 729140) for 1-2 h at room temperature, collected by centrifugation (13,000 rpm, 6 min, RT) and resuspended in 800 pL binding buffer. The OD of a 1:10 dilution of prepared nanoparticles should be 0.4-0.5 AU at 535 run. Test antibodies are diluted to 45 pg / mL in 108 pL of phosphate buffered saline (PBS, Invitrogen, 14190) or histidine-arginine buffer (20 mM histidine, 200 mM arginine, pH 6) in a 96 well polypropylene plate (Greiner, 650201). 12 pL of prepared gold nanoparticles are added to each well, mixed by pipetting, and the samples are incubated for 20 min at room temperature. The samples are transferred in duplicate to a Nunc 384 well assay plate (Thermo Fisher Scientific, 242757) and absorbance values between 450 run and 700 nm are detected using a PHERAstar plate reader (BMG Labtech). The wavelength at maximum absorbance is determined using MARS software 51 (BMG Labtech) and the redshift over the buffer control is calculated; a redshift >10 nm is indicative of self-association. As shown in Figure 10B and Figure 10D, the results show that none of the tested PAPPA mAbs has a risk of self-association. This indicates that the antibodies have favourable biophysical characteristics that would allow them to be formulated at a high concentration. Three control antibodies have been included- Ctrl mAbl negative control gives low signal in both buffers. Ctrl mAb2 - only gives a positive signal in PBS buffer and Ctrl mAb3 positive control that gives a significant signal in both buffers.

[0716] Example 9: Assessment of risk of fast in vivo clearance

[0717] The risk of fast in vivo clearance of selected antibodies was assessed using a baculovirus particle ELISA. This method was adapted from the following source: Hotzel et al, mAbs, 2012, 4(6), 753-60. 96 well Nunc maxisorp F plates (Thermo Fisher Scientific, 44-2404-21) are coated overnight at 4°C with either 50pL baculovirus particles at le8 particles / mL in sodium carbonate buffer (50 mM, pH 9.6), or 50pL sodium carbonate buffer (50 mM, pH 9.6) only. The assay plates are washed once with phosphate buffered saline (PBS), then blocked for 1 h at room temperature with 300 pL assay buffer consisting of 0.5% bovine serum albumin (BSA, Sigma, A9576) in PBS (Invitrogen, 14190).The plates are washed three times with PBS. Test antibodies are diluted to 10 nM and 100 nM in assay buffer, 50 pL of sample was added to the assay plates and incubated for 1 h at room temperature. The plates are washed three times with PBS, then incubated with 50 pL of a solution of HRP-conjugated anti-human Fey (1:5000 dilution, Sigma, A0170) for 30 min at room temperature. The plates are washed three times with PBS, incubated with 50 pL TMB SureBlue Reserve (KPL, 53-00-03) for 2-5 min and quenched with 50 pL 0.5 M sulfuric acid (H2SO4). The absorbance at 450 nm was measured on an EnVision plate reader (Perkin Elmer). The BV score was determined by averaging the absorbance values at both 10 nM and 100 nM sample, and dividing this by the buffer only control; a BV score above 5 indicates a risk of fast in-vivo clearance. As shown in Figure 11A and Figure 1 IB, the results show that none of the tested PAPPA mAbs shows a risk of fast clearance due to non-specific binding to baculovirus particles. Three control antibodies have been included- Ctrl mAbl positive control that gives a significant signal in plates both coated and non-coated with BV particles. Ctrl mAb2 that only binds to coated BV particles and Ctrl mAb3 as negative control.

[0718] Example 10: AB1 inhibits PAPPA-induced AKT phosphorylation

[0719] MCF cells treated with IGF1 at 50 ng / mL exhibited upregulation in the phosphorylation of AKT at both phosphorylation sites: Ser473 and Thr308. However, the introduction of IGFBP4 in the treatment, which was mixed with IGF1, resulted in the blockade of AKT phosphorylation. The addition of PAPPA to the mixture of IGF1 and IGFBP4 successfully rescued the AKT phosphorylation. Furthermore, the introduction of the PAPPA tool mAb or AB 1 into the mixture of PAPPA, IGF1, and IGFBP4 re-blocked the PAPPA-induced AKT phosphorylation, while the introduction of isotype control mAb showed no effect (FIG 12).

[0720] Example 11: AB1 decreases free PAPPA levels in cynomolgus plasma

[0721] 2 male naive (1093 and 1101) and 2 male non-naive (1105 and 1106) cynomolgus monkeys aged 2-4 years were housed and maintained according to established procedures with access to 200 g / day of LabDiet 5007 (PMI Nutrition International) diet and mains quality tap water ad libitum throughout the study. Animals in Group 1 (1101 and 1105) were administered AB1 using a first dosing regimen. Animals in Group 2 ( 1093 and 1106) were administered AB 1 using a second dosing regimen. Blood samples were collected into tubes containing K2EDTA anticoagulant from the femoral or cephalic vein by venipuncture. Immediately following collection, blood samples were inverted to ensure mixing and placed on wet ice. As soon as practically possible, samples were centrifuged (3000 rpm, 10 min, 4°C) and the resultant plasma aliquoted for subsequent analyses.Measurement of AB 1 concentrations in cynomolgus monkey plasma

[0722] The plasma concentration of AB 1 was measured by an in-house developed ligand-binding universal PK assay on Gyrolab. The mAb was captured using a mouse anti-human IgG monoclonal antibody and detected using Alexa-647 labelled sheep anti-human IgG.

[0723] Free PAPPA levels in cynomolgus monkey plasma

[0724] Levels of free PAPPA (not bound to compound) in circulation was used as a marker of target engagement. The assay to determine free PAPPA levels in plasma consisted of two steps, first PAPPA bound to the compound was removed by immunoprecipitation, using magnetic beads capturing the IgM-TM of the compound, whereas the unbound “free” PAPPA remained in the plasma supernatant. Thereafter, levels of free PAPPA were determined in the supernatants using an in-house developed SIMOA® assay.

[0725] Results

[0726] Administration of AB1 to cynomolgus monkeys at two different dose regimens resulted in a reduction of free PAPPA levels (FIG. 13A) with a clear exposure-response relationship (FIG. 13B), confirming target engagement in vivo with AB1.

[0727] Example 12: Epitope binning of antibodies against PAPPA

[0728] Epitope binning using BLI (Bio-layer interferometry) was conducted to identify any overlapping epitopes in PAPPA. A series of inhibitor antibodies, isotype controls and blocking IgGs were diluted to lug / mL in assay buffer (PBS + 0.1% BSA + 0.02% Tween-20) and human PAPPA was diluted to lOOnM in the same assay buffer. 60uL of each of the antibody and PAPPA solutions were dispensed into an octet plate. Octet analysis was carried out using AHC2 biosensors which were exposed to the different solutions in the following order: Buffer / primary mAb / Isotype control / buffer / huPAPPA / buffer / competitor mAb / buffer. A signal was observed when the tested mAbs had independent epitopes, but not when the epitopes were overlapping.

[0729] Alternatively, antibodies were diluted to 100 nM and biotinylated human PAPPA was diluted to 5 ug / ml in assay buffer. 70uL of each of the antibody and biotinylated huPAPPA solutions were dispensed into an octet plate. Octet analysis was carried out using SAX 2.0 biosensors which were exposed to the different solutions in the following order: Buffer / Biotin-huPAPPA solution / buffer / primary mAb / buffer / competitor mAb. A signal was observed in the competitor mAB binding step when the tested mAbs had independent epitopes, but not when the epitopes were overlapping.

[0730] This process was conducted with AB 1, AB2, AB7, AB8, AB9, AB 12, AB 16, AB34 TM, AB34 TM YTE and tool PAPPA mAb and the results are shown in Table 10 below.

[0731] Table 10 Inhibitory antibodies grouped by shared epitope

[0732] Group Clone ID

[0733] 1 AB1, AB2, AB6, AB7, AB34 TM, AB34 TM YTE

[0734] 2 AB8

[0735] 3 AB9

[0736] 4 AB 12

[0737] 5 AB 16, Tool PAPPA mAb

[0738]

[0739] Example 13: Determining whether STC-1 competes for binding to PAPPA with a PAPPA antibody

[0740] Competition experiments using BLI (Bio-layer interferometry) were conducted to determine overlapping epitopes with STC-I. A series of inhibitor antibodies, isotype controls and STC-I were diluted to 25ug / mL in assay buffer (PBS + 0.1% BSA + 0.02% Tween-20) and biotinylated PAPPA was diluted to lOug / mL in the same assay buffer. 60uL of each of the antibodies, STC-I and PAPPA solutions were dispensed into an octet plate. Octet analysis was carried out using SAX 2.0 biosensors which were exposed to the different solutions in the following order: Buffer / PAPPA solution / buffer / STC-I solution / buffer / inhibitory mAb solution / buffer. A signal was observed where STC-I and the mAb had independent epitopes; no signal was observed where the mAb and STC-I had overlapping epitopes. Figure 16A is an example of the results obtained for the described process for tool PAPPA mAb and AB1, where no signal was observed. This process was conducted with AB1, AB2 AB5, AB8, AB9, AB10, AB12, AB15, AB16 and tool PAPPA mAb and the results are shown in Table 11 below.

[0741] Alternatively, a series of inhibitor antibodies & isotype controls were diluted to 100 nM and STC-I was diluted to 300 nM in assay buffer. Biotinylated huPAPPA was diluted to 5ug / mL in the same assay buffer. 70uL of each of the antibodies, STC-I and Biotin-huPAPPA solutions were dispensed into an octet plate. Octet analysis was carried out using SAX 2.0 biosensors which were exposed tothe different solutions in the following order: Buffer / Botin-huPAPPA solution / buffer / STC-I solution / buffer / inhibitory mAb solution. A signal was observed in the inhibitory mAB binding step where STC-I and the mAb had independent epitopes; no signal was observed where the mAb and STC-I had overlapping epitopes. This process was conducted with AB1, AB34 TM, AB34 TM YTE, tool PAPPA mAb and a PAPPA benchmark mAb and the results are shown in Figure 16B and Table 11 below.

[0742] Table 11 Inhibitory antibodies grouped by competitor properties to STC-1

[0743] STC-I competitors Clone ID

[0744] Yes AB 16, Tool PAPPA mAb,

[0745] Benchmark PAPPA mAb

[0746] No AB1, AB2 AB5, AB8, AB9, AB10,

[0747] AB12, AB15, AB34 TM, AB34

[0748] TM YTE

[0749]

[0750] Example 14: Binding to PAPPA LG domain

[0751] Binding to the LG domain of PAPPA was investigated using BLI ((Bio-layer interferometry). A series of inhibitor antibodies and isotype controls were diluted to 25ug / mL in assay buffer (PBS + 0.1% BSA + 0.02% Tween-20) and biotinylated PAPPA [82-324] was diluted to lOug / mL in the same assay buffer. 60uL of each of the antibodies, and PAPPA[82-324] solutions were dispensed into an octet plate. Octet analysis was carried out using SAX 2.0 biosensors which were exposed to the different solutions in the following order: Buffer / PAPPA [82-324] solution / buffer / inhibitory mAb solution / buffer. A binding signal was observed where the antibody bound to the PAPPA LGD i.e., its epitope was within the domain. This process was conducted with AB 1, AB2 and tool PAPPA mAb and the results are shown in Table 12 below. Figure 15A is a representation of the BLI traces obtained when PAPPA LGD was exposed to AB1 and tool PAPPA mAb. A binding signal between AB 1 and PAPPA LGD was observed while no binding was observed for tool PAPPA mAb or the control mAb.

[0752] Alternatively, antibodies were diluted to 5ug / mL in assay buffer. huPAPPA [82-324] was diluted to 50 nM in the same assay buffer & subsequently subjected to a 7-point 2-fold titration. 70uL of each of the antibodies, and huPAPPA[82-324] solutions were dispensed into an octet plate. Octet analysis was carried out using AHC 2.0 biosensors which were exposed to the different solutions in the following order: Buffer / inhibitory mAb solution / buffer / PAPPA [82-324] solution / buffer.A binding signal was observed where the antibody bound to the PAPPA LGD i.e., its epitope was within the domain. This process was conducted with AB34 TM, AB34 TM YTE and the PAPPA tool mAb and the results are shown in Table 12 below. Figure 15B is a representation of the BLI traces obtained when AB34 TM, AB34 TM YTE and tool PAPPA mAb were exposed to huPAPPA LG domain. A binding signal between AB34 and PAPPA LGD was observed while no binding was observed for tool PAPPA mAb or the control mAb.

[0753] Table 12 Antibodies grouped by binders to PAPPA LGD

[0754] Binders to PAPPA Clone ID

[0755] LGD

[0756] Yes AB1, AB2, AB34 TM, AB34

[0757] TM YTE

[0758] No Tool PAPPA mAb

[0759]

[0760] Example 15: Cryo-EM Structure Determination of AB2 and PAPPA

[0761] 3 pl of the PAPPA(82- 1183): : fab complex was applied on to a freshly glow-discharged holey gold grid (UltrAuFoil Rl.2 / 1.3) at 4 °C, 100% humidity in a Vitrobot Mk IV system (Thermo Fisher Scientific). The grids were immediately blotted for 3.5 sec prior to plunge cooling in liquid ethane.

[0762] Cryo-EM micrographs were acquired on a Titan Krios G4i (Thermo Fisher) operated at 300 kV and equipped with a bottom-mounted Falcon 4 detector. Movies were acquired in one continuous session in an automated manner using EPU software at 120,000x nominal magnification. Exposures of 4.98 s were taken resulting in a total dose of 60 electrons / A2, with defoci values ranging from -0.8 to -2.4 pm. A total of 11,117 movies were acquired and were pre-processed using the CryoSPARC Live package. Images with substantial drift, astigmatism, or poor CTF fit were rejected, and as a result, 10, 187 curated motion-corrected averages were used for particle extraction. Initially 1.6 million particles were extracted by template picking.

[0763] Subsequent steps were performed in CryoSPARC. In brief, particles were aligned and averaged using reference-free 2D class averaging where falsely picked non-particles were rejected based on visual appearance. This resulted in 355,739 particles which were subjected to a 3-class ab initio reconstruction job, which found a subset of 131,158 particles that corresponded to the complex of interest. These particles were subjected to non-uniform refinement, resulting in a 3.4 A resolution reconstruction in which an atomic model could be fitted.Figure 14 is a representation of the resulting reconstruction depicting PAPPA[82-1183] bound to the Fab of AB2. The reconstruction also depicts the binding epitope for AB2, located on the LGD domain of PAPPA. The model suggests two distinct epitopes, Q131-D150 and I227-R250, as the main binding sites for AB2.

[0764] Example 16: Inhibition of IGFBP4 cleavage via inhibition of native PAPPA

[0765] The ability of antibodies to inhibit recombinant IGFBP4 cleavage via inhibition of native PAPPA was investigated using the human ADPKD cystic cell line WT9-12.

[0766] Cellular assay for cleaved IGFBP4

[0767] WT 9-12 (CRL-2833, ATCC), a human ADPKD cell line, were maintained in DMEM (31966, Gibco, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (A5256801, Gibco, Thermo Fisher Scientific) at 37°C in a humidified incubator with 5% CO2. For experiments, cells were seeded at 35,000 cells / cm2in 96-well tissue culture plates (Costar 3595, Coming). Cells were allowed to adhere and grow for 96 h. After 96 h, growth medium was removed and replaced with serum-free, specialized medium DMEM / F-12 (30-2006, ATCC) supplemented with the hTERT-immortalized RPTEC Growth Kit (ACS-4007, ATCC). PAPPA antibody was added at a 10-point dose range (pM to nM) and cells were pre-treated for 1 h. An IGFBP4:IGF-I complex (IGFBP4: 804-GB-025; IGF-I: 291-G1-200; R&D Systems) was pre-incubated for 30 min at 20 x the intended final concentration and then added to cells to achieve a final concentration of 800 ng / mL. After 72 h incubation, conditioned media were collected and stored at -80°C until analysis of cleaved IGFBP4 levels.

[0768] Cleaved IGFBP4 measurements using MSI) assays

[0769] Cleaved IGFBP4 levels in conditioned media were measured by in-house developed ligand-binding MSD immunoassays. The cleaved N-terminus (NT)-fragments were captured to the bottom of MSD-plates by antibodies specific for the neo-epitopes that are exposed after PAPPA -mediated cleavage of IGFBP4. Detection antibodies recognizing the NT-fragments of IGFBP4 and ECL reagents were used to generate a signal. In house generated recombinant NT IGFBP4 proteins (mouse or human) were used as calibration curves. The assay was qualified with regards to specificity, precision, recovery and parallelism according to internal standards.Results

[0770] Figure 17 shows that treatment with anti-PAPPA mAbs AB1, AB34 TM, AB34 TM YTE and the PAPPA tool antibody leads to concentration-dependent inhibition of IGFBP4 cleavage from the recombinant IGFBP4:IGF1 complex by native PAPPA in ADPKD cystic cells.

[0771] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present invention has been described in connection with specific preferred instances, it should be understood that the invention as claimed should not be unduly limited to such specific instances. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

CLAIMS1. A binding molecule that binds to pregnancy-associated plasma protein A (PAPPA) and comprises a heavy chain variable (VH) region having a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable (VL) region having a set of CDRs LCDR1, LCDR2 and LCDR3, wherein:(a) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 108 or 122 (e.g., SEQ ID NO: 108), HCDR2 of SEQ ID NO: 109 and HCDR3 of SEQ ID NO: 110, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113;(b) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 1 or 121 (e.g., SEQ ID NO: 121), HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6;(c) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 9 or 121 (e.g., SEQ ID NO: 121), HCDR2 of SEQ ID NO: 10 and HCDR3 of SEQ ID NO: 11, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13 and LCDR3 of SEQ ID NO: 14;(d) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 17, HCDR2 of SEQ ID NO: 18 and HCDR3 of SEQ ID NO: 19, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21 and LCDR3 of SEQ ID NO: 22;(e) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 25, HCDR2 of SEQ ID NO: 26 and HCDR3 of SEQ ID NO: 1 and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 29 and LCDR3 of SEQ ID NO: 30;(f) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 33, HCDR2 of SEQ ID NO: 34 and HCDR3 of SEQ ID NO: 35, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 37 and LCDR3 of SEQ ID NO: 38;(g) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 41, HCDR2 of SEQ ID NO: 42 and HCDR3 of SEQ ID NO: 43, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 44, LCDR2 of SEQ ID NO: 45 and LCDR3 of SEQ ID NO: 46; or(h) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 49, HCDR2 of SEQ ID NO: 50 and HCDR3 of SEQ ID NO: 51, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 52, LCDR2 of SEQ ID NO: 53 and LCDR3 of SEQ ID NO: 54.

2. The binding molecule of claim 1, wherein:(a) the VH region amino acid sequence comprises SEQ ID NO: 114, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprises SEQ ID NO: 115, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;(b) the VH region amino acid sequence comprises SEQ ID NO: 7, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprises SEQ ID NO: 8, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;(c) the VH region amino acid sequence comprises SEQ ID NO: 15, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprises SEQ ID NO: 16, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;(d) the VH region amino acid sequence comprises SEQ ID NO: 23, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprises SEQ ID NO: 24, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;(e) the VH region amino acid sequence comprises SEQ ID NO: 31, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprises SEQ ID NO: 32, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;(f) the VH region amino acid sequence comprises SEQ ID NO: 39, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprises SEQ ID NO: 40, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto;(g) the VH region amino acid sequence comprises SEQ ID NO: 47, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprises SEQ ID NO: 48, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; or(h) the VH region amino acid sequence comprises SEQ ID NO: 55, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; andthe VL region amino acid sequence comprises SEQ ID NO: 56, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

3. The binding molecule of claim 1 or claim 2, wherein:(a) the VH region amino acid sequence comprises SEQ ID NO: 114 and the VL region amino acid sequence comprises SEQ ID NO: 115;(b) the VH region amino acid sequence comprises SEQ ID NO: 7 and the VL region amino acid sequence comprises SEQ ID NO: 8;(c) the VH region amino acid sequence comprises SEQ ID NO: 15 and the VL region amino acid sequence comprises SEQ ID NO: 16;(d) the VH region amino acid sequence comprises SEQ ID NO: 23 and the VL region amino acid sequence comprises SEQ ID NO: 24;(e) the VH region amino acid sequence comprises SEQ ID NO: 31 and the VL region amino acid sequence comprises SEQ ID NO: 32;(f) the VH region amino acid sequence comprises SEQ ID NO: 39 and the VL region amino acid sequence comprises SEQ ID NO: 40;(g) the VH region amino acid sequence comprises SEQ ID NO: 47 and the VL region amino acid sequence comprises SEQ ID NO: 48; or(h) the VH region amino acid sequence comprises SEQ ID NO: 55 and the VL region amino acid sequence comprises SEQ ID NO: 56.

4. The binding molecule of any one of the previous claims, wherein the binding molecule does not bind to PAPPA-2.

5. The binding molecule of any one of the previous claims, wherein:(a) the binding molecule binds to human PAPPA; and / or(b) the binding molecule binds to cynomolgus PAPPA.

6. The binding molecule of any one of the previous claims, wherein:(a) the binding molecule comprises an Fc region;(b) the binding molecule comprises an Fc region and wherein the binding molecule comprises a L234F / L235E / P33 IS triple mutation in the Fc region; or(c) the binding molecule comprises an Fc region and wherein the binding molecule comprises a L234F / L235E / P331S triple mutation in the Fc region and M252Y / S254T / T256E mutations in the Fc region.1227. The binding molecule of any one of the previous claims, wherein the binding molecule is an anti-PAPPA antibody, or an antigen-binding fragment thereof.

8. The binding molecule of claim 7, wherein the anti-PAPPA antibody, or antigen-binding fragment thereof is a human antibody, or an antigen-binding fragment thereof.

9. The binding molecule of claim 7 or claim 8, wherein the anti-PAPPA antibody, or antigenbinding fragment thereof is a monoclonal antibody, or an antigen-binding fragment thereof.

10. The binding molecule of any one of claims 7-9, which is:(a) an IgG immunoglobulin or an antigen-binding fragment thereof; or(b) an IgGl immunoglobulin or an antigen-binding fragment thereof.

11. An isolated polynucleotide encoding the binding molecule of any one of claims 1-10.

12. A vector comprising:(a) the polynucleotide of claim 11 ; or(b) a polynucleotide encoding the VH region as defined in any one of claims 1-3, and a polynucleotide encoding the VL region as defined in any one of claims 1-3.

13. A host cell comprising the polynucleotide of claim 11 or the vector of claim 12.

14. A method of producing a binding molecule of any one of claims 1-10 comprising expressing a polynucleotide of claim 11 or a vector of claim 12 in a host cell.

15. A pharmaceutical composition comprising the binding molecule of any one of claims 1-10, and a pharmaceutically acceptable carrier or diluent.

16. A method of treating a PAPPA-mediated disease, disorder or condition in a subject, the method comprising administering to the subject the binding molecule of any one of claims 1-10, or the pharmaceutical composition of claim 15.

17. The method of claim 16, wherein the PAPPA-mediated disease is autosomal dominant polycystic kidney disease (ADPKD).

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