Activating EPHA2 antigen-binding agents and uses thereof

Abstract

Disclosed herein are antigen-binding molecules, T cell receptors (TCR) or chimeric antigen receptors (CAR) that can at least specifically recognize or bind to EphA2. In particular, disclosed herein are antigen-binding molecules that can activate EphA2. Also disclosed are methods of medical treatment and prophylaxis.

Description

TITLE OF THE INVENTIONACTIVATING EPHA2 ANTIGEN-BINDING AGENTS AND USES THEREOFRELATED APPLICATIONS

[0001] This application claims priority to Australian Provisional Patent Application No. 2024903184 entitled “Activating EphA2 Antigen-Binding Agents and Uses Thereof’ filed 2 October 2024, the contents of which are incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present invention relates to an antigen-binding molecules, T cell receptors (TCR) or chimeric antigen receptors (CAR) that can at least specifically recognize or bind to EphA2. In particular, the antigen-binding molecules activate EphA2. The present invention also relates to methods of medical treatment and prophylaxis.BACKGROUND

[0003] Ephrin receptors (Eph) represent the most important class of receptor tyrosine kinases (RTKs). The first Eph receptor described, EphA1 , was identified in liver cancer cells while screening for RTKs in 1987 (Hirai H, et al., A novel putative tyrosine kinase receptor encoded by the EPH gene. Science. 1987;238(4834):1717-20). Since then, 14 Eph receptors and eight related ligands (ephrins) have been identified in the human genome. The Eph family of RTKs have been implicated in normal development and disease including many human cancers (Kania A, Klein R. Mechanisms of ephrin- Eph signalling in development, physiology and disease. Nat Rev Mol Cell Biol 2016; 17: 240-256). Numerous studies have shown that Eph receptors can function in either oncogenic or tumoursuppressive capacities (Miao H, Wang B. EphA receptor signalling-complexity and emerging themes. Semin Cell Dev Biol 2012; 23: 16-25 and Pasquale EB. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer 2010; 10: 165-180).

[0004] It is now established that oncogenic functions typically occur in a kinase-independent manner, involving Eph receptor crosstalk with known oncogenic and mitogenic receptors. In comparison, Eph tumour-suppressive functions typically occur in a kinase-dependent, ephrin ligand- induced manner. These dual receptor functions add significant complexity to the biology of Eph family RTKs, particularly in the context of cancer. Despite this complexity, Eph and ephrins remain viable cell surface anti-cancer targets.

[0005] EphA2, originally termed ECK, was one of the first Eph receptors to be described and has been intensively studied in both development and disease (Hirai H, et al., A novel putative tyrosine kinase receptor encoded by the eph gene. Science. 1987;238(4834):1717-20). In particular, EphA2 has been implicated in the pathogenesis of many human cancers, where increasing receptor expression levels act in concert with reduced kinase function to promote tumour progression (Wykosky J, Debinski W. The EphA2 receptor and ephrinAI ligand in solid tumours: function and therapeutic targeting. Mol Cancer Res 2008; 6: 1795-1806). Furthermore, EphA2 has been shown to broadly promote oncogenesis by contributing to chemoresistance, self-renewal, and angiogenesis.

[0006] Prostate cancer remains a significant disease for which more effective tumour-specific therapies are needed to treat aggressive disease. EphA2 is expressed at high levels in prostate adenocarcinoma compared to benign prostatic epithelium, where its expression is low. In addition, high EphA2 expression has been shown to predict poor prognosis following radical prostatectomy in prostate cancer patients (Kurose H, et al. Elevated expression of EphA2 is associated with poor prognosis after radical prostatectomy in prostate cancer. Anticancer Res 2019; 39: 6249-6257 and Sachdeva A, et al. Non-canonical EphA2 activation underpins PTEN-mediated metastatic migration and poor clinical outcome in prostate cancer. Br J Cancer 2022; 127: 1254-1262). These studies imply that EphA2 primarily functions as an oncogene in the context of this disease. Mechanistically, EphA2 binds to the high-affinity ligand ephrin-A1 , leading to receptor-ligand cluster formation, resulting in kinase activation and downstream signalling events (Bartley TD, et al. B61 is a ligand for the ECK receptor proteintyrosine kinase. Nature 1994; 368:558-560 and Himanen JP, et al. Architecture of Eph receptor clusters. Proc Natl Acad Sci U S A 2010; 107:10860-10865).

[0007] A seminal study by Wang and colleagues found that activation of EphA2 inhibited prostate cancer cell migration, whereas receptor overexpression promoted migration in a ligandindependent, non-canonical manner (Miao H, et al. EphA2 mediates ligand-dependent inhibition and ligand-independent promotion of cell migration and invasion via a reciprocal regulatory loop with Akt. Cancer Cell 2009; 16: 9-20). This migratory, pro-oncogenic effect requires the phosphorylation of EphA2 on Serine 897 via crosstalk with AKT. Functionally, it appears that EphA2 activation inhibits AKT and ERK signalling in a reciprocal loop, diminishing EphA2-S897 phosphorylation and causing receptor-mediated endocytosis and EphA2 degradation. Such augmentation of EphA2 receptor activation against cancer may offer an attractive alternative to current treatment strategies.SUMMARY OF THE INVENTION

[0008] The present invention is broadly directed to an EphA2-activating antigen-binding molecule, inclusive of a human or humanized, recombinant EphA2 antibody, and methods of using the same.

[0009] In one aspect, the present invention generally provides an EphA2-binding molecule that activates (or otherwise enhances) EphA2. In some embodiments of this type, the EphA2-binding molecule comprises three heavy chain complementarity determining regions (CDR-H1 , CDR-H2, and CDR-H3) and three light chain complementary determining regions (CDR-L1 , CDR-L2, and CDR-L3) having an amino acid sequence as set forth in Table 1 and Table 2, respectively; or at least 85% identical thereto:TABLE 1CLONE 4B3 HEAVY CHAIN CDR1 , CDR2 AND CDR3 AMINO ACID SEQUENCESHFR = Heavy chain framework regionTABLE 2CLONE 4B3 LIGHT CHAIN CDR1 , CDR2 AND CDR3 AMINO ACID SEQUENCESLFR = Light chain framework region

[0010] In some embodiments, the activating EphA2-binding molecule comprises the VH polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 71 or an amino acid sequence at least 85% identical thereto; and / or the VL polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 72 or an amino acid sequence at least 85% identical thereto. In some embodiments the activating EphA2-binding molecule is an antibody or antibody fragment. Preferably, in some embodiments the activating EphA2-binding molecule comprises a heavy chain amino acid sequence and light chain amino acid sequence as set forth, below:Ab clone 4B3-VH heavy chain amino acid sequenceEVQLLQSGPELVKPGASVKLSCKASGYTFTDYDMNWMKQSHGKSLEWIGDLNPNNGGASYNQKFRG KATLTVDKSS STAYMELRSLTSEDSAVYYCARMGMTYFDYWGQGTTLTVS SAKTTAPSVYPLAPVC GGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLS SSVTVTSNTWPSQTIT CNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFI FPPKI KDVLMI SLSPMV TCVWDVSEDDPDVQI SWFVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNN RALPSPI EKTI SKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSDGRTEQNY KNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSWHEGLHNHLTTKTI SRSLGK [ SEQ ID NO : 71 ]Ab clone 4B3-VL light chain amino acid sequenceHSVLTKS PAIMSAS PGEKVTI SCSASS SVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGS GTSYSLTI STMEAEDAATYYCQQYHSYPLTFGAGTKLELKGVADAAPTVS I FPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQDGVLNSWTDQDSKDSTYSMS STLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC [ SEQ I D NO : 72 ] .

[0011] In some embodiments the EphA2-binding molecule is a recombinant, human or humanized antibody or antibody fragment.

[0012] In another aspect of the invention, the EphA2-binding molecule is a T cell receptor (TCR) comprising a CDRH1 that comprises an amino acid sequence set forth in SEQ ID NOs: 1-5, CDRH2 that comprises an amino acid sequence set forth in SEQ ID NOs: 6-10, and CDRH3 that comprises an amino acid sequence set forth in SEQ ID NO: 11-15 and a CDRL1 that comprises an amino acid sequence set forth in SEQ ID NO: 16-20, CDRL2 that comprises an amino acid sequence set forth in SEQ ID NO: 21-25, and CDRL3 that comprises an amino acid sequence set forth in SEQ ID NO: 26-30 or at least 85% identical thereto.

[0013] In some embodiments, the EphA2-binding molecule activates EphA2.

[0014] In yet another aspect of the invention, the antigen-binding molecule is a chimeric antigen receptor (CAR) that comprises an EphA2-binding domain.

[0015] In some aspects, the invention provides an isolated nucleic acid encoding the EphA2- binding molecule. In another aspect, the invention provides a genetic construct comprising an isolated nucleic acid. In some embodiments, the nucleic acid is an expression vector.

[0016] In some embodiments, the invention provides a cell comprising an isolated nucleic acid or an expression vector. In another aspect of the invention, a host cell comprises an isolated nucleic acid and / or a genetic construct and / or an expression vector. In yet another aspect, the host cell is or comprises a T cell.

[0017] In still yet another aspect, provided herein is a method of producing an isolated EphA2- binding molecule comprises culturing the host cell and isolating said EphA2-binding molecule from the cultured host cell. In some embodiments the method comprises culturing a cell comprising a nucleic acid or an expression vector, under conditions suitable for expression of the EphA2-binding molecule from the nucleic acid(s) or expression vector(s).

[0018] In another aspect, the invention provides a composition comprising the EphA2-binding molecule and a pharmaceutically acceptable carrier diluent or excipient. In some embodiments, the composition additionally comprises an immunotherapeutic agent, such as a checkpoint inhibitor.

[0019] In another aspect, provided herein is a method of treating or preventing cancer in a subject, said method including the step of administering a therapeutically effective amount of the activating EphA2 antigen-binding molecule, as described above and / or elsewhere herein, to the subject.

[0020] In some aspects, provided herein is the use of the activating EphA2 antigen-binding molecule in the manufacture of a medicament for the prevention and / or treatment of a cancer in a subject. In some embodiments, the cancer is a solid tumour. In a preferred embodiment, the solid tumour is prostate cancer.

[0021] In some embodiments of these types, the activating EphA2 antigen-binding molecule is the 4B3 antibody clone as described above and elsewhere herein.

[0022] In certain aspects, provided herein is a method of detecting EphA2 or a cell expressing EphA2. In some embodiments, the method includes the step of forming a complex between the activating EphA2-binding molecule and EphA2 to thereby detect EphA2 or the cell expressing EphA2. In some embodiments the cell is or comprises a cancer cell.

[0023] In some aspects, the invention provides an isolated nucleic acid comprising, consisting or consisting essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 73-78 or a nucleic acid sequence at least 85% identical thereto. In some preferred embodiments, the present invention provides an isolated nucleic acid comprising each of the nucleotide sequences set forth in SEQ ID NOs: 73-78.BRIEF DESCRIPTION OF THE FIGURES

[0001] Figure 1 shows EphA expression in prostate cancer cell lines. (A) Expression levels of EphA receptor tyrosine kinases and ephrin-A (efnA) ligands in prostate cancer cell lines relative to the house-keeping gene -actin were determined by quantitative real-time PCR (QPCR). PC-3 cells, its metastatic derivatives PC-3M and PC-3MM2 and DU145 prostate cancer cells express high levels of EphA2 but show little to no expression of any of the other EphA receptors and ephrin-A ligands. (B) EphA2 and EphA3 expression was confirmed by western blot analysis. -actin is the loading control. (C) Immunofluorescence staining of EphA2 (green) in PC-3 cells shows expression of the receptor on the cell surface. Nuclei were labelled with DAPI (blue). Scale bar represents 20 pm. (D) Western blot showing EphA2 receptor phosphorylation and inhibition of AKT, MEK / ERK and FAK / SRC signalling after stimulation with 1 pg / mL clustered ephrin-A1-Fc (efnA1-Fc) in PC-3 prostate cancer cells. Extended time course of data shown in Figure 1A. EphA2 receptor degradation became apparent after 3 hours of efnA1-Fc stimulation. At the same time phosphorylated EphA2 receptor levels alsodecreased, while inhibited downstream pathways continued to return to baseline levels. -actin was used as the housekeeping loading control in conjunction with total protein controls of tested phosphoproteins.

[0002] Figure 2 shows activation of EphA2 receptors causes rearrangement of the cytoskeleton, retraction of cell protrusions and cell rounding. (A) Western blot analysis showing EphA2 receptor phosphorylation and inhibition of AKT, MEK / ERK and FAK / SRC signalling after stimulation with 1 pg / mL clustered ephrin-A1-Fc (efnA1-Fc) in PC-3 prostate cancer cells. (B), (C) Confocal microscopy images of the F-actin cytoskeleton labelled with rhodamine-phalloidin (red) in PC-3 cells in response to stimulation with clustered efnA1-Fc compared to Fc control in the absence (B) and the presence (C) of Cytochalasin D (CytoD). Cell nuclei were labelled with DAPI (blue). Cell outlines, based on corresponding brightfield images, are indicated as white lines in the CytoD-treated cells. (D) Confocal microscopy images of microtubules (a-tubulin, green) in unstimulated and efnA1 -Fc-treated PC-3 cells. Cell outlines and nuclei are visualized using F-actin staining (rhodamine-phalloidin, red) and DAPI (blue), respectively. Anisotropy of microtubules was quantified using the Imaged plugin FibrilTool. Data represent means ± SE (n>40 individually analysed cells). ***p<0.001 (unpaired t-test). (E) Quantification of PC-3 cell adhesion to uncoated, fibronectin-, Matrigel-, clustered Fc-control- or efnA1 -Fc-coated cell culture surfaces. Data represent means ± SE (n = 4 independent experiments). *p<0.05, ***p<0.001 , ns. - not significant (repeated-measure ANOVA with post-hoc Tukey’s multiple comparisons tests). (F) Fluorescent microscopy images of PC-3 cell adhesion to glass coverslips coated in alternating stripes of clustered efnA1 -Fc (10 pg / mL) and either clustered Fc control (10 pg / mL) or Matrigel (1 :100) as an alternative substrate for adhesion. Cells were visualized with F-actin (rhodamine-phalloidin, red) and DAPI (blue) labelling. (G), (H) Time-lapse brightfield microscopy images of PC-3 cell invasion into Matrigel containing either 1 pg / mL Fc control or efnA1-Fc. Initial cell- to-Matrigel boundary at t = 0 is indicated as a black line. Coloured asterisks and arrow heads follow individual cells and cell protrusions, respectively, over time.

[0003] Figure 3 shows characterization of EphA2-specific monoclonal antibodies 1 F7 and 4B3 in vitro and in vivo. (A) Binding kinetics of EphA2-specific mAbs to EphA2 using Bio-Layer Interferometry technology. The EphA2-specific mAbs (i.e., the activating EphA2 antibody 4B3, and the inhibitory EphA2 antibody 1 F7) and recombinant ephrin-A1-Fc (efnA1) were sequentially bound to EphA2-Fc-loaded biosensors as indicated and binding curves acquired using the Forte Bio OctetRed system. (B) Flow cytometry analysis of PC-3 cells. Overall ephrin-A (efnA) cell surface expression was assessed using recombinant EphA3-Fc, which binds promiscuously to all five ephrin-A ligands. The EphA2 activating 4B3 antibody strongly labelled EphA2 on human PC-3 cells. Unstained and isotype- labelled cells are the negative controls. (C) Two biological replicate western blot results of EphAreceptor activation and AKT inhibition after a 20-minute stimulation with ephrin-A1-Fc (efnA1-Fc), clustered ephrin-A1-Fc (cl-efnA1-Fc), 4B3 and 1 F7 alone and in combination. Replicate 1 is identical to Figure 4A; replicate 2 confirms these data. Bar charts show the mean (n = 2) and individual data points of quantified western blotting results for phospho-EphA and phospho-AKT protein levels. (D) Complete bioluminescence imaging data for the survival experiment shown in Figure 2E. PC-3- luciferase cells were engrafted orthotopically into the prostate of NRG mice. Starting from day 10, mice received thrice weekly intraperitoneal injections of 4B3 mAb, 1 F7 mAb, or PBS vehicle control. (E) Larger field of view of the H&E-stained lung sections shown in Figure 2H comparing metastatic spread from the orthotopic tumour to the lung at 7 weeks after engraftment between treatment groups. Tumour nodules are outlined in blue.

[0004] Figure 4 shows in vivo efficacy of activating 4B3 and inhibitory 1 F7 mAbs on tumour growth, survival and metastatic spread in an orthotopic PC-3-luciferase prostate cancer model. (A), (B) Western blot analysis of EphA receptor activation and AKT inhibition after stimulation with ephrin-A1- Fc (efnA1-Fc), cross-linked ephrin-A1-Fc (cl-efnA1-Fc), 1 F7 and 4B3 alone, in combination and clustered (cl) as indicated. 1 F7 blocks efnA1 -mediated EphA2 pathway activation (A), while clustered 4B3 induces EphA2 pathway activation (B). -actin is the loading control. Bar graphs in B show the quantification of the relative signal intensities of phospho-EphA and phospho-AKT, with the bars representing the means ± SE (n = 3 independent experiments). (C) Schematic of experimental design with exemplary histological and macroscopic images (collage of two images to capture the full view) of the orthotopic PC-3 prostate cancer model. NRG mice were orthotopically injected with 2.5x105PC-3- luciferase cells. From day 10, mice received thrice weekly intraperitoneal injections of PBS vehicle control or 4B3, or 1 F7 mAbs (8 mg / kg) until endpoint. An IgG isotype control group was included as additional control in later experiments. Tumour burden was imaged weekly by intravital bioluminescence imaging. Tumour (T), bladder (Bl) and seminal vesicles (SV). (D) Representative intravital bioluminescence images of PC-3-luciferase tumour signals six weeks after engraftment showing augmented tumour burden in the inhibitory 1 F7 treatment group. (E) Chart shows total luciferase bioluminescence total flux (pixel / seconds) over time. Mean ± SE (n = 15 mice per group, pooled from three experiments). Mixed model analysis of log-transformed bioluminescence total flux demonstrated a significant difference between the inhibitory 1 F7 and PBS groups over time, which was determined by calculating the fixed effect parameter estimate of treatment (1 F7-PBS) x time. (F) Kaplan-Meier survival analysis (n = 5 mice / group). *p<0.05 (log-rank (Mantel-Cox) test for pairwise comparisons). (G) Representative lung ex vivo bioluminescence images of lung metastatic burden 7 weeks after orthotopic PC-3-luciferase engraftment. The box and whisker plot shows the log-scaled median, interquartile range ± maximum / minimum values of ex vivo total bioluminescence flux (pixel / second) of excised lungs; Individual data points represent signals of individual lungs. Data werepooled from three independent animal experiments, except for the isotype control, which had only been included in two of the three experiments, n = 14 lungs (PBS), n = 13 (IgG), n = 12 (1 F7), n = 13 (4B3). While lungs from 4B3-treated animals showed a trend towards lower values, one-way ANOVA of log- transformed data with Sidak’s post-hoc test for multiple comparisons found no significant differences due to large variances within each group. (H) Examples of H&E-stained lung sections comparing metastatic spread from the orthotopic tumour to the lung at 7 weeks after engraftment. Tumour nodules are outlined in blue.

[0005] Figure 5 shows in vivo efficacy of 4B3 mAb in an intravenous metastatic model of PC- 3-luciferase cells. (A) Schematic of the experimental design. NRG mice received intraperitoneal injections of 4B3, 1 F7, or IgG control mAbs (8 mg / kg) or PBS vehicle controls at 2 days and 4 h prior to and 2 days after tail vein injection with PC-3-luciferase cells. In one experimental setup (Protocol 1) mice continued to receive treatments thrice weekly via intraperitoneal injection until endpoint. For Protocol 2, mice received no further injections of antibody after the initial three doses. Tumour burden was monitored using weekly intravital bioluminescence imaging. (B), (C) Representative bioluminescence images at 8 and 9 weeks and quantification of total luminescence flux (pixel / second) from week 5 to 8 after PC-3 cell engraftment (B) and Kaplan Meier survival analysis (C) of mice treated according to Protocol 1. The box and whisker plot shows the log-scaled median, interquartile range ± maximum / minimum values of the total bioluminescence flux (pixels / second). Mixed model analysis of log-transformed bioluminescence total flux demonstrated a significant difference between treatment groups, over time and treatments x time. Kaplan Meier survival curves were analysed using log-rank test to compare all curves and log-rank (Mantel-Cox) test for pairwise comparisons (n = 7-8 mice per group as indicated), *p<0.05 and **P<0.01. (D), (E) Bioluminescence images at 9 and 10 weeks after injection of tumour cells and quantification of total bioluminescence flux (pixel / seconds) from week 6 to 11 (D) and Kaplan Meier survival analysis (E) of mice treated according to Protocol 2. The box and whisker plot shows the log-scaled median, interquartile range ± maximum / minimum values of the total bioluminescence flux (pixels / second). Mixed model analysis of log-transformed bioluminescence total flux demonstrated a significant difference between treatment groups, overtime and treatments x time.). Kaplan Meier survival curves were analysed by log-rank test to compare all curves and log-rank (Mantel-Cox) test for pairwise comparisons (n = 7-8 mice per group as indicated), **p<0.01 and ***P<0.001 . (F) Western blot analysis of EphA2 receptor activation and AKT inhibition in HUVEC cells after stimulation with ephrin-A1-Fc (efnA1-Fc), clustered ephrin-A1-Fc (X(efnA1-Fc)), 4B3 alone and in combination with the inhibitory 1 F7 antibody was rested as indicated. 1 F7 blocks EphA2 receptor activation by ephrin-A1-Fc. -actin is the loading control. (G) Vascular permeability was measured by Miles Assay. Two representative images from the flanks of two mice showing dye effusion in response to 1 F7, 4B3 and control IgG mAbs and PBS. The bar graph shows the relative amount of Evans Bluedye extravasation quantified spectroscopically after formamide extraction from tissue biopsy cores. Mean ± SE (n = 8). *P < 0.05, **P < 0.01 , ***P < 0.001 , ns. - not significant (Repeated measure, oneway ANOVA with post-hoc Tukey's test for multiple pairwise comparisons).

[0006] Figure 6 shows quantitative phosphoproteomics and enrichment analyses show EphA2 modulates phosphorylation of cell-cell adhesion and cytoskeletal proteins. (A) Schematic representation of phosphoproteomic workflow. PC-3 cells were differentially labelled with light (12C) and heavy (13C) Arg and Lys isotopes for 6 passages, then activated with clustered ephrin-A1-Fc (efnA1- Fc) or treated with clustered Fc control for 20 min. Following cell lysis and clean-up, equal amounts of lysates from each condition were combined and digested with LysC and trypsin. Phosphopeptides were enriched with TiC>2 and analyzsed by LC-MS / MS. n = 4 independently SILAC-labelled, stimulated and processed biological replicates. The light and heavy amino acid labelling was alternated between replicates (‘label swap’): replicates 1 and 3 were ‘forward labelled’ (Fc control: light, ephrin-A1-Fc: heavy) and replicates 2 and 4 were ‘reverse labelled’. (B) Heat map showing the Iog2-transformed activated :control ratios across the four replicates for the 30 most regulated phosphopeptides. (C) Regulated phosphoproteins were categorized into protein classes according to their function by manually curating information from HRPD, Panther and UniProt databases and the literature. (D), (E) GO enrichment analysis of Cellular Compartment (D) and Biological Process and Molecular Function terms (E). The ClueGo app for Cytoscape was used to calculate enrichment of terms associated with EphA2-regulated proteins compared to our own background dataset and to cluster related and redundant terms. Enrichment was evaluated using one-sided hypergeometric testing. FDR was controlled using the Benjamini-Hochberg correction for multiple hypothesis testing. Node size reflects the number of regulated phosphoproteins linked to a particular term; node colour indicates the Benjamini-Hochberg corrected p-value as a measure of significance as indicated.

[0007] Figure 7 shows motif and upstream kinase analysis of regulated phosphosites identifies RXXS / T-directed AGC kinases and proline-directed CMGC kinases as mediators of EphA2 signalling. (A) Enriched motifs were identified using the Motifs Analysis Tool from PhosphoSitePlus. 13-mer phosphosite-centered peptide sequences of regulated phosphosites were analysed using the Motif-All algorithm (significance threshold of 1 e05, support threshold of 0.05). A minimum motif occurrence threshold of five was manually applied to significantly overrepresented motifs. Motif logos were generated from the sequences of all 128 regulated phosphosites and from the phosphosites conforming with particular enriched motifs as indicated. (B), (C) Schematics showing proteins with an EphA2-regulated (RX)RXXS / T* AGC kinase motif (B) and S / T*P proline-directed CMGC kinase motif (C) organized by cellular functions. Phosphopeptide motifs are indicated by coloured squares; regulation (up / down) is indicated by red and green arrows. (D) For the analysis of kinase-substraterelationship the PhosphoSitePlus, HRPD, UniProt and NetworKIN databases were queried for known and predicted upstream kinases of regulated phosphorylation sites. Resulting kinase-substrate relationships are shown as black edges for known connections and as blue edges for connections predicted by the NetworKIN database. Kinases are organized according to kinase families. Only kinases with at least 3 substrate connections are shown. CMGC kinases are primarily proline-directed kinases. AGC kinases comprise kinases with a substrate specificity for RXXS / T motifs. Phosphorylation sites with an RXXS / T* or an S / T*P motif are indicated by red and blue outlines around phosphosite nodes, respectively. Kinase node sizes reflect the number of regulated phosphosite connected to a kinase. mTOR, AKT, ERK, GSK-313 and RSK are the most likely candidate kinases for mediating signalling downstream of EphA2.

[0008] Figure 8 shows validation of phosphoproteomic data. (A), (B) Proteome Profiler Human Phospho-Kinase Array and western blot analysis validating pathway regulation downstream of EphA2 kinase activation in PC-3 cells. PC-3 cells were left untreated or incubated with clustered human IgG (Fc control, Fc) or clustered ephrin-A1-Fc (A1) for 20 min to activate the EphA2 receptor. Representative images of array and western blot membranes are shown, -actin is the loading control. Increased PARD3 tyrosine phosphorylation was shown by immunoprecipitating PARD3 and subsequent probing with an anti-phospho-tyrosine antibody. Bar graph shows the quantification of the Proteome Profiler array as Iog2(fold change (efnA1-Fc:Fc control)). Mean ± SE, (n = 4 biological replicates). (C) Western blot analysis testing the regulation of EphA2-regulated proteins and pathways identified in PC- cells across a panel of prostate, breast, colon and brain cancer cell lines. Cell lines were left untreated or incubated with clustered human IgG (Fc control, Fc) or clustered ephrin-A1-Fc (A1) for 20 min. SHB phosphorylation on Y246 is consistently upregulated by EphA2 receptor activation across all tested cell lines. Downregulation of Afadin (S1718 / S1799) and NDRG1 was observed only in cell lines which responded with a robust downregulation of Akt phosphorylation on S473 (PC-3, BT- 519 and MN1).

[0009] Figure 9 shows functional validation identifies SHB as a mediator of EphA2-induced Erk-pathway inhibition and afadin as an EphA2-regulated phosphoprotein that mediates cell invasion in PC-3 prostate cancer cells. (A) Western blot analysis of the effect of transient SHB siRNA knockdown on Akt and Erk pathway inhibition in response to EphA2 activation. Cell lines were incubated with clustered human IgG (Fc control, Fc) or clustered ephrin-A1-Fc (efnA1-Fc) for 20 min. A representative western botting image is shown, p -actin is the loading control. Relative phosphorylated protein levels of phospho-EphA, phospho-Erk and phospho-AKT were quantified and are shown as bar charts. Mean ± SE (n = 3 biological replicates shown as individual data points). SHB Knockdown was confirmed by quantitative PCR (qPCR) and relative mRNA expression levels determined using the 2AACtmethod withP -actin as the housekeeping gene and the Lipofectamine control as the reference sample. Mean ± SE (n = 3 biological replicates). Knockdown of SHB diminished the EphA2-mediated Erk1 / 2 pathway inhibition, while the inhibitory effect on the Akt pathway remained intact. (B) Representative confocal immunofluorescence images of afadin (AFDN, red) and EphA2 (green) cellular localization in response to EphA2 stimulation with clustered ephrin-A1-Fc (efnA1-Fc). EphA2 activation regulates the intracellular localization of afadin. Cell nuclei are labelled with DAPI (blue). Scale bars are 20 pm. (C) Transient knockdown of afadin (AFDN) with two different siRNA sequences was confirmed by western blot analysis, p-actin is the loading control. Bar chart shows knockdown efficiency as determined by densitometry of western blotting results. Mean ± SE (n = 3 biological replicates). **p-value<0.01 , ***p- value<0.001 (One-way ANOVA with post-hoc Sidak test for multiple pairwise comparisons). (D) Transwell invasion assay comparing invasion of afadin (AFDN) knockdown cells compared to untransfected and negative control siRNA-transfected PC-3 cells. Mean ± SE (n = 5). *p-value<0.05 (One-way ANOVA with post-hoc Sidak test for multiple pairwise comparisons).

[0010] Figure 10 shows summary of EphA2-regulated phosphoproteins. Schematic showing EphA2-regulated phosphoproteins as rounded rectangular nodes (light blue) and regulated phosphorylation sites as circular nodes. Proteins are grouped based on their known functions and are depicted as a modified Venn-style diagram to allow for multiple functions. The colour and intensity of phosphosite nodes indicates the Iog2-transformed fold-change (efnA1-Fc:Fc control) values for the significantly EphA2-regulated phosphoproteins as determined by quantitative phosphoproteomics.

[0011] Figure 11 shows effect of EphA2-specific mAbs 4B3 and 1 F7 on prostate cancer metastasis in vivo and on endothelial cells in vitro. (A) Complete imaging data and quantification for the experiment shown in Figure 3B. PC-3-luciferase cells were injected into the lateral tail vein. PBS, control IgG, 4B3 and 1 F7 anti-EphA2 mAbs (8 mg / kg) were administered 2 days and 4 h before and two days after tumour cell injection and was then continued thrice weekly until endpoint. Tumour burden was monitored using intravital bioluminescence imaging of luciferase activity from PC-3-luciferase metastases throughout the body starting from five weeks (wks) after tumour cell injection. Quantification of bioluminescence total flux (pixel / s) is shown as individual plots for weeks 5 to 8. Geometric mean ± geometric SD (n = 7 / 8 mice per group). Individual data points representing individual mice. *p- value<0.05, **p-value<0.01 , ***p-value<0.001 (one-way ANOVA with post-hoc Tukey’s test for multiple pairwise comparisons). (B) Complete imaging data and quantification for the survival experiment shown in Figure 3D. NRG mice received a total of three doses of 1 F7 mAbs, 4B3 mAbs, IgG isotype mAbs (8 mg / kg) or PBS vehicle control at 2 days and 4 h prior to and 2 days after tail vein injection with PC-3- luciferase cells. Intravital bioluminescence imaging data of luciferase activity in metastases throughout the body were imaged starting from 5 weeks (wks) after tumour cell injection. Mice shaded grey andmarked with # mice were excluded due to reaching endpoint without detectable tumour burden early in the experiment and hence not meeting inclusion criteria. An additional three mice were excluded prior to commencement of imaging early in experiment as assessment of detectable tumour burden on imaging as an inclusion criteria was not possible. Quantification of bioluminescence total flux (pixel / s) is shown as individual scatter plots for weeks 5 to 11 . Geometric mean ± geometric SD (n = 7 / 8 / 9 mice per group at the start of imaging (week 5); sample sizes decrease over time due to mice reaching endpoint). Individual data points representing individual mice. *p-value<0.05, **p-value<0.01 , ***p- value<0.001 (One-way AN OVA with post-hoc Tukey’s test for multiple pairwise comparisons). (C) Flow cytometry analysis of ephrin-A (efnA) and EphA2 expression in HUVEC human endothelial primary cells and the 2H-11 mouse endothelial cell line. EphA3-Fc, which binds promiscuously to all five ephrin- A ligands, was used to measure overall efnA expression. 1 F7 and 4B3 mAbs were used to label EphA2. 1 F7 effectively labelled EphA2 in cells of human and mouse origin; 4B3 selectively bound to human EphA2 and showed little to no cross-reactivity with mouse EphA2 on 2H-11 endothelial cells. (D) Western blot analysis with quantification of EphA2 receptor activation and AKT inhibition in 2H-11 cells after a 20 minute stimulation with ephrin-A1-Fc (efnA1 -Fc), clustered ephrin-A1-Fc (cl-efnA1-Fc), 1 F7 and 4B3 alone and in combination as indicated. 1 F7 blocks EphA2 receptor activation by ephrin-A1- Fc. p-actin is the loading control. Two biological replicate experiments are shown. Bar charts show mean (n = 2) and individual data points of quantified western blotting results for phospho-EphA and phospho-AKT protein levels.

[0012] Figure 12 shows summary of phosphoproteomic analysis of EphA2 signalling in PC-3 prostate cancer cells. (A) Summary of phosphoproteome coverage, phosphosites distribution and phosphosite multiplicity of detected phosphopeptides. (B) MaxQuant normalized Iog2- transformed heavy:light (H:L) SILAC ratios of efnA1-activated:Fc-control (forward experiment; replicates 1 & 3) and Fc-control:efnA1 -activated (reverse experiment; replicates 2 & 4) were plotted against summed Iog2- transformed intensities of phosphosites detected with a localization probability > 0.75. Significantly- regulated phosphopeptides with a Benjamini Hochberg-corrected p-value of <0.05 and a q-value of <0.01 are highlighted in black compared to the grey colour of phosphopeptides that were not significantly different between Fc control and efnA1 -Fc-treated samples. (C) Heat map showing the Iog2-transformed SILAC ratios of efnA1-Fc:Fc-control of all 113 EphA2-regulated phosphopeptides that were significantly regulated in at least three of the four biological replicates. Each column represents one of the four replicates. Grey squares indicate where a particular phosphopeptide was not detected.

[0013] Figure 13 shows pathway and motif enrichment analyses and kinase -substrate relationship analysis of regulated phosphosites and proteins. (A) Enrichment analysis of KEGG and Reactome Pathway associations of regulated proteins. The ClueGo app for Cytoscape was used tocalculate enrichment of terms associated with EphA2-regulated proteins compared to our own background dataset and to cluster related and redundant terms. Enrichment was evaluated using onesided hypergeometric testing. FDR was controlled using the Benjamin i-Hochberg correction for multiple hypothesis testing. Node size reflects the number of regulated phosphoproteins linked to a particular term; node colour indicates the Benjamini-Hochberg corrected p-value as a measure of significance as indicated. (B) Complete list of enriched motifs identified using the Motifs Analysis Tool from PhosphoSitePlus. Enriched motifs were identified using the Motifs Analysis Tool from PhosphoSitePlus. 13-mer phosphosite-centered peptide sequences of regulated phosphosites were analysed using the Motif-All algorithm (significance threshold of 1e-05, support threshold of 0.05). A minimum motif occurrence threshold of five was manually applied to significantly overrepresented motifs. (C) Complete kinase-substrate relationship analysis. PhosphoSitePlus, HRPD, UniProt and NetworKIN databases were queried for known and predicted upstream kinases of regulated phosphorylation sites. Resulting kinase-substrate relationships are shown as black edges for known connections and as blue edges for connections predicted by the NetworKIN database. Kinases are organized according to kinase families. Phosphorylation sites with an RXXS / T* or S / T*P motif are indicated by red and blue outlines around phosphosite nodes, respectively. Kinase node sizes reflect the number of regulated phosphosites connected to a particular kinase.

[0014] Figure 14 shows Proteome Profiler Human Phospho-Kinase Array layout and analysis. (A) Layout of the Proteome Profiler Phospho-Kinase Array used in this study. (B) Bar graph of the complete quantification of the Proteome Profiler array. Data is represented as Iog2-fold change (efnA1- Fc:Fc control). Mean ± SE (n = 4 biological replicates).

[0015] Figure 15 shows validation of phosphoproteomics findings. (A) Western blot analysis of the effect of transient Grb10 siRNA knockdown on Akt and Erk pathway inhibition in response to EphA2 activation. Cell lines were incubated with clustered human IgG (Fc control, Fc) or clustered ephrin-A1-Fc (efnA1-Fc) for 20 min. -actin is the loading control. Bar chart shows relative phosphorylated protein levels of phospho-EphA, phospho-Erk and phospho- AKT. Mean ± SE (n = 2 biological replicates). Grb10 knockdown was confirmed by western blot. Grb10 siRNA knockdown had no effect on EphA2-mediated inhibition of Erk1 / 2 and Akt pathway inhibition in response to ephrin-A1 stimulation. (B) Confocal fluorescence images of Duolink proximity ligation assay assessing the interaction of EphA2 and afadin in PC-3 cells. Cells were incubated with anti-afadin and anti-EphA2 (1 F7) antibodies followed by secondary antibodies conjugated to PLA PLUS and MINUS oligonucleotide probes. A ligation reaction and rolling cycle amplification can occur only if the two target proteins are within <40 nm of each other. Binding of complementary fluorescent-labelled oligonucleotides to the reaction products then allows visualization of the protein interaction. An increasein fluorescent spots indicative of protein-protein interaction / close proximity was observed in cells treated with 1 pg / mL clustered ephrin-A1-Fc for 5 min compared to untreated cells. No antibody and single antibody labelling reactions are negative technical PLA controls.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0016] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0017] The articles “a” and “an” are used herein to refer to one or to more than one ( / .e., to at least one) of the grammatical object of the article. By way of example, “an element means one element or more than one element.

[0018] The term “activates", “activating" or “activation" as used herein refers to a compound that, when administered, causes the receptor in a resting state to become active. For example, in some embodiments the EphA2-binding molecule activates EphA2. The activation of EphA2 is marked by phosphorylation of the kinase domain and can lead to receptor internalization and degradation through the recruitment of metalloproteases to the cell membrane.

[0019] As used herein, the term “adjuvant’ broadly refers to an immunological or pharmacological agent that modifies or enhances the immunological response to a composition in vitro or in vivo. For example, an adjuvant might increase the presence of an antigen over time, help absorb an antigen-presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines. By changing an immune response, an adjuvant might permit a smaller dose of the immune interacting agent or preparation to increase the dosage effectiveness or safety. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent or preparation. Examples of adjuvants include, but are not limited to, an immune modulatory protein, Adjuvant 65, a-GalCer, aluminium phosphate, aluminium hydroxide, calcium phosphate, -Glucan Peptide, CpG DNA, GPI-0100, lipid A and modified versions thereof (e.g., monophosphorylated lipid A), lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D- isoglutamine, Pam3CSK4, Quil-A, and trehalose dimycolate.

[0020] As used herein, the term “administered", “administering" or “administration" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. Such an agent can contain, forexample, an isolated EphA2-binding molecule, CAR, encoding nucleic acid, genetic construct, cell or composition disclosed herein into an animal subject by a particular chosen route.

[0021] The term “amino acid" is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogues, derivatives and congeners thereof; amino acid analogues having variant side chains; and all stereoisomers of any of any of the foregoing.

[0022] As used herein, the term “antibody" may refer to both an intact antibody and an antigenbinding fragment thereof. Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. 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 (FR). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term “antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multi-specific antibodies (e.g., bispecific antibodies), single-chain antibodies and antigenbinding antibody fragments.

[0023] The term “antibody heavy chain," refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

[0024] The term “antibody light chain," refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (K) and lambda (A) light chains refer to the two major antibody light chain isotypes.

[0025] The term “recombinant antibody" refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNAor amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.

[0026] The term “antibody fragment refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing / destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen-binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0027] The term “antigen" or “Ag" refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumour sample, a cell or a fluid with other biological components.

[0028] An “antigen-binding molecule" refers to a molecule which is capable of binding to a target antigen, and encompasses monoclonal antibodies, polyclonal antibodies, monospecific antibodies and multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments, T cell receptors and CAR T cells, as long as they display binding to the relevant target molecule.

[0029] The term “binding" or “interacting" refers to an association, which may be a stable association, between two molecules, e.g., between a peptide and a binding partner or agent, e.g., small molecule, due to, for example, electrostatic, hydrophobic, ionic and / or hydrogen-bond interactions under physiological conditions.

[0030] The term “biological sample,” “tissue sample,” or simply “sample" each refers to a collection of cells obtained from a tissue of a subject. The source of the tissue sample may be solid tissue, as from a fresh, frozen and / or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or development of the subject.

[0031] As used herein, the term “binding domain" or “antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term “binding domain" or “antibody molecule" encompasses antibodies and antibody fragments. In an embodiment, an antibody molecule is a multi-specific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, a multi-specific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0032] As used herein, the term “cancer", “tumour1’, “malignant" and “malignancy" refer to diseases or conditions, or to cells or tissues associated with the diseases or conditions, characterized by aberrant or abnormal cell proliferation, differentiation and / or migration often accompanied by an aberrant or abnormal molecular phenotype that includes one or more genetic mutations or other genetic changes associated with oncogenesis, expression of tumour markers, loss of tumour suppressor expression or activity and / or aberrant or abnormal cell surface marker expression. The term “cancer" includes, but is not limited to, solid tumours and blood-borne tumours and may include diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term “cancer" further encompasses primary and metastatic cancers. The term “anti-cancer effect’’ refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumour volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or amelioration ofvarious physiological symptoms associated with the cancerous condition. An "anti-cancer effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies in prevention of the occurrence of cancer in the first place. The term "anti-tumour effect’ refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumour volume, a decrease in the number of tumour cells, a decrease in tumour cell proliferation, or a decrease in tumour cell survival.

[0033] The term “encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0034] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0035] The term “endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0036] In this specification, “EphA2" refers to EphA2 from any species and includes EphA2 isoforms, fragments, variants (including mutants), or homologues from any species. As used herein, a “fragment”, “variant”, or “homologue” of a protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of the reference protein (e.g., a reference isoform). In some embodiments, fragments, variants, isoforms, and homologues of a reference protein may be characterised by ability to perform a function performed by the reference protein. A fragment of EphA2 may have a minimum length of one of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 550, 600, 650, 700, 750, 800, 850, 900, 950 or up to about 1 ,000 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 550, 600, 650, 700, 750, 800, 850, 900, 950, or up to about 1 ,000 amino acids.

[0037] As generally used herein, an "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which a T cell receptor or antibody is capable of binding.

[0038] The term “exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0039] The term “expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0040] “Derived from" as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connotate or include a process or source limitation on a first molecule that is derived from a second molecule.

[0041] The term “immunoglobulin" includes any antigen-binding protein product of a mammalian immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG and IgE and antigen-binding fragments thereof. Included in the term “immunoglobulin” are immunoglobulins that are recombinant, chimeric or humanized or otherwise comprise altered or variant amino acid residues, sequences and / or glycosylation, whether naturally occurring or produced by human intervention (e.g., by recombinant DNA technology).

[0042] As used herein, by “isolated" is meant material, such as an EphA2-binding molecule, that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in recombinant, chemical synthetic, enriched, purified or partially purified form.

[0043] The term “isolated nucleic acid" refers to a polynucleotide of natural or synthetic origin or some combination thereof, which (1) is not associated with the cell in which the “isolated nucleic acid” is found in nature, and / or (2) is operably linked to a polynucleotide to which it is not linked in nature.

[0044] By "operably linked" is meant that said additional nucleotide sequence(s) (e.g., regulatory nucleic acid sequences) is / are positioned relative to the nucleic acid of the invention preferably to initiate, regulate or otherwise control transcription. Typically, the selected nucleic acid sequence and regulatory nucleic acid sequence (e.g., promoter and / or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette).

[0045] As used herein, the phrase “pharmaceutically acceptable" refers to those agents, compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0046] As used herein, the phrase “pharmaceutically acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0047] The terms “polynucleotide", and “nucleic acid" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labelling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0048] As used herein a “protein" is an amino acid polymer, wherein the amino acids may include D-amino acids, L-amino acids, natural and / or non-natural amino acids. As typically used herein, a “peptide" is a protein comprising no more than fifty (50) contiguous amino acids. As typically used herein, a “polypeptide” is a protein comprising more than fifty (50) contiguous amino acids. The term “protein” should also be understood to encompass protein-containing molecules such as glycoproteins and lipoproteins, although without limitation thereto.

[0049] The term “scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0050] The term “sequence identity" is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

[0051] As used herein, “specific binding" refers to the ability of an antibody to bind to a predetermined antigen or the ability of a peptide to bind to its predetermined binding partner. Typically, an antibody or peptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 107M or less, and binds to the predetermined antigen / binding partnerwith an affinity (as expressed by KD) that is at least 10 fold less, at least 100 fold less or at least 1000 fold less than its affinity for binding to a non-specific and unrelated antigen / binding partner (e.g., BSA, casein).

[0052] As used herein, the term "subject" includes but is not limited to mammals inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). In some embodiments, the subject is a human.

[0053] The term “therapeutically effective amount’ describes a quantity of a specified agent, such as an EphA2-binding molecule or CAR, sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a composition comprising one or more EphA2-binding molecules and / or CARs described herein, necessary to reduce, alleviate and / or prevent a cancer or cancer associated disease, disorder or condition, inclusive of cancer metastasis and recurrence. In some embodiments, a “therapeutically effective amount" is sufficient to reduce or eliminate a symptom of a cancer. In other embodiments, a “therapeutically effective amount" is an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent cancer growth, recurrence and / or metastasis.

[0054] As used herein, “treating", “treat’ or “treatment’ refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of cancer after the cancer and / or its symptoms have at least started to develop. Treatment or alleviation of a cancer may be effective to prevent progression of the cancer e.g., to prevent worsening of the condition or to slow the rate of development of a more severe disease state. As used herein, “preventing", “prevent’ or “prevention" refers to therapeutic intervention, course of action or protocol initiated priorto the onset of cancer and / or a symptom of cancer so as to prevent, inhibit or delay development or progression of the cancer or the symptom.

[0055] The term “vector" refers to the means by which a nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that may or may not be able to replicate autonomously or integrate into a chromosome of a host cell.1. General Overview

[0056] The present invention is at least partly based on the production of monoclonal antibodies or T cell receptors (TCR) directed to EphA2 and the subsequent creation of chimeric antigen receptors (CARs) based on the binding domains of these monoclonal antibodies. These monoclonalantibodies may be particularly suitable for the treatment and / or prevention of cancer, such as prostate cancer. Additionally, T cells expressing these CARs may be suitable for adoptive immunotherapy in subjects with cancer.

[0057] The present disclosure provides EphA2 antigen-binding molecules, such as anti- EphA2 antibodies and antigen-binding fragments thereof, compositions comprising the EphA2 antigenbinding molecules, and pharmaceutical compositions comprising the EphA2 antigen-binding molecules. The disclosure particularly provides EphA2 antigen-binding molecules having novel and / or improved properties as compared to existing anti-EphA2 antibodies. For example, the EphA2 antigenbinding molecules have the advantageous effect of activating EphA2.

[0058] Also provided by the disclosure are methods of treating a disease or condition by administering an activating EphA2 antigen-binding molecule. The methods provided by the disclosure are particularly useful for the treatment of a proliferative disease or cancer.

[0059] In one aspect, the invention provides novel activating EphA2-binding molecules comprising at least one complementarity determining region (CDR) having an amino acid sequence set forth in any one of SEQ ID NOs: 1-30 or an amino acid sequence at least 85% identical thereto.2. Ephrin type A receptor 2 (EphA2) antigen-binding molecules

[0060] In one aspect, activating antigen-binding molecules are provided. In every embodiment, the antigen-binding molecule comprises at least a first antigen-binding site specific for EphA2. The binding molecules are therefore termed EphA2 antigen-binding molecules or EphA2- binding molecules.

[0061] The EphA2-binding molecules of the present invention may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to EphA2. Antigen-binding regions of antibodies, such as single chain variable fragment (scFv), Fab and F(ab’)2 fragments may also be used / provided. An “antigen-binding region” is any fragment of an antibody which is capable of binding to the target for which the given antibody is specific.

[0062] In some embodiments, the EphA2-binding molecule provided herein is a recombinant, human or humanized antibody or antibody fragment. As broadly used herein, “humanized” antibodies may include antibodies entirely or at least partly of human origin, inclusive of modified antibodies or antibody fragments obtained from a non-human “foreign” species. In some embodiments, antibodies and antibody fragments may be modified so as to be administrable to one species having been produced in, or originating from, the same or another “foreign” species without eliciting a deleteriousimmune response to the “foreign” antibody. Human or non-human antibody fragments such as comprising complementarity determining regions (CDRs) or variable regions (i.e., VH and VL domains) may be “grafted” onto a human antibody scaffold or backbone to produce a “humanized” antibody or antibody fragment. In some embodiments, human or non-human CDRs or VL and VL domains are recombinantly grafted with a human antibody constant region.EphA2 Antigens.

[0063] The activating EphA2 antigen-binding molecules described herein bind specifically to EphA2 antigens. As used herein, “EphA2” antigens refer to EphA2 family members and homologues.

[0064] EphA2 is also referred to as epithelial cell kinase and tyrosine-protein kinase receptor ECK. EphA2 includes all known and naturally occurring EphA2 molecules inclusive of full-length EphA2 protein and fragments, variants and derivatives thereof. EphA2 includes, but is not limited to, mammalian EphA2. In some specific embodiments the EphA2 antigen is human EphA2. UniProtKB Accession No. P29317 describes a canonical human EphA2 protein, including its sequences and domain features, and is hereby incorporated by reference in its entirety. SEQ ID NO: 79 provides the full length EphA2 protein sequence.MELQAARACFALLWGCALAAAAAAQGKEWLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIY MYSVCNVMSGDQDNWLRTNWVYRGEAERI FIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDY GTNFQKRLFTKIDTIAPDEITVS SDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVR VYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAWPPGGEEPRMHCAVDGEWLVPIGQC LCQAGYEKVEDACQACS PGFFKFEASESPCLECPEHTLPS PEGATSCECEEGFFRAPQDPASMPC TRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPH GLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVS INQTEPPKVRLEGRSTTSLSVS WSI PPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSK VHEFQTLSPEGSGNLAVIGGVAVGWLLLVLAGVGFFIHRRRKNQRARQS PEDVYFSKSEQLKPL KTYVDPHTYEDPNQAVLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTS SGKKEVPVAI KTLKA GYTEKQRVDFLGEAGIMGQFSHHNI IRLEGVI SKYKPMMI ITEYMENGALDKFLREKDGEFSVLQ LVGMLRGIAAGMKYLANMNYVHRDLAARNI LVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKI P IRWTAPEAI SYRKFTSASDVWSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSA IYQLMMQCWQQERARRPKFADIVSILDKLI RAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVS EWLESIKMQQYTEHFMAAGYTAI EKWQMTNDDIKRI GVRLPGHQKRIAYSLLGLKDQVNTVGI P I [ SEQ ID NO : 79 ] .

[0065] In humans, EphA2 is encoded by the EphA2 gene (also known as ECK). The function of EphA2 is described e.g., in Lindberg RA, Mol. Cell. Biol. 1990), which is hereby incorporated byreference in its entirety. EphA2 is a -108 kDa single-pass type I transmembrane protein that functions as a receptor tyrosine kinase which binds promiscuously membrane-bound ephrin family ligands residing on adjacent cells, leading to contact-dependent bidirectional signalling into neighbouring cells. EphA2 is known to be aberrantly expressed in a variety of human cancers including breast, ovarian, cervical, colorectal, vulvar, gastric, lung, oral, endometrial, esophageal, renal cell carcinomas, and gliomas. Increased expression of EphA2 can promote tumour cellular proliferation, angiogenesis, and invasion.

[0066] The N-terminal 23 amino acids of SEQ ID NO: 79 constitutes a signal peptide, and so the mature form of EphA2 (i.e., after processing to remove the signal peptide) has the amino acid set forth in SEQ ID NO: 80. Positions 24 to 537 of SEQ ID NO: 79 form the extracellular domain (SEQ ID NO: 81), positions 538 to 558 form a transmembrane domain (SEQ ID NO: 82), and positions 559 to 976 form the cytoplasmic domain (SEQ ID NO 83). The extracellular domain comprises an Eph ligandbinding domain (positions 28 to 206 of SEQ ID NO: 79, shown in SEQ ID NO: 84); and two fibronectin type-ill domains (positions 328 to 432 of SEQ ID NO: 79, and positions 438 to 529 of SEQ ID NO: 79, shown in SEQ ID NO: 85 and 86, respectively). The cytoplasmic domain comprises a protein kinase domain (at position 613 to 875 of SEQ ID NO: 79, shown in SEQ ID NO: 87). The cytoplasmic domain also comprises a sterile alpha motif (SAM) (positions 904 to 968 of SEQ ID NO: 79, shown in SEQ ID NO: 88).

[0067] In some embodiments, the EphA2 is EphA2 from a mammal (e.g., a primate (rhesus, cynomolgus, non-human primate, or human) and / or rodent (e.g., rat or murine)). Isoforms, fragments, variants or homologues of EphA2 may optionally be characterised as having at least 85%, preferably one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature EphA2 isoform from a given species (e.g., human).

[0068] Isoforms, fragments, variants, or homologues may optionally be functional isoforms, fragments, variants, or homologues, e.g., having a functional property / activity of the reference EphA2, as determined by analysis by a suitable assay for the functional property / activity. For example, an isoform, fragment, variant, or homologues of EphA2 may e.g., display association with ephrin A5, or retain kinase activity.

[0069] In various embodiments, the EphA2-binding molecule additionally binds to at least one antigen additional to an EphA2 antigen.Regions of interest on the target molecule

[0070] In a first aspect, EphA2-binding molecules are provided. In every embodiment, the EphA2-binding molecule includes at least a first antigen-binding site specific for EphA2 antigen; the binding molecules are therefore termed EphA2 antigen-binding molecules or EphA2-binding molecules.

[0071] The EphA2-binding molecules described herein bind specifically to EphA2. The EphA2-binding molecules of the present invention may bind to a particular region of interest of EphA2. In some embodiments the EphA2-binding molecule may bind to a linear epitope of EphA2, consisting of a contiguous sequence of amino acids (i.e., an amino acid primary sequence). In some embodiments, the EphA2-binding molecule may bind to a conformational epitope of EphA2, consisting of a discontinuous sequence of amino acids of the amino acid sequence.

[0072] In some embodiments, the EphA2-binding molecule binds to the extracellular region of EphA2 (e.g., the region shown in SEQ ID NO: 81). In some embodiments, the EphA2-binding molecule binds to one or both of the fibronectin type III domains (e.g., the regions shown in SEQ ID NO: 85 and 86).

[0073] The region of a peptide or polypeptide to which an antibody binds can be determined by the skilled person using various methods well known in the art, including X-ray crystallography, any analysis of antibody antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based “protection” methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21 (3): 145-156, which is hereby incorporated by reference in its entirety.

[0074] In some embodiments, the EphA2-binding molecule of the present invention is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 79

[0075] In some embodiments, the EphA2-binding molecule of the present invention is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 80.

[0076] In some embodiments, the EphA2-binding molecule of the present invention is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 83.

[0077] In some embodiments, the EphA2-binding molecule of the present invention is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 85.

[0078] The ability of an EphA2-binding molecule to bind to a given peptide or polypeptide can be analysed by methods well known to the skilled person, including analysis by ELISA, immunoblot (e.g., western blot), immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al.,Methods Mol. Biol. (2012) 907: 411-442), or Bio-Layer Interferometry (see, e.g., Lad et al., (2015) J. Biomol. Screen 20(4): 498-507).

[0079] In embodiments where the EphA2-binding molecule is capable of binding to a peptide or polypeptide comprising a reference amino acid sequence, the peptide or polypeptide may comprise one or more additional amino acids at one or both ends of the reference amino acid sequence. In some embodiments the peptide / polypeptide comprises, for example, 1 -5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40 or 20-50 additional amino acids at one or both ends of the reference amino acid sequence.

[0080] In some embodiments, the additional amino acid(s) provided at one or both ends (i.e., the N-terminal and C-terminal ends) of the reference sequence correspond to the positions at the ends of the reference sequence in the context of the amino acid sequence of EphA2.

[0081] In some embodiments the EphA2-binding molecule is capable of binding to a polypeptide which is bound by an antibody comprising the VH and VL sequences of the antibody clone 4B3, as described herein.Variable Regions

[0082] The EphA2-binding molecules described herein have variable region domain amino acid sequences of an antibody, including VH and VL antibody domain sequences. VH and VL sequences are described in greater detail below.(I) VH Regions

[0083] The VH amino acid sequences in the EphA2-binding molecules described herein are antibody heavy chain variable domain sequences. In a typical antibody arrangement in both nature and in the EphA2-binding molecules described herein, a specific VH amino acid sequence associates with a specific VL amino acid sequence to form an antigen-binding site. In various embodiments, VH amino acid sequences are mammalian sequences, including human sequences, synthesized sequences, or combinations of non-human mammalian, mammalian, and / or synthesized sequences, as described in further detail below. In various embodiments, VH amino acid sequences are mutated sequences of naturally occurring sequences.(II) VL Regions

[0084] The VL amino acid sequences useful in the EphA2-binding molecules described herein are antibody light chain variable domain sequences. In a typical arrangement in both natural antibodiesand the antibody constructs described herein, a specific VL amino acid sequence associates with a specific VH amino acid sequence to form an antigen-binding site. In various embodiments, the VL amino acid sequences are mammalian sequences, including human sequences synthesized sequences, or combinations of human, non-human mammalian, mammalian, and / or synthesized sequences, as described in further detail below.

[0085] In various embodiments, VL amino acid sequences are mutated sequences of naturally occurring sequences. In certain embodiments, the VL amino acid sequences are lambda (A) light chain variable domains sequences. In certain embodiments, the VL amino acid sequences are kappa (K) light chain variable domain sequences. In a preferred embodiment, the VL amino acid sequences are kappa (K) light chain variable domain sequences.

[0086] The present invention provides EphA2-binding molecule capable of binding to EphA2. In some embodiments the EphA2-binding molecule is capable of binding the same region of EphA2, or an overlapping region of EphA2, to the region of EphA2 which is bound by an antibody comprising the VH and VL sequences of the antibody clone 4B3, as described herein.Complementarity Determining Regions

[0087] The VH and VL amino acid sequences comprise highly variable sequences termed “complementarity determining regions” (CDRs), typically three CDRs (CDR1 , CDR2, and CDR3). In a variety of embodiments, the CDRs are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CDRs are human sequences. In various embodiments, the CDRs are naturally occurring sequences that have been mutated to alter the binding affinity of the antigen-binding site for a particular antigen or epitope. In certain embodiments, the naturally occurring CDRs have been mutated in an in vivo host through affinity maturation and somatic hypermutation. In certain embodiments, the CDRs have been mutated in vitro through methods including, but not limited to, PCR-mutagenesis and chemical mutagenesis. In various embodiments, the CDRs and synthesised sequences including, but not limited to, CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries.

[0088] CDR identification and numbering may be according to any known CDR numbering system inclusive of Kabat (Kabat et aL, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)), Chothia (Chothia et al., J. Mol. Biol. 196:901-917 (1987)), AbM and Contact.

[0089] In various embodiments, CDRs identified as binding an antigen of interest are further mutated (i.e., “affinity matured”) to achieve a desired binding characteristic, such as an increasedaffinity for the antigen of interest relative to the original CDR. For example, targeted introduction of diversity into the CDRs, including those CDRs identified to bind an antigen of interest, can be introduced using degenerate oligonucleotides. Various randomization schemes can be employed. For example, “soft-randomization” can be used that provides a high bias towards the identity of wild-type sequence at a given amino acid position, such as allowing a given position in CDRs to vary among all twenty amino acids while biasing towards the wild-type sequence by doping the four bases at each codon position at non-equivalent level. As an illustrative example of soft-randomization, if achieving approximately 50% of the wild-type sequence is desired, each base of each codon is kept 70% wildtype and 10% each of other nucleotides and the degenerate oligonucleotides are used to make a focused phage library around the selected CDRs with the resulting phage particles used for phage panning under various stringent selection conditions depending on the need.Framework Regions and CDR Grafting

[0090] The VH and VL amino acid sequences comprise “framework region” (FR) sequences. FRs are generally conserved sequence regions that act as a scaffold for interspersed CDRs, typically in a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 arrangement (from N-terminus to C-terminus). In a variety of embodiments, the FRs are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the FRs are human sequences. In various embodiments, the FRs are naturally occurring sequences. In various embodiments, the FRs are synthesized sequences including, but not limited, rationally designed sequences.

[0091] In a variety of embodiments, the FRs and the CDRs are both from the same naturally occurring variable domain sequence. In a variety of embodiments, the FRs and the CDRs are from different variable domain sequences, wherein the CDRs are grafted onto the FR scaffold with the CDRs providing specificity for a particular antigen. In certain embodiments, the grafted CDRs are all derived from the same naturally occurring variable domain sequence. In certain embodiments, the grafted CDRs are derived from different variable domain sequences. In certain embodiments, the grafted CDRs are synthesized sequences including, but not limited to, CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries. In certain embodiments, the grafted CDRs and the FRs are from the same species. In certain embodiments, the grafted CDRs and the FRs are from different species. In a preferred grafted CDR embodiment, an antibody is “humanized”, wherein the grafted CDRs are non-human mammalian sequences including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, and goat sequences, and the FRs are human sequences. Humanized antibodies are discussed in more detail in U.S. Pat. No. 6,407,213, the entirety of which is hereby incorporated byreference for all it teaches. In various embodiments, portions or specific sequences of FRs from one species are used to replace portions or specific sequences of another species’ FRs.Exemplary amino acid sequences of the EphA2-bindinq molecules

[0092] In various embodiments, the EphA2-binding molecule comprises a particular VH CDR3 (CDR-H3) sequence and a particular VL CDR3 (CDR-L3) sequence.

[0093] In some embodiments, the EphA2-binding molecule comprises the CDR-H3 and CDR-L3 of the antibody clone 4B3. VH CDR and VL CDR amino acids sequences of the antibody clones are disclosed in Table 1 and Table 2. In one currently preferred embodiment, the EphA2 antigen binding molecule comprises the CDR-H3 and CDR-L3 of antibody clone 4B3.

[0094] In some embodiments, the EphA2-binding molecule comprises all three VH CDRs from one of the antibody clone 4B3. In one currently preferred embodiment, the EphA2 antigen binding molecule comprises all three VH CDRs from antibody clone 4B3. In some embodiments, the EphA2- binding molecule comprises all three VL CDRs from one of the antibody clone 4B3.

[0095] In some embodiments, the EphA2-binding molecule comprises all six CDRs from one of the antibody clone 4B3.

[0096] In some embodiments the antigen-binding molecule comprises a VH and a VL region which is, or which is derived from, the VH / VL region of an EphA2-binding antibody described herein (i.e., anti-EphA2 antibody clone 4B3).

[0097] Non-limiting examples of CDR amino acid sequences are set forth in SEQ ID NOs: 1- 30 and / or Table 1 and Table 2. CDR identification and numbering was performed using abYsis version 3.4.1 and IMGT / V-QUEST. Antibodies according to the invention may comprise 1 , 2 or 3 VL CDR amino acid sequences (e.g., CDR1 , CDR2 and / or CDR3) and / or 1 , 2, or 3 VH CDR amino acid sequences (e.g., CDR1 , CDR2 and / or CDR3), such as those set forth in SEQ ID NOs: 1-30 and / or Table 1 and Table 2.

[0098] In some embodiments, the EphA2-binding molecule comprises:(a) a heavy chain immunoglobulin variable region (VH) polypeptide comprising a CDR1 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 1 to 5; a CDR2 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 6 to 10; and a CDR3 having an amino acid sequence at least 85% identical to any one of SEQ ID NO: 11 to 15; and / or(b) a light chain immunoglobulin variable region (VL) polypeptide comprising a CDR1 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 16 to 20; a CDR2 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 21 to 25; and a CDR3 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 26 to 30.

[0099] With regard to such embodiments, the VH polypeptide suitably comprises an amino 1acid sequence set forth in SEQ ID NO: 71 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and / or the VL polypeptide suitably comprises an amino acid sequence set forth in SEQ ID NO: 72 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0100] The CDRs and FRs of the VH regions and VL regions of the antibody described herein are below defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res., (2015) 43 (Database issue): D413-22), which uses the IMGT V-DOMAIN numbering rules as described in LeFranc et aL, Dev. Comp. Immunol. (2003) 27: 55-77. In some embodiments, the antigen-binding molecule comprises a VH region according to (1) or (2) below:(1) (4B3) a VH region incorporating the following CDRs:HC-CDR1 having the amino acid sequence of SEQ ID NO: 5;HC-CDR1 having the amino acid sequence of SEQ ID NO: 10;HC-CDR1 having the amino acid sequence of SEQ ID NO: 15; or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC-CDR2, or HC-CDR3 are substituted with another amino acid; or

[0101] In some embodiments, the antigen-binding molecule comprises a VH region according to (2), below:(2) (4B3) a VH region incorporating the following FRs:HC-FR1 having the amino acid sequence of SEQ ID NO: 35;HC-FR2 having the amino acid sequence of SEQ ID NO: 40;HC-FR3 having the amino acid sequence of SEQ ID NO: 45;HC-FR4 having the amino acid sequence of SEQ ID NO: 50; or a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC- FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.

[0102] In some embodiments the antigen-binding molecule comprises a VH region comprising the CDRs according to one of (1) above, and the FRs according to (2) above.

[0103] In some embodiments, the antigen-binding molecule comprises a VL region according to (3) below:(3) (4B3) a VL region incorporating the following CDRs:LC-CDR1 having the amino acid sequence of SEQ ID NO: 20;LC-CDR2 having the amino acid sequence of SEQ ID NO: 25;LC-CDR3 having the amino acid sequence of SEQ ID NO: 30; or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC- CDR2, or LC-CDR3 are substituted with another amino acid.

[0104] In some embodiments, the antigen-binding molecule comprises a VL region according to (4), below:(4) (4B3) a VL region incorporating the following FRs:LC-FR1 having the amino acid sequence of SEQ ID NO: 55;LC-FR2 having the amino acid sequence of SEQ ID NO: 60;LC-FR3 having the amino acid sequence of SEQ ID NO: 65;LC-FR4 having the amino acid sequence of SEQ ID NO: 70; or a variant thereof in which one or two or three amino acids in one or more of LC-FR1 , LC- FR2, LC-FR3, or LC-FR4 are substituted with another amino acid.

[0105] In some embodiments the antigen-binding molecule comprises a VL region comprising the CDRs according to one of (1) and (2) above, and the FRs according to (3) or (4) above.

[0106] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (5) below:(5) a VH region comprising the CDRs according to (3) and the FRs according to (5).

[0107] Non-limiting examples of CDR amino acid sequences are set forth in SEQ ID NOs: 1- 30 and / or Table 1 and Table 2. CDR identification and numbering was performed using abYsis version 3.4.1 and IMGT / V-QUEST. Antibodies according to the invention may comprise 1 , 2 or 3 VL CDR amino acid sequences (e.g., CDR1 , CDR2 and / or CDR3) and / or 1 , 2, or 3 VH CDR amino acid sequences (e.g., CDR1 , CDR2 and / or CDR3), such as those set forth in SEQ ID NOs: 1-30 and / or Table 1 and Table 2.

[0108] In some embodiments, the EphA2-binding molecule comprises:(a) a heavy chain immunoglobulin variable region (VH) polypeptide comprising a CDR1 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs:1 to 5; a CDR2 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 6 to 10; and a CDR3 having an amino acid sequence at least 85% identical to any one of SEQ ID NO: 11 to 15; and / or(b) a light chain immunoglobulin variable region (VL) polypeptide comprising a CDR1 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 16 to 20; a CDR2 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 21 to 25; and a CDR3 having an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 26 to 30.

[0109] With regard to such embodiments, the VH polypeptide suitably comprises an amino acid sequence set forth in SEQ ID NO: 71 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and / or the VL polypeptide suitably comprises an amino acid sequence set forth in SEQ ID NO: 72 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0110] In some embodiments, the invention provides fragments of the isolated antibodies of the invention and the CARs of the invention.

[0111] The VH and VL region of an antigen-binding region of an antibody together constitute the Fv region. In some embodiments, the antigen-binding molecule according to the present invention comprises or consists of, an Fv region which binds to EphA2. In some embodiments, the VH and VLregions of the Fv are provided as a single polypeptide joined by a linker region, i.e., a single chain Fv (scFv).

[0112] Suitably, the EphA2-binding molecule binds an epitope of an EphA2 protein.Variable domain variants

[0113] The invention also includes variants of the EphA2-binding molecules disclosed herein. In some embodiments, the variant is an EphA2-binding molecule comprising an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 1-30 (referred to herein as a CDR “variant”). In some other embodiments, the variant comprises an amino acid sequence at least 85% identical to the VH and / or VL amino acid sequence of one or both of SEQ ID NO: 71 and 72.

[0114] Suitably, an EphA2-binding molecule comprising at least one of the CDR or other variant(s) is capable of binding an EphA2 protein.

[0115] In particular embodiments, a variant has at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of the reference protein (e.g., a reference isoform), such as those set forth in any one of SEQ ID NOs: 1-72. The protein “variant” disclosed herein may have one or more amino acids deleted, inserted, or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted or deleted without changing biological activity of the peptide (conservative substitutions). In some embodiments, fragments, variants, isoforms and homologues or a reference protein may be characterised by ability to perform a function performed by the reference protein.Constant Regions

[0116] In the EphA2-binding molecules, the EphA2-binding molecule can have a constant region domain sequence. Constant region domain amino acid sequences, as described herein, are sequences of a constant region domain of an antibody. Constant regions can refer to CH1 , CH2, CH3, CH4, or CL constant domain.

[0117] In a variety of embodiments, the constant region sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the constant region sequences are human sequences. In certain embodiments, the constant region sequences are from an antibody light chain. In particular embodiments, the constant region sequences are from a lambda or kappa light chain. In certainembodiments, the constant region sequences are from an antibody heavy chain. In some embodiments, the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of an IgG (e.g., IgG 1 , lgG2, lgG3, lgG4), IgA (e.g., Ig A1 , lgA2), IgD, IgE, or IgM. In a specific embodiment, the constant region sequences are from an IgG isotype. In a preferred embodiment, the constant region sequence is a human immunoglobulin G1 (lgG1) constant sequence (IGHG: UniProtKB Accession No. P01857, SEQ ID NO 90)Immunoglobulin heavy chain constant gamma 1

[0118] In some embodiments, the immunoglobulin heavy chain constant sequence is a human immunoglobulin G2 (lgG2) constant sequence (IGHG2: UniProt Accession No. P01859) SEQ ID NO: 91.Immunoglobulin heavy chain constant gamma 2

[0119] Exemplary constant regions and modifications thereof are described in International Patent Publication No. WO2018 / 075692, which is hereby incorporated by reference in its entirety.CH1 and CL Regions

[0120] CH1 amino acid sequences, as described herein, are sequences of the second domain of an antibody heavy chain, with reference from the N-terminus to C-terminus of a native antibody heavy chain architecture. In certain embodiments, the CH1 sequences are endogenous sequences. In a variety of embodiments, the CH1 sequences are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH1 sequences are human sequences. In certain embodiments, the CH1 sequences are from an lgA1 , lgA2, IgD, IgE, IgG 1 , lgG2, lgG3, lgG4, or IgM isotype. In some preferred embodiments, the CH1 sequences are from an lgG2 isotype. In an exemplary embodiment of this type, the CH1 sequence is UniProt Accession No. P01859, amino acids 1 to 98:ASTKGPSVFPIA.PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL S SWTVP S SNFGTQTYTCNVDHKPSNTKVDKV [ S EQ I D NO : 92 ] .

[0121] In some embodiments, a CH1 region comprises or consists of the sequence SEQ ID NO: 92, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 92.

[0122] Positions 99 to 116 of the lgG2 sequence set forth in SEQ ID NO: 91 form a hinge region between CH1 and CH2 regions and set forth, below:ERKCCVECPPCPAP PVAG [ SEQ I D NO : 93 ] ,

[0123] In another preferred embodiment, the CH1 sequences are from an lgG1 isotype. In some embodiments of this type, the CH1 sequence is UniProt accession number P01857 amino acids 1 to 98, as shown below:ASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQS S GLYSLS SWTVPS S SLGTQTYI CNVNHKPSNTKVDKKV [ SEQ I D NO : 96 ] ,

[0124] In some embodiments, a CH1 region comprises or consists of the sequence SEQ ID NO: 96, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 96.

[0125] In some embodiments, the hinge region between CH1 and CH2 regions comprises, consists, or consists essentially of the sequence corresponding positions 99 to 120 of the lgG1 sequence set forth in SEQ ID NO: 90:EPKSCDKTHTCPPCPAPELLGG [ SEQ I D NO : 97 ] ,

[0126] The CL amino acid sequences useful in the EphA2-binding molecules described herein are antibody light chain constant domain sequences, with reference to a native antibody light chain architecture. In certain embodiments, the CL sequences are endogenous sequences. In a variety of embodiments, the CL sequences are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, CL sequences are human sequences.

[0127] In certain embodiments, the CL amino acid sequences are lambda (1) light chain constant domain sequences. In particular embodiments, the CL amino acid sequences are human lambda light chain constant domain sequences. In preferred embodiments, the lambda (1) light chain constant domain sequence is UniProt Accession No. P0CG04.

[0128] In some embodiments, the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant sequence (IGLA; CA), e.g., IGLC1 , IGLC2, IGLC3, IGLC6, or IGLC7. In some embodiments a CL region comprises or consists of the sequence set forth in SEQ ID NO: 100, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 100.Immunoglobulin lambda constant regionGQPKANPTVTLFPPSSEELQANKATLVCLI SDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAA SSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS [ SEQ ID NO : 100 ] ,

[0129] In certain embodiments, the CL amino acid sequences are kappa (K) light chain constant domain sequences. In a preferred embodiment, the CL amino acid sequences are human kappa (K) light chain constant domain sequences. In a preferred embodiment, the kappa light chain sequence is the human sequence identified by UniProt Accession No. P01834, and set forth below.Immunoglobulin kappa light chain constant region (human)RTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLS SPVTKS FNRGEC [ SEQ I D NO : 101 ] ,

[0130] In some embodiments, the kappa light chain sequence is the mouse sequence identified by UniProt Accession No. P01837, and set forth below.Immunoglobulin kappa light chain constant region (mouse)RADAAPTVSI FPPSSEQLTSGGASWCFLNNFYPKDINVKWKI DGSERQNGVLNSWTDQDSKDSTYSM SSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC [ SEQ I D NO : 102 ] ,

[0131] The VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1) region of an antigen-binding region of an antibody together constitute the Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising a VH, a CH1 , a VL and a CL (e.g., CK or CA). In some embodiments, the Fab region comprising a VH and a CH1 (e.g., a VH-CH1 fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and a CL (e.g., a VH-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and a CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH (e.g., a CL-CH1 fusion polypeptide; that is, in some embodiments the Fab region is a CrossFab region. In some embodiments, the VH, CH1 , VL, and CL regions of the Fab or CrossFab are provided as a single polypeptide joined by linker regions, i.e., as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).

[0132] In certain embodiments, the CH1 sequence and the CL sequences are both endogenous sequences. In certain embodiments, the CH1 sequence and the CL sequences separately comprise respectively orthogonal modifications in endogenous CH1 and CL sequences, as discussed below and elsewhere herein. CH1 and CL sequences can also be portions thereof, either of an endogenous or modified sequence, such that a domain having the CH1 sequence, or portion thereof, can associate with a domain having the CL sequence, or portion thereof.CH1 and CL Orthogonal Modifications

[0133] In certain embodiments, the CH1 sequence and the CL sequences separately comprise respectively orthogonal modifications in endogenous CH1 and CL sequences. Orthogonal mutations, in general, are described in more detail below and elsewhere herein.

[0134] In particular embodiments, the orthogonal modifications in endogenous CH1 and CL sequences are an engineered disulfide bridge selected from engineered cysteines at position 138 of the CH1 sequence and position 116 of the CL sequence, at position 128 of the CH1 sequence and position 119 of the CL sequence, or at position 129 of the CH1 sequence and position 210 of the CL sequence, as numbered and discussed in more detail in U.S. Pat. No. 8,053,562 and U.S. Pat.No.9, 527, 927, each incorporated herein by reference in its entirety. In a preferred embodiment, the engineered cysteines are at position 128 of the CH1 sequence and position 118 of the CL Kappa sequence, as numbered by the Eu index.

[0135] In a series of preferred embodiments, the mutations that provide non-endogenous cysteine amino acids are a F118C mutation in the CL sequence with a corresponding A141 C in the CH1 sequence, or a F118C mutation in the CL sequence with a corresponding L128C in the CH1 sequence, or a S162C mutations in the CL sequence with a corresponding P171 C mutation in the CH1 sequence, as numbered by the Eu index.

[0136] In a variety of embodiments, the orthogonal mutations in the CL sequence and the CH1 sequence are charge-pair mutations. In specific embodiments the charge-pair mutations are a F118S, F118A or F118V mutation in the CL sequence with a corresponding A141 L in the CH1 sequence, or a T129R mutation in the CL sequence with a corresponding K147D in the CH1 sequence, as numbered by the Eu index and described in greater detail in Bonisch et al. (Protein Engineering, Design & Selection, 2017, pp.1-12), herein incorporated by reference for all that it teaches. In a series of preferred embodiments, the charge-pair mutations are a N138K mutation in the CL sequence with a corresponding G166D in the CH1 sequence, or a N138D mutation in the CL sequence with a corresponding G166K in the CH1 sequence, as numbered by the Eu index.CH2 Regions

[0137] In the EphA2-binding molecules described herein, the EphA2-binding molecules can have a CH2 amino acid sequence. CH2 amino acid sequences, as described herein, are CH2 amino acid sequences of the third domain of an antibody heavy chain, with reference from the N-terminus to C-terminus of a native antibody heavy chain architecture. In a variety of embodiments, the CH2 sequences are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH2 sequences are human sequences. In certain embodiments, the CH2 sequences are from an lgA1 , lgA2, IgD, IgE, IgG 1 , lgG2, lgG3, lgG4, or IgM isotype. In a preferred embodiment, the CH2 sequences are from an lgG1 isotype.

[0138] In certain embodiments, the CH2 sequences are endogenous sequences.

[0139] In some embodiments, the CH2 region comprises, consists, or consists essentially of, amino acid positions 117 to 219 of the lgG2 sequence (UniProt Accession No. P01859) set forth in SEQ ID NO: 91 , and as shown belowPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLT WHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTK [ SEQ ID NO : 94 ] ,

[0140] In particular embodiments, the CH2 region comprises, consists, or consists essentially of, amino acid positions 111-223 of the lgG1 sequence (UniProt Accession No. P01857) set forth in SEQ ID NO: 90, and as shown below.PSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAK [ SEQ ID NO : 98 ] .

[0141] In a series of embodiments, a EphA2-binding molecule has more than one paired set of CH2 domains that have CH2 sequences, wherein a first set has CH2 amino acid sequences from a first isotype and one or more orthologous sets of CH2 amino acid sequences from another isotype. The orthologous CH2 amino acid sequences, as described herein, are able to interact with CH2 amino acid sequences from a shared isotype, but not significantly interact with the CH2 amino acid sequences from another isotype present in the EphA2-binding molecule. In particular embodiments, all sets of CH2 amino acid sequences are from the same species. In preferred embodiments, all sets of CH2 amino acid sequences are human CH2 amino acid sequences. In other embodiments, the sets of CH2 amino acid sequences are from different species. In particular embodiments, the first set of CH2 amino acid sequences is from the same isotype as the other non-CH2 domains in the EphA2-binding molecule. In a specific embodiment, the first set has CH2 amino acid sequences from an IgG isotype and the one or more orthologous sets have CH2 amino acid sequences from an IgM or IgE isotype. In certain embodiments, one or more of the sets of CH2 amino acid sequences are endogenous CH2 sequences. In other embodiments, one or more of the sets of CH2 amino acid sequences are endogenous CH2 sequences that have one or more mutations. In particular embodiments, the one or more mutations are orthogonal knob-hole mutations, orthogonal charge-pair mutations, or orthogonal hydrophobic mutations. Orthologous CH2 amino acid sequences useful for the EphA2-binding molecules are described in more detail in international PCT applications W02017 / 011342 and WO2017 / 106462, herein incorporated by reference in their entirety.CH3 Regions

[0142] CH3 amino acid sequences, as described herein, are sequences of the C-terminal domain of an antibody heavy chain, with reference from the N-terminus to C-terminus of a native antibody heavy chain architecture.

[0143] In a variety of embodiments, the CH3 sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences.In a preferred embodiment, the CH3 sequences are human sequences. In certain embodiments, the CH3 sequences are from an lgA1 , lgA2, IgD, IgE, IgM, lgG1 , lgG2, lgG3, lgG4 isotype or CH4 sequences from an IgE or IgM isotype. In a specific embodiment, the CH3 sequences are from an IgG isotype. In a preferred embodiment, the CH3 sequences are from an lgG1 isotype.

[0144] In certain embodiments, the CH3 sequences are endogenous sequences.

[0145] In some embodiments, the CH3 region comprises, consists, or consists essentially of, amino acid positions 220 to 326 of the lgG2 sequence (UniProt Accession No. P01859) set forth in SEQ ID NO: 91 form the CH3 region, as shown below:GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI SVEWESNGQPENNYKTTPPMLDSDGS FFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK [ SEQ ID NO : 95 ] ,

[0146] In particular embodiments, the CH3 sequence is UniProt Accession No. P01857 amino acids 224-330. In various embodiments, a CH3 sequence is a segment of an endogenous CH3 sequence. In particular embodiments, a CH3 sequence has an endogenous CH3 sequence that lacks the N-terminal amino acids G224 and Q225. In some particular embodiments, a CH3 sequence has an endogenous CH3 sequence that lacks the C-terminal amino acids P328, G329, and K330. In particular embodiments, a CH3 sequence has an endogenous CH3 sequence that lacks both the N-terminal amino acids G224 and Q225 and the C-terminal amino acids P328, G329, and K330. In preferred embodiments, a EphA2-binding molecule has multiple domains that have CH3 sequences, wherein a CH3 sequence can refer to both a full endogenous CH3 sequence as well as a CH3 sequence that lacks N-terminal amino acids, C-terminal amino acids, or both.

[0147] In certain embodiments, the CH3 sequences are endogenous sequences that have one or more mutations. In particular embodiments, the mutations are one or more orthogonal mutations that are introduced into an endogenous CH3 sequence to guide specific pairing of specific CH3 sequences, as described in more detail below.

[0148] In certain embodiments, the CH3 sequences are engineered to reduce immunogenicity of the antibody by replacing specific amino acids of one allotype with those of another allotype and referred to herein as isoallotype mutations, as described in more detail in Stickler et al. (Genes Immun. 2011 ; 12(3): 213-221), which is herein incorporated by reference in its entirety. In particular embodiments, specific amino acids of the G1 m1 allotype are replaced. In a preferred embodiment, isoallotype mutations D356E and L358M are made in the CH3 sequence.

[0149] In some embodiments, an lgG1 CH3 amino acid sequence comprises the following mutational changes: P343V; Y349C; and a tripeptide insertion, 445P, 446G, 447K. In other preferred embodiments, domain B has a human lgG1 CH3 sequence with the following mutational changes: T366K; and a tripeptide insertion, 445K, 446S, 447C. In still other preferred embodiments, domain B has a human lgG1 CH3 sequence with the following mutational changes: Y349C and a tripeptide insertion, 445P, 446G, 447K.

[0150] In some embodiments, an lgG1 CH3 amino acid sequence comprises a 447C mutation incorporated into an otherwise endogenous CH3 sequence.Antigen Binding Sites

[0151] In some embodiments, a VL or VH amino acid sequence and a cognate VL or VH amino acid sequence are associated and form a first antigen binding site (ABS). The antigen binding site (ABS) is capable of specifically binding an epitope of an antigen. Antigen binding by an ABS is described in greater detail below and elsewhere herein.

[0152] In alternative embodiments (e.g., wherein the EphA2-binding molecule is a single domain antibody) a VH or VL amino acid sequence forms the first ABS.

[0153] In some embodiments, the EphA2 antigen binding molecule is multi-specific (e.g., a second ABS of the EphA2 antigen binding molecule specifically binds an antigen that is different than the EphA2 antigen specifically bound by the first ABS).Binding of Antigen by ABS

[0154] An ABS, and the EphA2-binding molecule comprising such ABS, is said to “recognize” the epitope (or more generally, the antigen) to which the ABS specifically binds, and the epitope (or more generally, the antigen) is said to be the “recognition specificity” or “binding specificity” of the ABS.

[0155] The ABS is said to bind to its specific antigen or epitope with a particular affinity. As described herein, “affinity” refers to the strength of interaction of non-covalent intermolecular forces between one molecule and another. The affinity (i.e., the strength of the interaction) can be expressed as a dissociation equilibrium constant (KD), wherein a lower KD value refers to a stronger interaction between molecules. KD values of antibody constructs are measured by methods well known in the art including, but not limited to, bio-layer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., BIACORE®), and cell binding assays. For purposes herein, affinities are dissociation equilibrium constants measured by bio-layer interferometry using Octet / FORTEBIO®.

[0156] “Specific binding,” as used herein, refers to an affinity between an ABS and its cognate antigen or epitope in which the KD value is below 106M, 107M, 108M, 109M, or 10 M.

[0157] The number of ABSs in an EphA2-binding molecule as described herein defines the “valency” of the EphA2-binding molecule. A EphA2-binding molecule having a single ABS is “monovalent”. A EphA2-binding molecule having a plurality of ABSs is said to be “multivalent”. A multivalent EphA2-binding molecule having two ABSs is “bivalent”. A multivalent EphA2-binding molecule having three ABSs is “trivalent”. A multivalent EphA2-binding molecule having four ABSs is “tetravalent”.

[0158] In various multivalent embodiments, all of the plurality of ABSs have the same recognition specificity. Such a EphA2-binding molecule is a “monospecific” “multivalent” binding construct. In other multivalent embodiments, at least two of the plurality of ABSs have different recognition specificities. Such EphA2-binding molecules are multivalent and “multispecific”. In multivalent embodiments in which the ABSs collectively have two recognition specificities, the EphA2- binding molecule is “bispecific”. In multivalent embodiments in which the ABSs collectively have three recognition specificities, the EphA2-binding molecule is “trispecific”.

[0159] In multivalent embodiments in which the ABSs collectively have a plurality of recognition specificities for different epitopes present on the same antigen, the EphA2-binding molecule is “multiparatopic”. Multivalent embodiments in which the ABSs collectively recognize two epitopes on the same antigen are “biparatopic”.

[0160] In various multivalent embodiments, multivalency of the EphA2-binding molecule improves the avidity of the EphA2-binding molecule for a specific target. As described herein, “avidity” refers to the overall strength of interaction between two or more molecules, e.g. a multivalent EphA2- binding molecule for a specific target, wherein the avidity is the cumulative strength of interaction provided by the affinities of multiple ABSs. Avidity can be measured by the same methods as those used to determine affinity, as described above. In certain embodiments, the avidity of a EphA2-binding molecule for a specific target is such that the interaction is a specific binding interaction, wherein the avidity between two molecules has a KD value below 10-6M, 10-7M, 10-8M, 10-9M, or 10-10M. In certain embodiments, the avidity of a EphA2-binding molecule for a specific target has a KD value such that the interaction is a specific binding interaction, wherein the one or more affinities of individual ABSs do not have has a KD value that qualifies as specifically binding their respective antigens or epitopes on their own. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABSs for separate antigens on a shared specific target or complex, such as separate antigens found on an individual cell. In certain embodiments, the avidity is the cumulativestrength of interaction provided by the affinities of multiple ABSs for separate epitopes on a shared individual antigen.Orthogonal Modifications

[0161] In the EphA2-binding molecules described herein, a EphA2-binding molecule can have constant region domains comprising orthogonal modifications. Constant region domain amino acid sequences are described in greater detail above and elsewhere herein.

[0162] “Orthogonal modifications” or synonymously “orthogonal mutations” as described herein are one or more engineered mutations in an amino acid sequence of an antibody domain that increase the affinity of binding of a first domain having orthogonal modification for a second domain having a complementary orthogonal modification. In certain embodiments, the orthogonal modifications decrease the affinity of a domain having the orthogonal modifications for a domain lacking the complementary orthogonal modifications. In certain embodiments, orthogonal modifications are mutations in an endogenous antibody domain sequence. In a variety of embodiments, orthogonal modifications are modifications of the N-terminus or C-terminus of an endogenous antibody domain sequence including, but not limited to, amino acid additions or deletions. In particular embodiments, orthogonal modifications include, but are not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations, as described in greater detail below. In particular embodiments, orthogonal modifications include a combination of orthogonal modifications selected from, but not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations. In particular embodiments, the orthogonal modifications can be combined with amino acid substitutions that reduce immunogenicity, such as isoallotype mutations, as described in greater detail above or elsewhere herein.Orthogonal Engineered Disulfide Bridges

[0163] In a variety of embodiments, the orthogonal modifications comprise mutations that generate engineered disulfide bridges between a first and a second domain. As described herein , “engineered disulfide bridges” are mutations that provide non-endogenous cysteine amino acids in two or more domains such that a non-native disulfide bond forms when the two or more domains associate. Engineered disulfide bridges are described in greater detail in Merchant et al. (Nature Biotech (1998) 16:677-681), the entirety of which is hereby incorporated by reference for all it teaches. In certain embodiments, engineered disulfide bridges improve orthogonal association between specific domains. In a particular embodiment, the mutations that generate engineered disulfide bridges are a K392C mutation in one of a first or second CH3 domains, and a D399C in the other CH3 domain. In a preferredembodiment, the mutations that generate engineered disulfide bridges are a S354C mutation in one of a first or second CH3 domains, and a Y349C in the other CH3 domain. In another preferred embodiment, the mutations that generate engineered disulfide bridges are a 447C mutation in both the first and second CH3 domains that are provided by extension of the C-terminus of a CH3 domain incorporating a KSC tripeptide sequence.Orthogonal Knob-Hole Mutations

[0164] In a variety of embodiments, orthogonal modifications comprise knob-hole (synonymously, knob-in-hole) mutations. As described herein, knob-hole mutations are mutations that change the steric features of a first domain’s surface such that the first domain will preferentially associate with a second domain having complementary steric mutations relative to association with domains without the complementary steric mutations. Knob-hole mutations are described in greater detail in U.S. Pat. No.5, 821 ,333 and U.S. Pat. No. 8,216,805, each of which is incorporated herein in its entirety. In various embodiments, knob-hole mutations are combined with engineered disulfide bridges, as described in greater detail in Merchant et al. (Nature Biotech (1998) 16:677-681), incorporated herein by reference in its entirety. In various embodiments, knob-hole mutations, isoallotype mutations, and engineered disulfide mutations are combined.

[0165] In certain embodiments, the knob-in-hole mutations are a T366Y mutation in a first domain, and a Y407T mutation in a second domain. In certain embodiments, the knob-in-hole mutations are a F405A in a first domain, and a T394W in a second domain. In certain embodiments, the knob-in- hole mutations are a T366Y mutation and a F405A in a first domain, and a T394W and a Y407T in a second domain. In certain embodiments, the knob-in-hole mutations are a T366W mutation in a first domain, and a Y407A in a second domain. In certain embodiments, the combined knob-in-hole mutations and engineered disulfide mutations are a S354C and T366W mutations in a first domain, and a Y349C, T366S, L368A, and aY407V mutation in a second domain. In a preferred embodiment, the combined knob-in-hole mutations, isoallotype mutations, and engineered disulfide mutations are a S354C and T366W mutations in a first domain, and a Y349C, D356E, L358M, T366S, L368A, and aY407V mutation in a second domain.Orthogonal Charge-Pair Mutations

[0166] In a variety of embodiments, orthogonal modifications are charge-pair mutations. As used herein, charge-pair mutations are mutations that affect the charge of an amino acid in a domain’s surface such that the domain will preferentially associate with a second domain having complementary charge-pair mutations relative to association with domains without the complementary charge-pairmutations. In certain embodiments, charge-pair mutations improve orthogonal association between specific domains. Charge-pair mutations are described in greater detail in U.S. Pat. No.8, 592, 562, U.S. Pat. No.9, 248, 182, and U.S. Pat. No.9, 358, 286, each of which is incorporated by reference herein for all they teach. In certain embodiments, charge-pair mutations improve stability between specific domains. In a preferred embodiment, the charge-pair mutations are a T366K mutation in a first domain, and a L351 D mutation in the other domain.

[0167] In specific embodiments, the orthogonal mutations are charge-pair mutations at the VH / VL interface. In preferred embodiments, the charge-pair mutations at the VH / VL interface are a Q39E in VH with a corresponding Q38K in VL, or a Q39K in VH with a corresponding Q38E in VL, as described in greater detail in Igawa et al. (Protein Eng. Des. Sei., 2010, vol.23, 667-677), herein incorporated by reference for all it teaches.Trivalent and Tetravalent EphA2-bindinq molecules

[0168] In another series of embodiments, the EphA2-binding molecules have three antigen binding sites and are therefore termed “trivalent”. In a variety of embodiments, the EphA2-binding molecules have four antigen binding sites and are therefore termed “tetravalent”.EphA2-bindinq molecule Architecture

[0169] The antigen binding sites described herein, including specific CDR subsets, can be formatted into any binding molecule architecture including, but not limited to, full-length antibodies, Fes, Fvs, lgG1 fragments, F(ab')? fragments, scFvs, tandem scFvs, scDiabodies, DARTs, tandAbs, minibodies, camelid VHH, and other antibody fragments or formats known to those skilled in the art. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MAbs, 2017, 9(2): 182-212), herein incorporated by reference. The antigen binding sites described herein, including specific CDR subsets, can also be formatted into a “B-body” format, as described in more detail in U.S. Pat. Pub. No. 2018 / 0118811 and International Application Pub. No. WO2018 / 075692, each of which is herein incorporated by reference in its entirety.

[0170] The invention is also contemplated to include multivalent recombinant antibody fragments, including diabodies, triabodies and / or tetrabodies, comprising a plurality of scFvs, as well as dimerisation-activated demibodies (e.g., International PCT Patent Publication No. W02007 / 062466). By way of example, such antibodies may be prepared in accordance with the methods described in Holliger et al., 1993 Proc Natl Acad Sci USA 90:6444-6448; or in Kipriyanov, 2009 Methods Mol Biol 562:177-93 and herein incorporated by reference in their entirety.

[0171] In some embodiments, the invention provides fragments of the isolated antibodies of the invention and the CARs of the invention.

[0172] Fragments of the invention can be produced by those methods described herein. Alternatively, fragments can be produced, for example, by digestion of an antibody with proteinases such as endoLys-C, endoArg-C, endoGlu-C and V8-protease. The digested fragments can be purified by chromatographic techniques as are well known in the art.

[0173] Particular embodiments of the invention provide an immunogenic fragment of the EphA2 antigen-binding molecules of the invention. By “immunogenic” is meant capable of eliciting an immune response upon administration to an animal, such as a human, mouse or rabbit. The immune response may include the production, activation or stimulation of the innate and / or adaptive arms of the immune system inclusive of immune cells such as B and / or T lymphocytes, NK cells, granulocytes, macrophages and dendritic cells and / or molecules such as antibodies, cytokines and chemokines, although without limitation thereto.

[0174] Antibody fragments include Fab and F(ab')2 fragments, diabodies, triabodies, bispecific antibodies and single chain antibody fragments (e.g., scFvs), although without limitation thereto. In some embodiments, an antibody fragment may comprise at least a portion of a CDR1 , 2 and / or 3 amino acid sequence, such as set forth in SEQ ID NOs: 1-30 or a VH and / or VL amino acid sequence, such as set forth in SEQ ID NOs: 71 and 72. A preferred antibody fragment comprises at least one entire light chain variable region CDR and / or at least one entire heavy chain variable region CDR.Further modification

[0175] In a further series of embodiments, the EphA2-binding molecule has additional modifications.Antibody-Drug Conjugates

[0176] In various embodiments, the EphA2-binding molecule is conjugated to a therapeutic agent (i.e., drug) to form a EphA2-binding molecule-drug conjugate. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (e.g. radioisotopes), immune modulators (e.g., cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g. cytotoxic agents). In certain embodiments, the therapeutic agents are attached to the EphA2-binding molecule through a linker peptide, as discussed in more detail below.

[0177] Methods of preparing antibody-drug conjugates (ADCs) that can be adapted to conjugate drugs to the EphA2-binding molecules disclosed herein are described, e.g., in U.S. PatentNo. 8,624,003 (pot method), U.S. Patent No. 8,163,888 (one-step method), U.S. Patent No. 5,208,020(two-step method), U.S. Patent No. 8,337,856, U.S. Patent No. 5,773,001 , U.S. Patent No. 7,829,531 ,U.S. Patent No. 5,208,020, U.S. Patent No. 7,745,394, and International Patent Publication Nos.WO2017 / 136623, WO2017 / 015502, WO2017 / 015496, WO2017 / 015495, W02004 / 010957W02005 / 077090, W02005 / 082023, W02006 / 065533, W02007 / 030642, W02007 / 103288,WO2013 / 173337, WO2015 / 057699, WO2015 / 095755, WO2015 / 123679, WO2015 / 157286,WO2017 / 165851 , W02009 / 073445, WO2010 / 068759, W02010 / 138719, W02012 / 171020WO2014 / 008375, WO2014 / 093394, WO2014 / 093640, W02014 / 160360, WO2015 / 054659,WO2015 / 195925, WO2017 / 160754, Storz (MAbs. 2015; 7(6): 989-1009), Lambert et al. (Adv Ther, 201734: 1015), Diamantis et al. (British Journal of Cancer, 2016, 114, 362-367), Carrico et al. (Nat Chem Biol, 2007.3: 321-2), We et al. (Proc Natl Acad Sci USA, 2009.106: 3000-5), Rabuka et al. (Curr Opin Chem Biol., 201114: 790-6), Hudak et al. (Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. (Nat Protoc., 2012 7:1052-67), Agarwal et al. (Proc Natl Acad Sci USA. , 2013, 110: 46-51), Agarwal et al. (Bioconjugate Chem., 2013, 24: 846-851), Barfield et al. (Drug Dev. and D., 2014, 14:34-41), Drake et al. (Bioconjugate Chem., 2014, 25:1331-41), Liang et al. (J Am Chem Soc., 2014, 136:10850- 3), Drake et al. (Curr Opin Chem Biol., 2015, 28:174-80), and York et al. (BMC Biotechnology, 2016, 16(1):23), each of which is hereby incorporated by reference in its entirety.Additional Binding Moieties

[0178] In various embodiments, the EphA2-binding molecule has modifications that comprise one or more additional binding moieties. In certain embodiments the binding moieties are antibody fragments or antibody formats including, but not limited to, full-length antibodies, Fab fragments, Fvs, scFvs, tandem scFvs, Diabodies, scDiabodies, DARTs, tandAbs, minibodies, camelid VHH, and other antibody fragments or formats known to those skilled in the art. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MAbs, 2017, 9(2): 182-212), herein incorporated by reference in its entirety.

[0179] In some embodiments, the one or more additional binding moieties are attached to the C-terminus of the first or third polypeptide chain. In some embodiments, the one or more additional binding moieties are attached to the C-terminus of both the first and third polypeptide chain. In some embodiments, the one or more additional binding moieties are attached to the C-terminus of both the first and third polypeptide chains. In some embodiments, individual portions of the one or more additional binding moieties are separately attached to the C-terminus of the first and third polypeptide chains such that the portions form the functional binding moiety.

[0180] In some embodiments, the one or more additional binding moieties are attached to the N-terminus of any ofthe polypeptide chains (e.g., the first, second, third, fourth, fifth, orsixth polypeptide chains). In certain embodiments, individual portions of the additional binding moieties are separately attached to the N-terminus of different polypeptide chains such that the portions form the functional binding moiety.

[0181] In some embodiments, the one or more additional binding moieties are specific for a different antigen or epitope of the ABSs within the EphA2-binding molecule. In particular embodiments, the one or more additional binding moieties are specific for the same antigen or epitope of the ABSs within the EphA2-binding molecule. In certain embodiments, wherein the modification is two or more additional binding moieties, the additional binding moieties are specific for the same antigen or epitope. In certain embodiments, wherein the modification is two or more additional binding moieties, the additional binding moieties are specific for different antigens or epitopes.

[0182] In some embodiments, the one or more additional binding moieties are attached to the EphA2-binding molecule using in vitro methods including, but not limited to, reactive chemistry and affinity tagging systems, as discussed in more detail below. In certain embodiments, the one or more additional binding moieties are attached to the EphA2-binding molecule through Fc-mediated binding (e.g., Protein A / G). In some embodiments, the one or more additional binding moieties are attached to the EphA2-binding molecule using recombinant DNA techniques, such as encoding the nucleotide sequence of the fusion product between the EphA2-binding molecule and the additional binding moieties on the same expression vector (e.g., plasmid).Functional / Reactive Groups

[0183] In various embodiments, the EphA2-binding molecule has modifications that comprise functional groups or chemically reactive groups that can be used in downstream processes, such as linking to additional moieties (e.g., drug conjugates and additional binding moieties, as discussed in more detail above or elsewhere herein) and downstream purification processes.

[0184] In some embodiments, the modifications are chemically reactive groups including, but not limited to, reactive thiols (e.g., maleimide based reactive groups), reactive amines (e.g., N — hydroxysuccinimide based reactive groups), “click chemistry” groups (e.g., reactive alkyne groups), and aldehydes bearing formylglycine (FGIy). In certain embodiments, the modifications are functional groups including, but not limited to, affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and Strep systems, etc.). In some embodiments, the functional groups or chemically reactive groups have a cleavable peptide sequence. In particular embodiments, the cleavable peptide is cleaved by meansincluding, but not limited to, photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In particular embodiments, protease cleavage is carried out by intracellular proteases. In particular embodiments, protease cleavage is carried out by extracellular or membrane-associated proteases. ADC therapies adopting protease cleavage are described in more detail in Choi et al. (Theranostics, 2012; 2(2): 156-178), the entirety of which is hereby incorporated by reference.Reduced Effector Function

[0185] In certain embodiments, the EphA2-binding molecule has one or more engineered mutations in an amino acid sequence of an antibody domain that reduce the effector functions naturally associated with antibody binding. Effector functions include, but are not limited to, cellular functions that result from an Fc receptor binding to an Fc portion of an antibody, such as antibody-dependent cellular cytotoxicity (ADCC, also referred to as antibody-dependent cell-mediated cytotoxicity), complement fixation (e.g., C1q binding), antibody dependent cellular-mediated phagocytosis (ADCP), and opsonization. Exemplary engineered mutations that reduce the effector functions are described in more detail in U.S. Pub. No. 2017 / 0137530, Armour, et al. (Eur. J. Immunol. 29(8) (1999) 2613-2624), Shields, et al. (J. Biol. Chem. 276(9) (2001) 6591-6604), and Oganesyan, et al. (Acta Crystallographica D64 (2008) 700-704), each of which is herein incorporated by reference in its entirety.Methods of Purification

[0186] A method of purifying an EphA2-binding molecule is provided herein. Purification steps include, but are not limited to, purifying the EphA2-binding molecules based on protein characteristics, such as size (e.g., size exclusion chromatography), charge (e.g., ion exchange chromatography), or hydrophobicity (e.g., hydrophobicity interaction chromatography), and affinity. In one embodiment, cation exchange chromatograph is performed. Other purification methods known to those skilled in the art can be performed including, but not limited to, use of Protein A, Protein G, or Protein A / G reagents. Multiple iterations of a single purification method can be performed. A combination of purification methods can be performed.Assembly and Purity of Complexes

[0187] In the embodiments of the present invention, at least four distinct polypeptide chains associate togetherto form a complete complex, i.e., the EphA2-binding molecule. However, incomplete complexes can also form that do not contain the at least four distinct polypeptide chains. For example, incomplete complexes may form that only have one, two, or three of the polypeptide chains. In other examples, an incomplete complex may contain more than three polypeptide chains, but does notcontain the at least four distinct polypeptide chains, e.g., the incomplete complex inappropriately associates with more than one copy of a distinct polypeptide chain. The method of the invention purifies the complex, i.e., the completely assembled EphA2-binding molecule, from incomplete complexes. In some embodiments, the EphA2-binding molecule is purified using a cleavable purification tag, which is cleaved after affinity chromatography purification has occurred.

[0188] Methods to assess the efficacy and efficiency of the purification steps are well known to those skilled in the art and include, but are not limited to, SDS-PAGE analysis, ion exchange chromatography, size exclusion chromatography, and mass spectrometry. Purity can also be assessed according to a variety of criteria. Examples of criterion include, but are not limited to: 1) assessing the percentage of the total protein in an eluate that is provided by the completely assembled EphA2-binding molecule, 2) assessing the fold enrichment or percent increase of the method for purifying the desired products, e.g., comparing the total protein provided by the completely assembled EphA2-binding molecule in the eluate to that in a starting sample, 3) assessing the percentage of the total protein or the percent decrease of undesired products, e.g., the incomplete complexes described above, including determining the percent or the percent decrease of specific undesired products (e.g., unassociated single polypeptide chains, dimers of any combination of the polypeptide chains, or trimers of any combination of the polypeptide chains). Purity can be assessed after any combination of methods described herein.Methods of Manufacturing

[0189] The EphA2 antigen-binding molecules described herein may be produced as recombinant synthetic antibodies or antibody fragments by expressing a nucleic acid encoding the antibody or antibody fragment in an appropriate host cell. The EphA2-binding molecules described herein can readily be manufactured by expression using standard cell free translation, transient transfection, and stable transfection approaches currently used for antibody manufacture. In specific embodiments, Expi293 cells (ThermoFisher) can be used for production of the EphA2-binding molecules using protocols and reagents from ThermoFisher, such as ExpiFectamine, or other reagents known to those skilled in the art, such as polyethylenimine as described in detail in Fang et al. (Biological Procedures Online, 2017, 19:11), herein incorporated by reference in its entirety. Nonlimiting examples of recombinant antibody expression and selection techniques are provided in Chapter 17 of Coligan et al., Current Protocols In Immunology and Zuberbuhler et al., 2009, Protein Engineering, Design & Selection 22 169.

[0190] The expressed proteins can be readily separated from undesired proteins and protein complexes using various purification strategies including, but not limited to, use of Protein A, ProteinG, or Protein A / G reagents. Further purification can be affected using ion exchange chromatography as is routinely used in the art.

[0191] scFvs may be prepared in accordance with the methods described in United States Patent No 5,091 ,513, European Patent No 239,400, or the article by Winter & Milstein, 1991 , Nature 349:293, which are incorporated herein by reference.

[0192] Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Cys, Vai, etc.), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., He, Thr, and Vai), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0193] Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include “comparison window", “sequence identity", “percentage of sequence identity" and “substantial identity". Because respective nucleic acids / proteins may each comprise (1) only one or more portions of a complete nucleic acid / protein sequence that are shared by the nucleic acids / proteins, and (2) one or more portions which are divergent between the nucleic acids / proteins, sequence comparisons are typically performed by comparing sequences over a “comparison window / ’ to identify and compare local regions of sequence similarity. A “comparison window / ’ refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wl, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporatedherein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-2015).

[0194] Derivatives ofthe antibody, antibody fragments or variants thereof disclosed herein are also provided.

[0195] As used herein, “derivative" antibodies, antibody fragments or variants thereof have been altered, for example by conjugation or complexing with other chemical moieties, by post- translational modification (e.g. phosphorylation, ubiquitination, glycosylation), chemical modification (e.g. cross-linking, acetylation, biotinylation, oxidation or reduction and the like), conjugation with labels (e.g. fluorophores, enzymes, radioactive isotopes, drugs) and / or inclusion of additional amino acid sequences as would be understood in the art.

[0196] In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2015) for more extensive methodology relating to chemical modification of proteins.

[0197] Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and / or purification of the isolated fusion protein. Non-limiting examples include metal-binding (e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g. GFP, RFP), epitope tags such as myc, FLAG and haemagglutinin tags.

[0198] The isolated proteins (e.g., EphA2 antibodies, antibody fragments and CARs), variants, fragments and / or derivatives of the present invention may be produced by any means known in the art, including but not limited to, chemical synthesis, recombinant DNA technology and proteolytic cleavage to produce peptide fragments.

[0199] Chemical synthesis is inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International PCT Patent Publication Nos. WO 99 / 02550 and WO 97 / 45444.

[0200] In one preferred embodiment, the EphA2 antibodies, antibody fragments and / or CAR proteins of the present invention are recombinant proteins.

[0201] Recombinant proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. NY USA 1995- 2008), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et aL, (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 1 , 5 and 6.T cell receptors

[0202] The present invention also provides T cell receptors (TCR) comprising CDRs that recognise epitopes derived from EphA2 antigens and presented in association with several frequently- occurring human leukocyte antigens. These TCRs may be particularly suitable for the production of genetically engineered T cells and their administration to humans to prevent and / or treat a cancer in a subject.

[0203] In another aspect, the invention provides an isolated alpha chain or beta chain of a TCR or a fragment thereof, comprising at least one CDR amino acid sequence according to any one of SEQ ID NOs: 1-30 or an amino acid sequence at least 85% identical thereto.

[0204] It will be understood that a TCR is the molecule found on the surface of T cells that is responsible for recognising antigenic peptides bound to MHC or HLA molecules. The TCR heterodimer typically includes an alpha chain and a beta chain in 95% of T cells, whereas 5% of T cells generally have TCRs consisting of gamma and delta chains.

[0205] With respect to the alpha and beta chains, these generally broadly comprise variable, joining and constant regions, and the beta chain also usually contains a short diversity region between the variable and joining regions, but this diversity region is often considered as part ofthe joining region. Each variable region comprises three CDRs (i.e., CDR1 , CDR2 and CDR3) embedded in a framework sequence, one being the hypervariable region named CDR3. There are typically several types of alpha chain variable (Va) regions and several types of beta chain variable (V ) regions known in the art and as distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence.

[0206] It will be appreciated that the TCR alpha chain ofthe present invention may be a hybrid TCR alpha chain comprising sequences derived from more than one species, such as human and mouse. By way of example, it has surprisingly been found that exchanging the constant regions of human TCRs with the murine counterpart may improve function as well as expression levels on human T cells (see, e.g., Sommermeyer and Uckert, J Immunol, 2010, which is incorporated by referenceherein). The TCR may therefore comprise human-derived variable regions and murine-derived constant regions.

[0207] In view of the foregoing, the term “T cell receptor"’ is used herein in a conventional manner to mean a molecule capable of recognising a peptide when presented by an MHC or HLA molecule. The molecule may be a heterodimer of two chains alpha (a) and beta (P) (or optionally gamma (y) and delta (6)) or it may be a single chain TCR construct.

[0208] In certain embodiments, the alpha chain and the beta chain of the TCR of the present aspect can be joined by a linker, such as those known in the art. By way of example, the linker can join the alpha and beta chains of the TCR of the invention by way of a disulphide bridge or bond.

[0209] In some embodiments, the TCR of the present aspect is or comprises a soluble TCR. It will be appreciated that soluble TCRs can be conjugated to immunostimulatory peptides and / or proteins, and / or moieties such as, but not limited, to CD3 agonists (e.g., anti-CD3 antibodies). The CD3 antigen is present on mature human T cells, thymocytes, and a subset of natural killer cells. It can be associated with the TCR so as to facilitate signal transduction of the TCR. Antibodies specific for the human CD3 antigen are well known in the art (see, e.g., International PCT Patent Publication No. WO 2004 / 106380; U.S. Patent Publication No. 2004 / 0202657, and 2009 / 0117102; U.S. Patent Nos. 6,750,325; 6,706,265 and 5,968,509; Great Britain Patent Publication No. GB).

[0210] Suitably, the soluble TCR may be included in one or more bi-specific immunotherapeutic agents such as ImmTACs (Immune mobilising TCRs against cancer) (Liddy, et al. (2012) Nat Med 8'. 980- 987) or BiTEs (Bispecific T cell engaging antibodies) (Baeuerle, et al. (2009). Curr Opin Mol Ther 11 (1): 22-30). ImmTACs represent bifunctional proteins that combine affinity monoclonal T cell receptor (mTCR) targeting with a therapeutic mechanism of action (e.g., an anti-CD3 scFv).Chimeric antigen receptors (CARs)

[0211] The present invention also provides chimeric antigen receptors (CARs) comprising the antigen-binding molecules or polypeptides of the present invention.

[0212] Therefore, in a related aspect of the invention provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain including at least one CDR having an amino acid sequence set forth in SEQ ID NOs: 1-30 and / or Table 1 and Table 2, or an amino acid sequence at least 85% identical thereto, a transmembrane domain, and an intracellular T cell signalling domain.

[0213] A CAR is an artificially constructed hybrid protein or polypeptide containing the antigenbinding domains of an antibody (e.g., single chain variable fragment (scFv)) linked to a T cell signalling domain. Characteristics of CARs include their ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC-restricted manner and exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigens independent of antigen processing, thus bypassing a major mechanism of tumour escape. Moreover, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains. CAR structure and engineering is reviewed, for example, in Dotti et al, Immunol Rev (2014) 257(1); and Guedan et al., Mol. Ther. Methods Clin. Dev. 2019, 12: 145-156, hereby incorporated by reference in their entirety. CARs comprise an antigenbinding region linked to a cell membrane anchor region (also known as the transmembrane domain) and a signalling region. An optional hinge region may provide separation between the antigen-binding region and cell membrane anchor region, and may act as a flexible linker. The CAR of the present invention comprises an antigen-binding region which comprises, consists, or consists essentially of polypeptide according to the invention.

[0214] The cell membrane anchor region is provided between the antigen-binding domain and the signalling region of the CAR and provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding region in the extracellular space, and signalling region inside the cell. In some embodiments, the CAR comprises of, or is derived from, the transmembrane region amino acid sequence for one of CD3-^, CD4, CD8, or CD28. Suitably, the transmembrane domain is derived from a membrane protein selected from CD8a, CD8 , 4-1 BB / CD137, CD28, CD34, CD4, FceRly, CD16, OX40 / CD134, CD3 CD3E, CD3y, CD36, TCRa, CD32, CD64, VEGFR2, FAS, FGFR2B and any combination thereof. In some particular embodiments, the transmembrane domain may be derived from a CD8 and / or CD28 transmembrane domain, which generally provide good receptor stability. As used herein, a region which is “derived from” a reference amino acid sequence comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical to the reference sequence. In some embodiments, the transmembrane domain comprises an amino acid sequence set forth in SEQ ID NO: 103 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical thereto.

[0215] The transmembrane domain (i.e., the cell membrane anchor region) of the chimeric receptors described herein can be in any form known in the art. As used herein, a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. Transmembrane domains compatible for use in the chimeric receptorsused herein may be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non- naturally occurring protein segment (e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane; see e.g., U.S. Patent No. 7,052,906 and International PCT Publication No. WO 2000 / 032776, which are incorporated by reference herein). To this end, the transmembrane domain may comprise a hydrophobic alpha helix.

[0216] Any intracellular or cytoplasmic T cell signalling domain (e.g., CD3-^ or FceRly) can be used to construct the chimeric receptors described herein, such as those comprising an immunoreceptor tyrosine-based activation motif (ITAM), for phosphorylation and activation of the CAR- expressing T cell. An “ITAM,” as used herein, is a conserved protein motif that is generally present in the tail portion of signalling molecules expressed in many immune cells. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used T cell signalling component is that of CD3-^ which contains three ITAMs. This transmits an activation signal to the T cell after antigen is bound. It will be appreciated, however, that the CD3-^ cytoplasmic signalling domain may not provide a fully competent activation signal and an additional co-stimulatory signalling domain, such as those hereinbefore described may be utilised. For example, chimeric CD28 and / or 4-1 BB / CD137 can be used with CD3-^ to transmit a proliferative / survival signal, or all three can be used together. Accordingly, the endodomain of the CAR of the invention may comprise a CD28 costimulatory domain (e.g., SEQ ID NO: 104), a 4-1 BB / CD137 co-stimulatory domain (e.g., SEQ ID NO: 105), and / or a CD3-^ intracellular signalling domain (e.g., SEQ ID NO: 106).

[0217] Signalling regions of CARs may also comprise co-stimulatory sequences derived from the signalling region of co-stimulatory molecules, to facilitate activation of CAR-expressing T cells upon binding to the target protein. Activation of a co-stimulatory signalling domain in a host cell (e.g., an immune cell) may induce the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The co-stimulatory signalling domain of any co-stimulatory molecule may be compatible for use in the chimeric receptors described herein. The type(s) of co-stimulatory signalling domain is selected can be based on factors such as the type of the immune cells in which the chimeric receptors would be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect). In other words, the term “co-stimulatory signalling domain”, as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response, such as an effector function. The co-stimulatory signalling domain of the chimeric receptor described herein can be a cytoplasmic signalling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, dentritic cells, neutrophils, or eosinophils

[0218] Exemplary co-stimulatory signalling domains for use in the chimeric receptors can be the cytoplasmic signalling domain of co-stimulatory proteins, including, without limitation, members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7 H1 / PD L1 , B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD- 1 , PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1 BB / TNFSF9 / CD137, 4-1 BB Ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF-R / TNFRSF13C, CD27 / TNFRSF7, CD27 Ligand / TNFSF7, CD30 / TNFRSF8, CD30 Ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 Ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR Ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-alpha / TNF-p, OX40 / TNFRSF4, 0X40 Ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1 A / TNFSF15, TNF, and TNF RII / TNFRSF1 B); members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other co-stimulatory molecules, such as CD2, CD7, CD53, CD82 / Kai-1 , CD90 / Thy1 , CD96, CD160, CD200, CD300a / LMIR1 , HLA class I, HLA-DR, Ikaros, integrin a4 / CD49d, integrin a4 1 , integrin a4 7 / LPAM 1 , LAG-3, TCL1A, TCL1 B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM 1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen 1 (LFA-1), and NKG2C. In some embodiments, the co- stimulatory signalling domain is of 4-1 BB, CD28, 0X40, ICOS, CD27, GITR, HVEM, TIM1 , LFA1 (CD11 a) or CD2, or any variant thereof. In some embodiments, the co-stimulatory signalling domain is derived from 4-1 BB (e.g., SEQ ID NO: 105) and / or CD28 (e.g., SEQ ID NO: 104).

[0219] Also within the scope of the present disclosure are variants of any ofthe co-stimulatory signalling domains described herein, such that the co-stimulatory signalling domain is capable of modulating the immune response of the immune cell. Additionally, it is envisaged that the chimeric receptors may comprise more than one co-stimulatory signalling domain (e.g., 2, 3, 4 or more). In some embodiments, the chimeric receptor comprises two or more of the same co-stimulatory signalling domains, for example, two copies of the co-stimulatory signalling domain of CD28. In some embodiments, the chimeric receptor comprises two or more co-stimulatory signalling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. In some cases, CARs are engineered to provide for co-stimulation of different intracellular signalling pathways. For example, signalling associated with CD28 co-stimulation preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, whereas the 4-1 BB-mediated signalling is through TNF receptor associated factor (TRAF) adaptor proteins. Signalling regions of CARs therefore sometimes contain co-stimulatory sequences derived from signalling regions of more than one co-stimulatory molecule. In some embodiments, the CAR of the present invention comprises one or more co- stimulatory sequences comprising or consisting of an amino acid sequence which comprises, consistsof, or is derived from amino acid sequences of the intracellular domain of one or more of CD28, 0X30, 4-1 BB, ICOS, and CD27.

[0220] An optional hinge region may provide separation between the antigen-binding domain and the transmembrane domain, and may act as a flexible linker. Any suitable hinge regions can be used, including those derived from lgG1 , CD8, and / or CD24 (as described in detail in Fujiwara et al., Cells, 2020, 9(5), 1182 and Guedan et al., Mol Ther Methods Clin Dev. 2019, 15(12): 145-156). In some embodiments, the CAR of the present invention comprises a hinge region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the hinge region of lgG1 .

[0221] It is envisaged that the CARs of the invention may be considered to be, for example, a first generation, second generation, third generation or fourth generation (i.e., associated with a T cell redirected for universal cytokine-mediated killing (TRUCKS)) CAR, as are known in the art. First generation CARs typically join an antibody-derived scFv to the CD3-zeta ( or z) intracellular signalling domain of the T cell receptor through hinge and transmembrane domains. Second generation CARs incorporate an additional domain (e.g., CD28, 4-1 BB, or ICOS) to supply a costimulatory signal. Third- generation CARs typically contain two costimulatory domains fused with the TCR CD3-^ chain. Third- generation costimulatory domains may include, for example, a combination of CD3-^, CD27, CD28, 4- 1 BB, ICOS, DAP-10 or 0X40. Accordingly, the CARs of the invention may contain an ectodomain commonly derived from a single chain variable fragment (scFv), a hinge, a transmembrane domain, and an endodomain with one (first generation), two (second generation), or three (third generation) signalling domains derived from CD3-^ and / or co-stimulatory molecules.

[0222] In some embodiments, the CAR is associated with a T cell redirected for cytokine activity (e.g., TRUCK), also known as a fourth generation CAR. TRUCKS are CAR-redirected T cells used as vehicles to trigger effector activity of the CAR T cells and in addition produce and release a transgenic cytokine (e.g., IL-12) that accumulates in the targeted tissue (e.g., a tumour tissue that expresses EphA2). The transgenic cytokine is made constitutively or released upon CAR engagement of the target. TRUCK cells may deposit a variety of therapeutic cytokines at the target site. This may result in therapeutic concentrations at the targeted site and avoid systemic toxicity of these same cytokines.

[0223] The CARs of the invention suitably have antigen specificity for EphA2. The phrases “have antigen specificity” and “elicit antigen-specific response” as used herein means that the CAR can specifically bind to and immunologically recognize an antigen, such that binding of the CAR to the antigen elicits an immune response. Without being bound to a particular theory or mechanism, it isbelieved that by eliciting an antigen-specific response against EphA2, the CARs described herein provide for one or more of any of the following: targeting and destroying EphA2-expressing cancer cells, reducing or eliminating cancer cells, facilitating infiltration of immune cells to tumour site(s), and enhancing / extending anti-cancer responses.

[0224] An embodiment of the invention provides a CAR comprising an antigen-binding domain of one of the monoclonal antibodies described herein. In particular embodiments, the CAR comprises an antigen-binding domain of the 4B3 monoclonal antibodies, which specifically bind to EphA2. In this regard, a preferred embodiment of the invention provides CARs comprising an antigen-binding domain comprising, consisting of, or consisting essentially of, a single chain variable fragment (scFv) of the antigen-binding domain of 4B3.

[0225] The antigen-binding domain may comprise a light chain variable region and / or a heavy chain variable region. In an embodiment of the invention, the heavy chain variable region comprises a CDR1 region, a CDR2 region, and a CDR3 region. In this regard, the antigen-binding domain may comprise one or more of a heavy chain CDR1 region comprising any one of SEQ ID NOs: 1-5 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; a heavy chain CDR2 region comprising any one of SEQ ID NOs: 60-10 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and a heavy chain CDR3 region comprising any one of SEQ ID NOs: 11-15, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. Preferably, the heavy chain comprises each of a CDR1 region, a CDR2 region, and a CDR3 region selected from SEQ ID NOs: 1- 5, 6-10, or 11-15, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0226] In an embodiment of the invention, the light chain variable region may comprise a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region. In this regard, the antigen-binding domain may comprise one or more of a light chain CDR1 region comprising any one of SEQ ID NOs: 16-20 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; a light chain CDR2 region comprising any one of SEQ ID NOs: 21-25 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and a light chain CDR3 region comprising any one of SEQ ID NOs: 26-30, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. Preferably, the heavy chain comprises each of a CDR1 region, a CDR2 region, and a CDR3 region selected from SEQID NOs: 15-20, 21-25, or 26-30, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0227] The heavy chain variable region of the antigen-binding domain may comprise, consist of, or consist essentially of, the sequence set forth in SEQ ID NO: 71 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. The light chain variable region of the antigen-binding domain may comprise, consist of, or consist essentially of, SEQ ID NO: 72 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. Accordingly, in an embodiment of the invention, the antigen-binding domain comprises a heavy chain variable region comprising SEQ ID NO: 71 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a light chain variable region comprising SEQ ID NO: 72 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. Preferably, the antigen-binding domain comprises both SEQ ID NOs: 71 and 72 or amino acid sequences at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0228] In an embodiment of the invention, the light chain variable region and the heavy chain variable region may be joined by a spacer or linker sequence. The linker may comprise any suitable amino acid sequence. The linker sequence may comprise glycine and serine amino acid residues. In some embodiments of the invention, the linker may comprise, consist, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 107 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0229] Additionally, the CAR may comprise a further spacer or linker sequence to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain thereof. A flexible spacer or hinge region allows the antigen-binding domain to orient in different directions to enable EphA2 binding. By way of example, hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is the hinge domain that joins the constant domains CH1 and CH2 of an antibody. Accordingly, the further spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a CD8 stalk or hinge, or a combination thereof.

[0230] It is envisaged that the antigen-binding domain can further include a leader or signal peptide sequence. The leader sequence may be a peptide sequence (e.g., about 5, about 10, about 15, about 20, about 25 or about 30 amino acids in length) present at the N-terminus of the newly synthesized protein (e.g., positioned adjacent the heavy chain variable region), which directs the proteininto the secretory pathway. The leader sequence may comprise any suitable leader sequence known in the art, such as those derived from CD8, granulocyte-macrophage colony-stimulating factor (GM- CSF) receptor, CD28, murine kappa chain and CD16 In an embodiment, the leader sequence is a CD8 leader sequence. In this regard, the antigen-binding domain may comprise a leader sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 108 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. In an embodiment of the invention, while the leader sequence may facilitate expression of the CAR on the surface of the cell, the presence of the leader sequence in an expressed CAR is not necessary in order for the CAR to function. Accordingly, upon insertion into the endoplasmic reticulum membrane during translation for expression of the CAR on the cell surface, the leader sequence may be cleaved off from the CAR. As such, in an embodiment of the invention, the CAR lacks a leader sequence.

[0231] The antigen-binding domain of a CAR is commonly fused via a spacer and / or hinge region and transmembrane domain to an endodomain, which comprises or associates with an intracellular or cytoplasmic T cell signalling domain. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T cell it is expressed on. The endodomain is the portion of the CAR involved in signal-transmission and in this manner may comprise one or more co-stimulatory domains and / or one or more intracellular T cell signalling domains.

[0232] Included in the scope of the invention are functional portions of the CARs described herein. The term “functional portion” when used in reference to a CAR refers to any part or fragment of the CAR of the invention, which part or fragment retains the biological activity of the CAR of which it is a part (the parent CAR). Functional portions encompass, for example, those parts of a CAR that retain the ability to recognize target cells, or detect, treat, or prevent a disease, to a similar extent, the same extent, or to a higher extent, as the parent CAR. In reference to the parent CAR, the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent CAR.

[0233] The functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., recognize target cells, detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent CAR.

[0234] Included in the scope of the invention are functional variants of the CARs described herein. The term “functional variant" as used herein refers to a CAR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent CAR, which functional variant retains the biological activity of the CAR of which it is a variant. Functional variants encompass, for example, those variants of the CAR described herein (the parent CAR) that retain the ability to recognize target cells to a similar extent, the same extent, or to a higher extent, as the parent CAR. In reference to the parent CAR, the functional variant can, for instance, be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99% or more identical in amino acid sequence to the parent CAR.

[0235] A functional variant can, for example, comprise the amino acid sequence of the parent CAR with at least one conservative amino acid substitution. Alternatively, or additionally, the functional variants can comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR.

[0236] The CARs of embodiments of the invention (including functional portions and functional variants) can be of any length, i.e., can comprise any number of amino acids, provided that the CARs (or functional portions or functional variants thereof) retain their biological activity (e.g., the ability to specifically bind to antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal, etc). For example, the CAR can be about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0237] Also provided is a cell comprising a CAR according to the invention. The CAR according to the present invention may be used to generate CAR-expressing immune cells, e.g., CAR T cells or CAR NK cells. Engineering of CARs into immune cells may be performed during culture, in vitro.

[0238] The antigen-binding region of the CAR of the present invention may be provided with any suitable format, e.g., scFv, scFab, etc.CAR-expressing cells

[0239] The present disclosure also provides a cell comprising or expressing a CAR according to the present disclosure. Also provided is a cell comprising or expressing a nucleic acid encoding a CAR according to the disclosure. Engineering of CARs into T cells may be performed during culture, invitro, for transduction and expression, such as happens during expansion of T cells for adoptive T cell therapy. Methods for engineering immune cells to express CARs are known to the skilled person and are described, for example, in Wang and Riviere, Mol Ther Oncolytics, (2016) 3: 16015, which is hereby incorporated by reference in its entirety. It will be appreciated that “at least one cell” encompasses a plurality of cells, e.g., a population of such cells.

[0240] The cell comprising or expressing a CAR according to the present disclosure may be a eukaryotic cell, e.g., a mammalian cell. The mammal may be a human, or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse, or other rodent (including any animal in the order Rodentia), cat dog, pig, sheep, goat, cattle (including cows, e.g., dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).

[0241] In some embodiments, the cell may be from, or may have been obtained from, a human subject. Where the CAR-expressing cell is to be used in the treatment of a subject, the cell may be from the subject to be treated with the CAR-expressing cell (i.e., the cell may be autologous), or the cell may be from a different subject (i.e., the cell may be allogeneic).

[0242] In particular embodiments, the cell is or comprises an immune cell. The cell may be a cell of hematopoietic origin, e.g., a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be, e.g., a T cell, B cell, NK cell, NKT cell, or innate lymphoid cell (ILC), or a precursor thereof. The cell may express, e.g., CD3 polypeptides (e.g., CD3y, CD3E, CD3^, or CD36), TCR polypeptides (TCRa or TCR ), CD27, CD28, CD4, or CD8.

[0243] Suitably, the immune cell is or comprises a T cell inclusive of CD4+ helper T cells and / or a CD8+ cytotoxic T cells (e.g., a cytotoxic T- lymphocyte (CTL)). In this regard, the T cell of the present aspect may be in a mixed population of CD4+ helper T cell / CD8+ cytotoxic T cells.

[0244] The use of CAR T cells is associated with advantages that they can be systemically administered, and will home to both primary and metastasized tumours (see, Manzo et al., Human Mol Genetics (2015) R67-73).

[0245] In some embodiments, the cell is an antigen-specific T cell. In embodiments of this type, an “antigen-specific T celf’ is a cell which displays certain functional properties of a T cell in response to the antigen for which the T cell is specific, or a cell expressing said antigen. In some embodiments, the properties are functional properties associated with effector T cells (e.g., cytotoxic T cells).

[0246] In some embodiments, an antigen-specific T cell may display one or more of the following properties: cytotoxicity, e.g., to a cell comprising / expressing antigen for which the T cell is specific; proliferation, IFN-y expression, CD107a expression, IL-2 expression, TNF expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression, e.g., in response to antigen for which the T cell is specific or a cell comprising / expressing antigen for which the T cell is specific. Antigen-specific T cells comprise a TCR capable of recognising a peptide of the antigen for which the T cell is specific when presented by the appropriate MHC molecule. Antigenspecific T cells may be CD4+ T cells and / or CD8+ T cells.

[0247] In some embodiments, the antigen for which the T cell is specific may be a peptide or polypeptide of a virus (e.g., Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Adenovirus, human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV)).

[0248] Advantageously, the isolated CAR of the present invention can be utilised in CAR gene transfer, an approach that is rapid, reliable and capable of generating large quantities of T cells (>108- 1010cells / patient) with specificity to EphA2, regardless of the patient's pre-existing immune repertoire. For example, retroviral or lentiviral transductions may require only 48 hours of culture with pre-activated T cells. Further, large numbers of autologous T cells can be obtained from leukapheresis or isolation of peripheral blood mononuclear cells (PBMC) from a blood sample from a subject. Thus, it may be possible to engineer 108-10® transformed or transfected T cells for infusion in a few days.

[0249] Accordingly, a host cell (e.g., a T cell) of the present invention can be used in the treatment of an EphA2-associated disease, disorder or condition, such as cancer, by means of adoptive transfer. To this end, T cells are typically isolated from a biological sample taken from a subject, inclusive of donor subjects, for use in the adoptive transfer of genetically modified cells.

[0250] Preferably, the T cells transduced ortransformed with the CAR of the present invention contain a mixture of naive, central memory and effector memory cells.

[0251] In alternative embodiments, the host cell is, or is derived from, a stem cell, such as a haemopoietic stem cell (HSC). To this end, the host cell may therefore be a gene-modified stem cell, which, upon differentiation, produces a T cell expressing a CAR of the invention.

[0252] In some embodiments, the host cell, such as a T cell, is genetically engineered to express a cytokine, chemokine and / or a receptor thereof.

[0253] To this end, CAR T cells may be designed in several ways that enhance tumour cytotoxicity and specificity, evade tumour immunosuppression, avoid host rejection, and prolong their therapeutic half-life. TRUCK (T cells Redirected for Universal Cytokine Killing) T cells for example, possess a CAR but are also engineered to express and release cytokines such as IL-12 that promote tumour killing. Because these cells are designed to release a molecular payload upon activation of the CAR once localized to the tumour environment, these CAR T cells are sometimes also referred to as “armoured CARs". Exemplary cytokines include IL-2, IL 3. IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, M-CSF, GM-CSF, IFN-a, IFN-y, TNF, TRAIL, FLT3 ligand, Lymphotactin, and TGF- .

[0254] “Self-driving” or “homing” CART cells are engineered to express a chemokine receptor in addition to their CAR. As certain chemokines can be upregulated in tumours, incorporation of a chemokine receptor aids in tumour trafficking to and infiltration by the adoptive T cell, thereby enhancing both specificity and functionality of the CAR T cell. Universal CAR T cells also possess a CAR, but are engineered such that they do not express endogenous TCR (T cell receptor) or MHC (major histocompatibility complex) proteins. Removal of these two proteins from the signalling repertoire of the adoptive T cell therapy prevents graft-versus-host-disease and rejection, respectively. Armoured CAR T cells are additionally so named for their ability to evade tumour immunosuppression and tumour- induced CAR T cell hypofunction. These particular CAR T cells possess a CAR, and may be engineered to not express inhibitory checkpoint receptors. Alternatively, these CAR T cells can be co-administered with a monoclonal antibody (mAb) that blocks checkpoint signalling. Administration of an anti-PDL1 antibody significantly restored the killing ability of CAR TILs (tumour-infiltrating lymphocytes). While PD1-PDL1 and CTLA-4-CD80 / CD86 signalling pathways have been investigated, it is possible to target other immune checkpoint signalling molecules in the design of an armoured CAR-T including LAG-3, Tim-3, IDO-1 , 2B4, and KIR. Other intracellular inhibitors of TILs include phosphatases (SHP1), ubiquitin-ligases (i.e., cbl-b), and kinases (i.e., diacylglycerol kinase). Armoured CAR T cells may also be engineered to express proteins or receptors that protect them against or make them resistant to the effects of tumour-secreted cytokines. For example, CTLs (cytotoxic T lymphocytes) transduced with the dominant negative form of the TGF- receptor are resistant to the immunosuppression by lymphoma secreted TGF- . These transduced cells showed notably increased anti-tumour activity in vivo when compared to their control counterparts.3. Nucleic acids and vectors

[0255] The present invention provides a nucleic acid, or a plurality of nucleic acids, encoding an antigen-binding molecule according to the present invention.

[0256] In some embodiments, the nucleic acid is purified or isolated, e.g., from other nucleic acids, or naturally-occurring biological material. In some embodiments the nucleic acid(s) comprise or consist of DNA and / or RNA.

[0257] Thus, in another aspect, the present invention contemplates isolated nucleic acids that encode, or are complementary to a nucleic acid sequence which encodes, the isolated proteins (e.g., antibody and CAR proteins, inclusive of fragments, variants and derivatives thereof) disclosed herein.

[0258] Nucleotide sequences encoding the isolated proteins of the invention may be readily deduced from one or more of the complete nucleic acid sequences provided herein (see, e.g., SEQ ID NOs: 73-78), although without limitation thereto.

[0259] This aspect also includes fragments, variants and derivatives of said isolated nucleic acid, such as those herein before described.

[0260] The term “nucleic acid" as used herein designates single- or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and / or thiouridine, although without limitation thereto.

[0261] Accordingly, in particular embodiments, the isolated nucleic acid is cDNA.

[0262] A “polynucleotide" is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “oligonucleotide" has less than eighty (80) contiguous nucleotides.

[0263] A “probe" may be a single or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

[0264] A “primer1’ is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid “template" and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SEQUENASE™

[0265] In some embodiments, nucleic acid variants encode a variant of an isolated protein of the invention.

[0266] In some other embodiments, nucleic acid variants share at least 40%, 45%, 50%, 55%, 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74% or 75%, more preferably at least 80%, 81%, 82%, 83%, 84%, or 85%, and even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with an isolated nucleic acid of the invention.

[0267] In some embodiments, the isolated nucleic acid of the present aspect consists of: (a) a nucleic acid that: (i) encodes a segment, domain, portion or region of an antibody and / or an isolated CAR protein described herein, such as those according to SEQ ID NOs: 1 to 72, and inclusive of variants or derivatives thereof; and (b) optionally one or more additional nucleic acid sequences. In this regard, the additional nucleic acid sequences can be heterologous nucleic acid sequences that can be at the 5’ (5-prime) and / or 3’ (3-prime) ends of the isolated nucleic acid sequence, although without limitation thereto.

[0268] The present invention also contemplates nucleic acids that have been modified such as by taking advantage of codon sequence redundancy. In a more particular example, codon usage may be modified to optimize expression of a nucleic acid in a particular organism or cell type.

[0269] The invention further provides use of modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (for example, thiouridine and methylcytosine) in nucleic acids of the invention.

[0270] It will be well appreciated by a person of skill in the art that the isolated nucleic acids of the invention can be conveniently prepared using standard protocols such as those described in Chapter 2 and Chapter 3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 1995-2008).

[0271] In yet another embodiment, complementary nucleic acids hybridise to nucleic acids of the invention under high stringency conditions.

[0272] “Hybridise" and “Hybridisation" is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA, RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing.

[0273] “Stringency" as used herein, refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and / or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridizing nucleotide sequences.

[0274] “Stringent conditions" designates those conditions under which only nucleic acid having a high frequency of complementary bases will hybridize.

[0275] Stringent conditions are well-known in the art, such as described in Chapters 2.9 and 2.10 of Ausubel et al., supra, which are herein incorporated by reference. A skilled addressee will also recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization.

[0276] Complementary nucleotide sequences may be identified by blotting techniques that include a step whereby nucleotides are immobilized on a matrix (preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step, typically using a labelled probe or other complementary nucleic acid. Southern blotting is used to identify a complementary DNA sequence; Northern blotting is used to identify a complementary RNA sequence. Dot blotting and slot blotting can be used to identify complementary DNA / DNA, DNA / RNA or RNA / RNA polynucleotide sequences. Such techniques are well known by those skilled in the art, and have been described in Ausubel et al., supra, at pages 2.9.1 through 2.9.20. According to such methods, Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridizing the membrane bound DNA to a complementary nucleotide sequence. An alternative blotting step is used when identifying complementary nucleic acids in a cDNA or genomic DNA library, such as through the process of plaque or colony hybridization. Other typical examples of this procedure are described in Chapters 8-12 of Sambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989).

[0277] Methods for detecting labelled nucleic acids hybridized to an immobilized nucleic acid are well known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection.

[0278] Nucleic acids may also be isolated, detected and / or subjected to recombinant DNA technology using nucleic acid sequence amplification techniques.

[0279] Suitable nucleic acid amplification techniques covering both thermal and isothermal methods are well known to the skilled addressee, and include polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q- replicase amplification, recombinase polymerase amplification (RPA) and helicase-dependent amplification, although without limitation thereto.

[0280] As used herein, an “amplification product’ refers to a nucleic acid product generated by nucleic acid amplification.

[0281] Nucleic acid amplification techniques may include particular quantitative and semi- quantitative techniques such as qPCR, real-time PCR, droplet PCR and competitive PCR, as are well known in the art.

[0282] In some embodiments, the nucleic acid may be in a genetic construct that facilitates delivery and expression of the nucleic acid. In some embodiments, the present invention provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present invention.

[0283] Accordingly, in yet another aspect, the invention provides a genetic construct comprising: (i) the isolated nucleic acid described herein; or (ii) an isolated nucleic acid comprising a nucleotide sequence complementary thereto. In one embodiment, the isolated nucleic acid is operably linked or connected to one or more regulatory sequences in a vector (e.g., an expression vector).

[0284] Suitably, the genetic construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology and / or expression of the nucleic acid or an encoded protein of the invention.

[0285] For the purposes of host cell expression, the genetic construct can be an expression construct. Suitably, the expression construct comprises the nucleic acid of the invention operably linked to one or more additional sequences in an expression vector. A “vector” as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. An “expression vector” may be either a selfreplicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome. In this regard, the vector may be capable of transferring a nucleic acid of the invention to a host cell, such as a T cell, such that the cell expresses an EphA2-binding molecule. To this end, the vector should ideally be capable of sustained high-level expression in T cells.

[0286] Such vectors may include a promotor sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers.

[0287] Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

[0288] Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.

[0289] Constitutive or inducible promoters as known in the art are contemplated by the invention.

[0290] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors, transposon-based vectors, and artificial chromosomes.

[0291] In particular embodiments, the expression vector is or comprises one or more viral delivery systems, such as adenovirus vectors, an adeno-associated virus (AAV) vectors, a herpesvirus vectors, a retrovirus vectors (e.g., gammaretroviral vectors (e.g., murine Leukemia virus (MLV) -de rived vectors)), a lentiviral vectors, vaccinia virus vectors, and baculoviral vectors.

[0292] In some embodiments, the vector may be a eukaryotic vector, e.g., a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g., comprising a cytomegalovirus (CMV) or SV40 promotor to drive protein expression.

[0293] In a further aspect, the invention provides a host cell transfected or transformed with a nucleic acid molecule or a genetic construct described herein.

[0294] Suitable host cells for expression may be prokaryotic or eukaryotic. For example, suitable host cells may include but are not limited to mammalian cells (e.g. HeLa, HEK293T, Jurkat cells), yeast cells (e.g., Saccharomyces cerevisiae), insect cells (e.g., Sf9, Trichoplusia ni) utilized with or without a baculovirus expression system, plant cells (e.g., Chlamydomonas reinhardtii, Phaeodactylum tricornutum) or bacterial cells, such as E. coli. Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, as for example described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 9 and 16.4. Generating EphA2 Antibodies

[0295] In still a further aspect, the invention resides in an antibody or antibody fragment which binds and / or is raised against:(i) the EphA2-binding molecule of the first mentioned aspect; and / or(ii) the CAR of the second mentioned aspect, inclusive of fragments, variants and derivatives thereof.

[0296] Suitably, said antibody or antibody fragment specifically binds said isolated EphA2- binding molecule or CAR. Preferably, the antibody or antibody fragment specifically or selectively binds or recognizes a full or partial amino acid sequence of a CDR, a VH domain and / or a VL domain described herein (e.g., SEQ ID NOs: 1-72). In this regard, the antibody or antibody fragment of the present aspect may be suitable for use in methods of detecting or isolating a T cell that expresses the CAR having that particular CDR, VH domain or VL domain in a sample. To this end, antibodies and antibody fragments of the invention may be particularly suitable for affinity chromatography purification of the isolated EphA2-binding molecules and CARs described herein. For example, reference may be made to affinity chromatographic procedures described in Chapter 9.5 of Coligan et al., supra.

[0297] Antibodies may be polyclonal or monoclonal, native or recombinant. Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al., supra; and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, which are both herein incorporated by reference.

[0298] Generally, antibodies of the invention bind to or conjugate with an isolated protein, fragment, variant, or derivative of the invention. For example, the antibodies may be polyclonal antibodies. Such antibodies may be prepared for example by injecting an isolated protein, fragment, variant or derivative of the invention into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al., supra, and in Harlow & Lane, 1988, supra.

[0299] Monoclonal antibodies may be produced using the standard method as for example, described in an article by Kohler & Milstein, 1975, Nature 256, 495, which is herein incorporated by reference, or by more recent modifications thereof as for example, described in Coligan et al., supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the isolated proteins, fragments, variants or derivatives of the invention.5. Pharmaceutical Compositions

[0300] In another aspect, pharmaceutical compositions are provided that comprise a EphA2- binding molecule as described herein and a pharmaceutically acceptable carrier or diluent. In typical embodiments, the pharmaceutical composition is sterile.

[0301] Accordingly, provided herein is a composition (e.g., a pharmaceutical composition) comprising a EphA2-binding molecule, or preparation thereof, formulated together with a pharmaceutical carrier, as well as methods of administering such pharmaceutical compositions.

[0302] In various embodiments, the pharmaceutical composition comprises the EphA2- binding molecule at a concentration of 0.1 mg / mL to 100 mg / mL. In specific embodiments, the pharmaceutical composition comprises the EphA2-binding molecule at a concentration of 0.5 mg / mL, 1 mg / mL, 1 .5 mg / mL, 2 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, or 10 mg / mL. In some embodiments, the pharmaceutical composition comprises the EphA2-binding molecule at a concentration of more than 10 mg / mL. In certain embodiments, the EphA2-binding molecule is present at a concentration of 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or even 50 mg / mL or higher. In particular embodiments, the EphA2-binding molecule is present at a concentration of more than 50 mg / mL.

[0303] In various embodiments, the pharmaceutical compositions are described in more detail in U.S. Pat No.8,961 ,964, U.S. Pat No.8, 945, 865, U.S. Pat. No. 8,420,081 , U.S. Pat. No. 6,685,940, U.S. Pat. No. 6,171 ,586, U.S. Pat. No. 8,821 ,865, U.S. Pat. No. 9,216,219, U.S. Pat. Appl. No. 10 / 813,483, and International Patent Publication Nos. WO2014 / 066468, WO2011 / 104381 , and WO2016 / 180941 , each of which is incorporated herein in its entirety.

[0304] In some embodiments, the composition may further comprise an adjuvant.

[0305] Methods of preparing these formulations or compositions include the step of bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0306] Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0307] Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate, vegetable oils (such as olive oil),synthetic oils, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulphates, organic acids such as acetates, propionates and malonates and pyrogen-free water. Further examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, and suitable mixtures thereof, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0308] Regardless of the route of administration selected, the agents of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

[0309] A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington’s Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference.6. Methods of Treatment

[0310] In another aspect, methods of treatment are provided, the methods comprising administering an activating EphA2-binding molecule as described herein to a patient with a disease or condition in an amount effective to treat the patient.Subjects

[0311] In some embodiments, the subject can be a mammal. In some embodiments, the mammal is a mouse. In a preferred embodiment, the mammal is a human.Combination therapy

[0312] The EphA2-binding molecule can be used alone or in combination with other therapeutic agents or procedures to treat or prevent a disease or condition. The EphA2-binding molecule can be administered either simultaneously or sequentially with a second therapeutic agent, dependent upon the disease to be treated.

[0313] In some embodiments, the anti-EphA2-binding molecules is used in combination with an agent or procedure that is used in the clinic or is within the current standard of care to treat or prevent a disease or condition, such as proliferative disease or cancer. In some embodiments, the EphA2-binding molecule is administered in combination with an immune checkpoint inhibitor, such as an anti- PD-L1 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-LAB3 antibody, anti-TIM1 antibody, anti-TIGIT antibody, anti-PVRIG antibody.Proliferative Diseases

[0314] In some embodiments, the treatment comprises administration one or more activating EphA2-binding molecule as described herein to a subject with a proliferative disease in an amount effective to treat the subject.

[0315] In some embodiments, the treatment comprises administration of an effective amount of one or more activating EphA2-binding molecules as described herein for the treatment of cancer and / or precancer. In some embodiments, the treatment comprises administration of an effective amount of one or more activating EphA2-binding molecules as described herein, in combination with another cancer therapeutic and / or treatment regimen (radiation, surgery, or the like, etc.). Cancers may include any aggressive or potentially aggressive cancers, tumours or other malignancies such as listed in the NCI Cancer Index at http: / / www.cancer.gov / cancertopics / alphalist, including all major cancer forms

[0316] In various embodiments, the cancer is a cancer of the bladder, blood, bone, bone marrow, brain, breast, colon, oesophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, head and neck, ovary, prostate, pancreas, skin, stomach, testis, tongue, or uterus.

[0317] In some embodiments, the cancerous or pre-cancerous tumour is a neoplasm, malignant tumour, carcinoma, undifferentiated tumour, giant and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, head and neck squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, gastrinoma, malignant, cholangiocarcinoma, hepatocellular carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in adenomatous polyp, adenocarcinoma, familial polyposis coli, solid carcinoma, carcinoid tumour, malignant, branchiolo-alveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, acidophil carcinoma, oxyphilic adenocarcinoma, basophil carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, non-encapsulating sclerosing carcinoma, adrenal cortical carcinoma, endometroid carcinoma, skin appendage carcinoma, apocrine adenocarcinoma, sebaceous adenocarcinoma, ceruminous adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma,cystadenocarcinomas, pancreatic neuroendocrine tumours (PanNETs), adenosquamous carcinomas of the pancreas, signet ring cell carcinomas of the pancreas, hepatoid carcinomas of the pancreas, colloid carcinomas of the pancreas, undifferentiated carcinomas of the pancreas, and undifferentiated carcinomas with osteoclast-like giant cells of the pancreas, acinar cell carcinomas of the pancreas, solid pseudopapillary neoplasms of the pancreas, pancreatoblastoma, rare exocrine cancers of the pancreas, pancreatic serous cystadenomas, pancreatic mucinous cystic neoplasms, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, infiltrating duct carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease, mammary, acinar cell carcinoma, adenosquamous carcinoma, adenocarcinoma w / squamous metaplasia, thymoma, malignant, ovarian stromal tumour, malignant thecoma, malignant granulosa cell tumour, malignant androblastoma, malignant Sertoli cell carcinoma, Leydig cell tumour, malignant lipid cell tumour, malignant paraganglioma, malignant extra-mammary paraganglioma, malignant pheochromocytoma, glomangiosarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, melanoma in giant pigmented nevus, epithelioid cell melanoma, blue nevus, malignant sarcoma, fibrosarcoma, fibrous histiocytoma, malignant myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, mixed tumour, malignant mullerian mixed tumour, nephroblastoma, hepatoblastoma, carcinosarcoma, mesenchymoma, malignant Brennertumour, malignant phyllodes tumour, malignant synovial sarcoma, mesothelioma, malignant dysgerminoma, embryonal carcinoma, teratoma, malignant struma ovarii, malignant choriocarcinoma, mesonephroma, malignant hemangiosarcoma, hemangioendothelioma, malignant Kaposi's sarcoma, hemangiopericytoma, malignant, lymphangiosarcoma, osteosarcoma, juxtacortical osteosarcoma, chondrosarcoma, chondroblastoma, malignant mesenchymal chondrosarcoma, giant cell tumour of bone, Ewing's sarcoma, odontogenic tumour, malignant ameloblastic odontosarcoma, ameloblastoma, malignant ameloblastic fibrosarcoma, pinealoma, malignant chordoma, glioma, malignant ependymoma, astrocytoma, protoplasmic astrocytoma, fibrillary astrocytoma, astro blastoma, glioblastoma, oligodendroglioma, oligodendroblastoma, primitive neuroectodermal, cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumour, meningioma, malignant, neurofibrosarcoma, neurilemmoma, malignant granular cell tumour, malignant lymphoma, Hodgkin's disease, Hodgkin's paragranuloma, malignant lymphoma, small lymphocytic, malignant lymphoma, large cell, diffuse, malignant lymphoma, follicular, mycosis fungoides, other specified Non-Hodgkin's lymphomas, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphoid leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblasticleukemia, myeloid sarcoma, or hairy cell leukemia. In some embodiments, the cancer is a solid cancer, such as glioblastoma multiforme. Suitably, the cancer expresses, such as overexpresses, EphA2.

[0318] In some embodiments, the cancer is a viral-induced cancer, for example, a cancer caused by an infection from a oncovirus or a tumour virus (which are also known as a “cancer virus”). In some embodiments, the cancer virus is a DNA virus. In some embodiments, the cancer virus is an RNA virus.

[0319] In some embodiments, the cancerous or pre-cancerous tumour is associated or caused by a cancer virus. Non-limiting examples of a cancer virus include: an Epstein-Barr virus (EBV), a hepatitis B virus, a hepatitis C virus, a human papilloma virus, a human T-lymphotropic virus 1 (HTLV- 1), a Kaposi sarcoma associated-herpesvirus (KHSV), a Merkel cell polyomavirus, or a cytomegalovirus.

[0320] In some embodiments, the cancerous or pre-cancerous tumour is associated or caused by a cancer virus that directly induces transformation of the infected host cell, thereby regulating the host cell’s growth and survival or alternatively initiating a DNA damage response which in turn increases genetic instability and accelerates the acquisition of the cancer-causing mutations in the genome of the host cell.

[0321] In some embodiments, the cancerous or pre-cancerous tumour is associated or caused by a cancer virus that induces chronic inflammation in a host. For example, infections with HBV and HCV can induce chronic liver inflammation associated with oxidative DNA damage followed by cirrhosis resulting in some cases in the development of hepatocellular carcinoma.

[0322] In some embodiments, the cancerous or pre-cancerous tumour is associated or caused by a cancer virus that is not oncogenic but inhibits the host’s immune system, disrupting immunosurveillance and thereby allowing for the emergence of mutated malignant cells, for example HIV-infected patients.

[0323] In some embodiments, the treatment comprises administration one or more EphA2- binding molecules as described herein to a subject with an infectious disease(s), such as infection with HIV, HCV, HBV, EBV, or HPV.

[0324] In some embodiments, the treatment comprises administration one or more EphA2- binding molecules as described herein to a subject with HIV or AIDs in an amount effective to treat the subject.Administration

[0325] The EphA2-binding molecule may be administered to a subject by any route known in the art. For example, the EphA2-binding molecule is administered to a human subject via, e.g., intravenous, subcutaneous, intramuscular, intradermal, intraarterial, intraperitoneal, intranasal, parenteral, pulmonary, topical, oral, sublingual, intratumoural, peritumoural, intralesional, intrasynovial, intrathecal, intra-cerebrospinal, or perilesional administration. The EphA2-binding molecule may be administered to a subject per se or as a pharmaceutical composition. Exemplary pharmaceutical compositions are described herein.

[0326] Methods of treating cancer may be prophylactic, preventative or therapeutic and suitable for treatment of cancer in mammals, particularly humans.

[0327] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for reducing, alleviating and / or preventing a cancer will be dependent on the subject being treated, the type and severity of any associated disease, disorder and / or condition (e.g., the number and location of any associated metastases), and the manner of administration of the therapeutic composition.

[0328] It will be appreciated that the method of the present aspect may include one or more further cancer treatments in addition to those recited above. Such cancer treatments may include drug therapy, chemotherapy, antibody, nucleic acid and other biomolecular therapies, radiation therapy, surgery, nutritional therapy, relaxation or meditational therapy and other natural or holistic therapies, although without limitation thereto. Generally, drugs, biomolecules (e.g., antibodies, inhibitory nucleic acids such as siRNA) or chemotherapeutic agents are referred to herein as “anti-cancer therapeutic agents" or “anti-cancer agents" .

[0329] In some embodiments, the subject is also administered an anti-cancer compound. Exemplary anti-cancer compounds include, but are not limited to, Alemtuzumab (Campath®), Alitretinoin (Panretin®), Anastrozole (Arimidex®), Bevacizumab (Avastin®), Bexarotene (Targretin®), Bortezomib (Velcade®), Bosutinib (Bosulif®), Brentuximab vedotin (Adcetris®), Cabozantinib (Cometriq™), Carfilzomib (Kyprolis™), Cetuximab (Erbitux®), Crizotinib (Xalkori®), Dasatinib (Sprycel®), Denileukin diftitox (Ontak®), Erlotinib hydrochloride (Tarceva®), Everolimus (Afmitor®), Exemestane (Aromasin®), Fulvestrant (Faslodex®), Gefitinib (Iressa®), Ibritumomab tiuxetan (Zevalin®), Imatinib mesylate (Gleevec®), Ipilimumab (Yervoy™), Lapatinib ditosylate (Tykerb®), Letrozole (Femara®), Nilotinib (Tasigna®), Ofatumumab (Arzerra®), Panitumumab (Vectibix®),Pazopanib hydrochloride (Votrient®), Pertuzumab (Peijeta™), Pralatrexate (Folotyn®), Regorafenib (Stivarga®), Rituximab (Rituxan®), Romidepsin (Istodax®), Sorafenib tosylate (Nexavar®), Sunitinib malate (Sutent®), Tamoxifen, Temsirolimus (Torisel®), Toremifene (Fareston®), Tositumomab and I3 ll-tositumomab (Bexxar®), Trastuzumab (Herceptin®), Tretinoin (Vesanoid®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®), Vorinostat (Zolinza®), and Ziv-aflibercept (Zaltrap®).

[0330] In some embodiments, the subject is also administered a chemotherapeutic agent. Examples of such chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma (1 ,1) and calicheamicin omega (1 ,1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene;edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0331] In some embodiments, the subject is also administered an immunotherapeutic agent. Immunotherapy refers to a treatment that uses a subject’s immune system to treat or prevent a condition, e.g. cancer vaccines, cytokines, use of target-specific antibodies, T cell therapy, NK cell therapy, and dendritic cell therapy.

[0332] In some embodiments, the subject is also administered an immune modulatory protein. Examples of immune modulatory proteins include, but are not limited to, B lymphocyte chemoattractant (“BLC”), C-C motif chemokine 11 (“Eotaxin-1 ”), Eosinophil chemotactic protein 2 ("Eotaxin-2"), Granulocyte colony-stimulating factor (“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”), 1-309, Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon gamma (“IFN-y”), lnterleukin-1 alpha (IL-1 a”), lnterleukin-1 beta (“IL-1 P”), Interleukin 1 receptor antagonist (“IL-1 ra”), lnterleukin-2 (“IL 2”), lnterleukin-4 (“IL-4”), lnterleukin-5 (“IL 5”), lnterleukin-6 (“IL-6”), lnterleukin-6 soluble receptor (“IL-6 sR”), lnterleukin-7 (“IL-7”), lnterleukin-8 (“IL-8”), Interleukin- 10 (“IL-10”), lnterleukin-11 (“IL-11”), Subunit beta of Interleukin-12 (“IL-12 p40” or “"IL-12 p70”), Interleukin-13 (“IL- 13”), Interleukin-15 (“IL-15”), Interleukin-16 (“IL-16”), Interleukin 17 (“IL-17”), Chemokine (C-C motif) Ligand 2 (“MCP-1”), Macrophage colony-stimulating factor (“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-C motif) ligand 2 (“MIP-1a”), Chemokine (C-C motif) ligand 4 (“MIP- 1 P”), Macrophage inflammatory protein-1 -delta ("MIP-16"), Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C-C motif) ligand 5, Regulated on Activation, Normal T cell Expressed and Secreted (“RANTES”), TEMP metallopeptidase inhibitor 1 (“TIMP-1”), TIMP metallopeptidase inhibitor 2 (“TIMP-2”), Tumour necrosis factor, (“TNF”), Tumour necrosis factor, lymphotoxin-beta (“TNF-P”), Soluble TNF receptor type 1 (“sTNFRI”), sTNFRIIAR, Brain-derived neurotrophic factor (“BDNF”), Basicfibroblast growth factor (“bFGF”), Bone morphogenetic protein 4 (“BMP-4”), Bone morphogenetic protein 5 (“BMP-5”), Bone morphogenetic protein 7 (“BMP-7”), Nerve growth factor (“b-NGF”), Epidermal growth factor (“EGF”), Epidermal growth factor receptor (“EGFR”), Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”), Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor (“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glial cell-derived neurotrophic factor (“GDNF”), Growth Hormone, Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growth factor (“HGF”), Insulin-like growth factor binding protein 1 (“IGFBP-1 ”), Insulin-like growth factor binding protein 2 (“IGFBP-2”), Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-like growth factor binding protein 4 ("IGFBP-4"), Insulin-like growth factor binding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1 ”), Insulin, Macrophage colony-stimulating factor (“M-CSFR”), Nerve growth factor receptor (“NGFR”), Neurotrophin-3 (“NT-3”), Neurotrophin-4 (“NT -4"), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”), Platelet-derived growth factor receptors (“PDGF-AA”), Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-box containing complex (“SCF”), Stem cell factor receptor (“SCFR”), Transforming growth factor alpha (“TGFa”), Transforming growth factor beta-1 (“TGFpi”), Transforming growth factor beta-3 (“TGFP3”), Vascular endothelial growth factor (“VEGF”), Vascular endothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelial growth factor receptor 3 (“VEGFR3”), VEGF-D 6Ckine, Tyrosine-protein kinase receptor FIFO (“Axl”), Betacellulin (“BTC”), Mucosae-associated epithelial chemokine (“CCL28”), Chemokine (C-C motif) ligand 27 (“CTACK”), Chemokine (C-X-C motif) ligand 16 (“CXCL16”), C-X-C motif chemokine 5 (“ENA- 78”), Chemokine (C-C motif) ligand 26 (“Eotaxin-3”), Granulocyte chemotactic protein 2 (“GCP-2”), GRO, Chemokine (C-C motif) ligand 14 (“HCC-1 ”), Chemokine (C-C motif) ligand 16 (“HCC-4”), lnterleukin-9 (“IL-9”), lnterleukin-17F (“IL-17F”), lnterleukin-18-binding protein (“IL-18 BPa”), lnterleukin-28A (“IL-28A”), Interleukin 29 (“IL-29”), Interleukin-31 (“IL-31 ”), C-X-C motif chemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“l-TAC”), Leukaemia inhibitory factor (“LIF”), Light, Chemokine (C motif) ligand (“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocyte chemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4 (“MCP-4”), Macrophage- derived chemokine (“MDC”), Macrophage migration inhibitory factor (“MIF”), Chemokine (C-C motif) ligand 20 (“MIP-3a”), C-C motif chemokine 19 (“MIP-3 ”), Chemokine (C-C motif) ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain (“MSPa”), Nucleosome assembly protein 1 -like 4 (“NAP- 2”), Secreted phosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulated cytokine (“PARC”), Platelet factor 4 (“PF4”), Stroma cell-derived factor-1 alpha (“SDF-1a”), Chemokine (C-C motif) ligand 17 (“TRC”), Thymus-expressed chemokine (“TECK”), Thymic stromal lymphopoietin (“TSLP 4-IBB”), CD 166 antigen (“ALCAM”), Cluster of Differentiation 80 (“B7-1”), Tumour necrosis factor receptor superfamily member 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster of Differentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40 Ligand”), Carcinoembryonic antigen- related cell adhesion molecule 1 (biliary glycoprotein) (“CEACAM-I”), Death Receptor 6 (“DR6”),Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein (“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”), Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosis antigen1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumour necrosis factor receptor superfamily member 1 (“GITR”), Tumour necrosis factor receptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule 3 (“ICAM-3”), IL-1 R4, IL-1 Rl, IL-1OR0, IL-17R, IL-2Ry, IL-21 R, Lysosome membrane protein2 (“LI MRU”), Neutrophil gelatinase-associated lipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium (“LYVE-1”), MHC class I polypeptide-related sequence A (“MICA”), MHC class I polypeptide-related sequence B (“MICB”), NRGI-0I, Beta-type platelet-derived growth factor receptor (“PDGF R0”), Platelet endothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A virus cellular receptor 1 (“TIM-1 ”), Tumour necrosis factor receptor superfamily member IOC (“TRAIL R3”), Trappin protein transglutaminase binding domain (“Trappin 2”), Urokinase receptor (“uPAR”), Vascular cell adhesion protein 1 (“VCAM-1”), XEDAR, Activin A, Agouti-related protein (“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1 , Angiostatin, Cathepsin S, CD40, Cryptic family protein IB (“Cripto-1 ”), DAN, Dickkopf-related protein 1 (“DKK-1 ”), E-Cadherin, Epithelial cell adhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), Fcg RIIB / C, FoUistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”), IL-13 Rl, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, Neuronal cell adhesion molecule (“NrCAM”), Plasminogen activator inhibitor- 1 (“PAI-1”), Platelet derived growth factor receptors (“PDGF-AB”), Resistin, stromal cell-derived factor 1 (“SDF-10”), sgpl30, Secreted frizzled-related protein 2 (“ShhN”), Sialic acid-binding immunoglobulin-type lectins (“Siglec-5”), ST2, Transforming growth factor-beta 2 (“TGF 2”), Tie-2, Thrombopoietin (“TPO”), Tumour necrosis factor receptor superfamily member 10D (“TRAIL R4”), Triggering receptor expressed on myeloid cells 1 (“TREM 1 ”), Vascular endothelial growth factor C (“VEGF-C”), VEGFR1 , Adiponectin, Adipsin (“AND”), Alphafetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4”), Beta-2-microglobulin (“02M”), Basal cell adhesion molecule (“BCAM”), Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3”), Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), Human Epidermal Growth Factor Receptor 2 (“ErbB2”), FoUistatin, Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand1 ("GROa), human chorionic gonadotropin ("0 HCG"), Insulin-like growth factor 1 receptor ("IGF-1 sR"), IL 1 sRII, IL-3, IL-18 R0, IL-21 , Leptin, Matrix metalloproteinase-1 (“MMP-1 ”), Matrix metalloproteinase-2 (“MMP-2”), Matrix metalloproteinase-3 (“MMP-3”), Matrix metalloproteinase-8 (“MMP-8”), Matrix metalloproteinase-9 (“MMP-9”), Matrix metalloproteinase- 10 (“MMP-10”), Matrix metalloproteinase- 13 (“MMP-13”), Neural Cell Adhesion Molecule (“NCAM-I”), Entactin (“Nidogen-1”), Neuron specific enolase (“NSE”), Oncostatin M (“OSM”), Procalcitonin, Prolactin, Prostate specific antigen (“PSA”), Sialic acid-binding Ig-like lectin 9 (“Siglec-9”), ADAM 17 endopeptidase (“TACE”), Thyroglobulin, Metalloproteinase inhibitor 4 (“TIMP-4”), TSH2B4, Disintegrin and metalloproteinase domaincontaining protein 9 (“ADAM-9”), Angiopoietin 2, tumour necrosis factor ligand superfamily member 13, Acidic leucine-rich nuclear phosphoprotein 32 family member B (“APRIL”), Bone morphogenetic protein2 (“BMP-2”), bone morphogenetic protein 9 (“BMP-9”), Complement component 5a (“C5a”), Cathepsin L, CD200, CD97, Chemerin, Tumour necrosis factor receptor superfamily member 6B (“DcR3”), Fatty acid-binding protein 2 (“FABP2”), Fibroblast activation protein, alpha (“FAP”), Fibroblast growth factor 19 (“FGF-19”), Galectin-3, Hepatocyte growth factor receptor (“HGFR”), IFN-a / p R2, Insulin-like growth factor 2 (“IGF-2”), Insulin-like growth factor 2 receptor (“IGF-2R”), lnterleukin-1 receptor 6 (“IL-1 R6”), Interleukin 24 (“IL-24”), Interleukin 33 (“IL-33”, Kallikrein 14, Asparaginyl endopeptidase (“Legumain”), Oxidized low-density lipoprotein receptor 1 (“LOX-1”), Mannose-binding lectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1 , translocation-associated (Drosophila) (“Notch-1 ”), Nephroblastoma overexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1 (“PD-1 ”), N-acetylmuramoyl-L- alanine amidase (“PGRP-5”), Serpin A4, Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin, Toll-like receptor 2 (“TLR2”), Tumour necrosis factor receptor superfamily member 10A (“TRAIL Rl”), Transferrin (“TRF”), WIF-1 ACE-2, Albumin, AMICA, Angiopoietin 4, B-cell activating factor (“BAFF”), Carbohydrate antigen 19-9 (“CA19-9”), CD163, Clusterin, CRT AM, Chemokine (C-X- C motif) ligand 14 (“CXCL14”), Cystatin C, Decorin (“DCN”), Dickkopf-related protein 3 (“Dkk-3”), Deltalike protein 1 (“DLL1”), Fetuin A, Heparin-binding growth factor 1 (“aFGF”), Folate receptor alpha (“FOLR1”), Furin, GPCR-associated sorting protein 1 (“GASP-I”), GPCR-associated sorting protein 2 (“GASP-2”), Granulocyte colony-stimulating factor receptor (“GCSFR”), Serine protease hepsin (“HAI- 2”), lnterleukin-17B Receptor (“IL-17B R”), Interleukin 27 (“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V (“LDL R”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST, Heparan sulphate proteoglycan (“Syndecan-1”), Tumour necrosis factor receptor superfamily member 13B (“TACI”), Tissue factor pathway inhibitor (“TFPI”), TSP-I, Tumour necrosis factor receptor superfamily, member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase Plasminogen Activator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascular endothelial) also known as CD144 (“VE- Cadherin”), WNTI-inducible-signalling pathway protein 1 (“WISP-1”), and Receptor Activator of Nuclear Factor k B (“RANK”).

[0333] In some embodiments, the subject is also administered an immune checkpoint inhibitor. Immune checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response. Examples of immune checkpoint proteins include, but are not limited to, CTLA4, PD-1 , PD-L1 , PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. Immune checkpoint inhibitors can be antibodies or antigen-binding fragments thereof that bind to and inhibit an immune checkpoint protein. Examples of immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, atezolizumab, ipilimumab, AMP- 224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.

[0334] In some embodiments, a composition provided herein (e.g., a vaccine composition provided herein) is administered prophylactically to prevent cancer. In some embodiments, the vaccine is administered to inhibit tumour cell expansion. The vaccine may be administered prior to or after the detection of cancer cells in a patient. Inhibition of tumour cell expansion is understood to refer to preventing, stopping, slowing the growth, or killing of tumour cells. In some embodiments, after administration of a vaccine comprising peptides, nucleic acids, antibodies or APCs described herein, a proinflammatory response is induced. The proinflammatory immune response comprises production of proinflammatory cytokines and / or chemokines, for example, IFN-y and / or IL-2. Proinflammatory cytokines and chemokines are well known in the art.

[0335] Combination therapy includes sequential, simultaneous and separate, and / or coadministration of the active compounds in such a way that the therapeutic effects of the first agent administered have not entirely disappeared when the subsequent treatment is administered. In some embodiments, the second agent may be co-formulated with the first agent or be formulated in a separate pharmaceutical composition.

[0336] Administration of the activating EphA2-binding molecule, CAR or variant thereof, or an encoding nucleic acid, or a genetic construct, or cell, or a composition comprising same, may be by any known parenteral, topical or enteral route inclusive of intravenous, intramuscular, intraperitoneal, intracranial, transdermal, oral, intranasal, anal and intra-ocular, although without limitation thereto.

[0337] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and / or microspheres.

[0338] Compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared byuniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0339] In another related aspect, the invention resides in use of the EphA2-binding molecule described herein, the CAR described herein, the isolated nucleic acid described herein, the genetic construct described herein and / or the host cell described herein in the manufacture of a medicament for the prevention and / or treatment of a cancer in a subject.

[0340] In some specific embodiments, the cancer is prostate cancer.7. Labels and conjugates

[0341] In still another aspect, the invention provides a method of detecting EphA2 or a cell expressing EphA2, said method including the step of forming a complex between the EphA2-binding molecule orthe CAR hereinbefore described and EphA2 to thereby detect EphA2 orthe cell expressing EphA2.

[0342] In some embodiments, the method includes the initial step of contacting the EphA2 or the cell expressing EphA2 with the EphA2 antigen-binding molecule or the CAR described above or elsewhere herein.

[0343] Thus, in some embodiments the antigen-binding molecule of the present invention may additionally comprise a detectable moiety.

[0344] In certain embodiments, the cell is or comprises a cancer cell.

[0345] It will therefore be understood that an activating EphA2-binding molecule or CAR disclosed herein may be used to assist medical diagnosis of cancer. Suitably, the method includes detecting EphA2, such as when expressed by cancer cells present in, or obtained from, a biological sample. In certain embodiments, the biological sample may be a pathology sample that comprises one or more fluids, cells, tissues, organs or organ samples obtained from a human. Non-limiting examples include blood, plasma, saliva, serum, lymphocytes, urine, faeces, amniotic fluid, cervical samples, cerebrospinal fluid, tissue biopsies, bone marrow and skin, although without limitation thereto.

[0346] In some embodiments, the antigen-binding molecule comprises a detectable moiety. For example, the EphA2 antigen-binding molecule and / or CAR is labelled with a fluorescent label, phosphorescent label, luminescent label, immunodetectable label (e.g., an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label. The antigen-binding molecule may be covalently or non- covalently labelled with the detectable moiety.

[0347] Fluorescent labels include e.g., fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP), chelates of rare earths (such as europium (Eu), terbium (Tb) and samarium (Sm)), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, TruRed, 7-amino-4- methyl coumarin, Alexa fluor (including Alexa fluor 488, Alexa fluor 430, Alexa fluor 532, Alexa fluor 546, Alexa fluor 555, etc.), Cy2, Cy3, Cy5, and Cy7.

[0348] Radiolabels include radioisotopes such as fluorine-18, copper-64, iodine-123, iodine-125, iodine-126, iodine-131 , iodine-133, bromine-77, technetium-99m, indium-111 , indium-113m, gallium-67, gallium-68, lutetium-177, zirconium-89, ruthenium-95, rhenium-99m, technetium-99m ruthenium-103, ruthenium-105, mercury-207, mercury-203, rhenium-101 , rhenium-105, scandium-47, tellurium-121 m, tellurium-122m, tellurium-125, thulium-165, thulium-167, thulium-16, copper-67, fluorine-18, yttrium-90, palladium-100, bismuth-217 and antimony-211.

[0349] Luminescent labels include as radioluminescent, chemiluminescent (e.g., acridinium ester, luminol, isoluminol) and bioluminescent labels. Immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers. Enzymatic labels include e.g., peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase and luciferase.

[0350] The label may be selected from a group including biotin, avidin, digoxigenin, an enzyme (e.g., alkaline phosphatase or horseradish peroxidase), a fluorophore (e.g., FITC, Texas Red, Coumarin), a radioisotope (e.g.,125l,131l,67Ga,111ln) and / or a direct visual label (e.g., a gold particle), although without limitation thereto.

[0351] In some embodiments the antigen-binding molecules of the present invention are conjugated to one or multiple chemical moieties. The chemical moieties may be moieties for providing a therapeutic effect. Antibody-drug conjugates are reviewed, e.g., in Parslow et al., Biomedicines. 2016 Sep; 4(3): 14; and Tsuchikama et aL, Nature Rev. Clin. Oncol. 2024, 21 , 204-223). In some embodiments, the chemical moiety may be a drug moiety (e.g., a cytotoxic agent). In some embodiments, the drug moiety may be a chemotherapeutic agent.

[0352] Suitably, detection of EphA2 includes the step of forming a detectable complex between an EphA2-binding molecule or CAR and EphA2 or a cell expressing EphA2. The complex so formed may be detected by any technique, assay or means known in the art including immunoblotting, immunohistochemistry, immunocytochemistry, immunoprecipitation, ELISA, flow cytometry, magnetic bead separation, biosensor-based detection systems such as surface plasmon resonance and imaging such as PET imaging, although without limitation thereto.

[0353] To facilitate detection the EphA2-binding molecule or CAR may be directly labelled as hereinbefore described or a labelled secondary antibody may be used. The labels may be as hereinbefore described.

[0354] In some embodiments, a detection kit may be provided which comprises an antibody or antibody fragment disclosed herein together with one or more detection reagents such as enzymes, enzyme substrates (e.g., Luminol, AMPPD, NBT), secondary antibodies and / or magnetic beads although without limitation thereto.

[0355] In a final aspect, the invention provides an isolated nucleic acid comprising, consisting or consisting essentially of a nucleic acid sequence set forth in any one of SEQ ID NOs: 1 to 12 and / or Table 3 or a nucleic acid sequence at least 85% identical thereto.

[0356] So that preferred embodiments may be described in detail and put into practical effect, reference is made to the following non-limiting experimental examples.EXPERIMENTALEphA2 activation inhibits oncogenic signaling and reduces invasion.

[0357] EphA family receptor mRNA and protein expression were assessed by qPCR and western blotting in a panel of six prostate cancer cell lines. Apart from EphA2 and EphA3, EphA family receptor levels were low with EphA2 being the most consistently elevated; the highest expressing line being PC-3 (Figure 1A and B). Immunofluorescence (IF) analysis of PC-3 cells confirmed elevated EphA2 protein levels in the cell membrane (Figure 1 C). Based on these findings, the inventors selected the PC-3 prostate cancer cell line for further functional analysis. The EphA2 kinase was readily activated in PC-3 cells following stimulation with the clustered high-affinity ligand ephrin-A1-Fc, and the results showed inhibition of the AKT, ERK and FAK / SRC signalling pathways (Figure 1 D, Figure 2A). EphA2 activation rapidly induced transient retraction of cell protrusions and cell rounding, which was accompanied by increased cortical F-actin staining, consistent with increased actomyosin contractility and concomitant reduction in microtubule polarity (Figure 2B and 2D). Consistently, Cytochalasin D, an inhibitor of actin dynamics, blocked ephrin-A1 -induced cell rounding (Figure 2C). Cell adhesion to ephrin-A1 -Fc-coated culture surfaces was also assessed. PC-3 cells preferentially adhered to ephrin- A1-Fc compared to uncoated and Fc-control-coated surfaces and was comparable to integrin-mediated adhesion to Matrigel (Figure 2E and 2F). The inventors investigated whether activation of EphA2 inhibited cell invasion by embedding PC-3 cells in Matrigel containing clustered ephrin-A1-Fc compared to Fc control protein (Figure 2G and 2H). Reduced invasion in the presence of ephrin-A1-Fc appears to be due to cell processes repulsion and retraction instead of establishing firm adhesions to pull thecell body forward, as observed in the control (Figure 2H). These data show that EphA2 kinase activation causes retraction of cell protrusions, thus promoting a non-invasive phenotype.EphA2 Antibody Characterisation.

[0358] In seeking to better understand the anti- and pro-oncogenic functions of EphA2, the inventors first characterized the binding kinetics and receptor-blocking function of two EphA2-specific mAbs clones: 4B3 and 1 F7. The lgG2a 4B3 mAb binds only to human EphA2, whereas the lgG1 1 F7 mAb binds to mouse and human EphA2. Octet analysis was used to define the binding kinetics to biotinylated EphA2-Fc protein alone and followed by ephrin A1-Fc (Figure 3A). These results show that 1 F7 and 4B3 bind distinct epitopes and that ephrin-A1 ligand binding does not interfere with 4B3 binding, whereas 1 F7 binding to ephrin-saturated EphA2 appears to be reduced, indicating that 1 F7 likely binds close to the ephrin binding site. 1 F7 and 4B3 binding to PC-3 cells was tested by flow cytometry and confirmed strong equivalent EphA2 receptor binding (Figure 3B). Next, the ability of 1 F7 and 4B3 to activate EphA2 alone or in combination with clustered ephrin A1 -Fc was assessed. While neither antibody alone induced significant activation, 4B3 permitted ephrinA1 -Fc induced EphA2 kinase phosphorylation, whereas 1 F7 effectively blocked receptor phosphorylation on activating sites Y588 / Y594 (Figure 3C, Figure 4A). A clear reduction in phosphorylated ATK levels was observed following ligand-induced EphA2 activation, which was also inhibited following 1 F7 treatment (Figure 3C, Figure 4A). Notably, combination of 1 F7 and 4B3 induced receptor activation and downstream signalling, consistent with their ability to bind independent epitopes on EphA2, thus potentially inducing receptor cross-linking. Furthermore, the inventors found that the 4B3 antibody, when crosslinked, is capable of inducing EphA2 receptor phosphorylation and inhibiting downstream phosphorylation of AKT on S473. In contrast, clustered 1 F7 antibody was not activating (Figure 4B). Together these data demonstrate contrasting functional effects of 4B3 and 1 F7 on EphA2 receptor activation.Efficacy of 4B3 and 1F7 on prostate tumour growth and metastasis in vivo.

[0359] The response to 4B3 and 1 F7 mAb treatment in vivo was assessed using a prostate cancer orthotopic xenograft model. PC-3-luciferase cells reliably formed orthotopic tumours in mice; treatment commenced on day 10 consisting of vehicle control, 1 F7 and 4B3 (IP, 8 mg / kg, three times weekly) (Figure 4C). Due to differences in their binding capacities to human and mouse EphA2, 4B3 will bind exclusively to EphA2 expressed on the human tumour xenografts, whereas 1 F7 is capable of binding EphA2 expressed on both human tumour and mouse host tissues. Imaging showed 1 F7 treatment increased the tumour burden, leading to a decrease in overall survival (Figure 4D-F; Figure 3D). Metastatic spread to the lungs was assessed seven weeks after orthotopic engraftment, showing a trend towards reduced spontaneous lung metastasis in the 4B3-treated cohort (Figure 4G and 4H;Figure 3E). To further investigate the effect of 4B3 on metastasis, the inventors adopted an experimental metastasis approach involving direct injection of PC-3 cells into the periphery via IV tail vein injection (Figure 5A). Antibodies were administered two days and four hours prior to PC-3 IV injection to have antibodies circulating at the time of PC-3 cell engraftment. Antibodies were subsequently continued three times per week to maintain antibody treatment for the duration of the experiment (Figure 5A, Protocol 1). 4B3 treatment delayed metastatic spread, significantly increasing overall survival, whereas 1 F7 treatment increased metastasis and significantly decreased survival (Figure 5B and C; Figure 6A). To distinguish between early treatment responses on extravasation and / or tissue invasion at metastatic sites versus later growth once metastatic sites are established , the treatment window of 4B3 and 1 F7 was reduced. Monoclonal antibodies were injected 2 days prior to, on engraftment day, and 2 days following PC-3 IV injection with the aim to have antibody present only during the early phases of metastatic engraftment (Figure 5A, Protocol 2).

[0360] Imaging data showed that 1 F7 still appeared to promote metastasis, but the response was reduced and did not affect overall survival. These data suggest that 1 F7 predominantly affects tumour growth / progression. In comparison, the anti-metastatic and significant pro-survival response of 4B3 treatment was maintained (Figure 5D, 5E; and Figure 6B). This finding suggests that the tumoursuppressive properties of 4B3 occurs early during metastasis formation, affecting tumour cell tissue invasion and / or the establishment of tumours at metastatic sites rather than subsequent tumour growth.Quantitative phosphoproteomic analysis identifies downstream effector proteins of ligand-induced EphA2 receptor activation.

[0361] EphA2 activation has been shown to inhibit key oncogenic pathways, including PI3K / AKT, MEK / ERK and FAK / SRC signalling pathways (Figure 2A). However, less is known about other downstream effector proteins involved in mediating EphA2 tumour-suppressive functions following kinase activation. The inventors used SILAC in combination with mass spectrometry to measure proteome-wide changes in phosphopeptide abundance following EphA2 activation in PC-3 cells (Figure 7A). 2735 phosphoproteins with at least 75% localization probability across four biological replicates were identified, comprising two forward- and two reverse-labelled experiments (Figure 8A, 8B). As expected after TiO2 phosphopeptide enrichment, the majority of the modified sites were Ser (85%), followed by Thr (13.4%) and Tyr residues (1.6%). The inventors identified 113 phosphopeptides, comprising 128 phosphosites and 73 phosphoproteins that were significantly regulated in at least three out of four biological replicates following EphA2 receptor activation (Figure 7, Figure 8A-C; Table 1). The majority of these regulated phosphosites (94 of 128) showed reduced phosphorylation, whereas 34 (including six from the EphA2 receptor) showed increased phosphorylation.TABLE 1List of significant EphA2-regulated phosphoproteins, phosphopeptides and phosphosites in PC-3 prostate cancer cells.

[0362] Consistent with earlier ERK and AKT western blot data, it was shown to reduce phosphorylation of proteins involved in these pathways, including ERK2, p90RSK, JUNB and FOSL downstream of ERK signaling and PRAS40, S6K, eEF2K, and eEF2 downstream of AKT signalling (Figure 8C; Table 1).Functional enrichment analyses ofEphA2 downstream signaling molecules.

[0363] Next, the inventors performed enrichment analyses to gain insight into the functional relevance of the identified EphA2-regulated phosphoproteins and hence the role of EphA2 receptor activation in prostate cancer. Regulated phosphoproteins were individually categorized into functional protein classes by manually curating data collated from the HPRD (Prsad TS et al., 2009), GeneCards (Stelzer G et al., 2016), UniProt (UniProt C, 2023) and Panther (Thomas PD et al., 2022) databases and the literature (Figure 7C; Table 4). A large proportion of proteins belong to protein classes related to cell signalling and cytoskeletal regulation, including actin-binding adaptors and actin-dynamics regulators. Gene Ontology (GO) enrichment analysis of EphA2-regulated proteins revealed enrichment of cellular compartments describing cell-cell and cell-substrate junctions, the actin cytoskeleton, contractile structures, the TORC1 complex, and membrane compartments, including rafts and endosomes (Figure 7D; Table 4). Similarly, GO annotations for biological processes and molecular functions showed significant enrichment of a large cluster of terms related to cytoarchitecture, including cell-cell adhesion, cell junction assembly, actin cytoskeleton binding and small GTPases. (Figure 7E; Table 5). Terms related to axon guidance, chemotaxis, and plasma membrane localization were also significantly over- represented (Figure 4E; Table 5). Consistent with EphA2 being a member of the RTK family, the inventors also found a large cluster of enriched annotations describing signalling responses to stimuli, such as hormones and growth factors. Similar functions were confirmed by the enrichment analysis of KEGG and Reactome pathways (Figure 8A; Table 6). Furthermore, a comparison of the EphA2-regulated dataset to proteomic analyses of the E-cadherin (Guo Z et aL, 2014) and integrin (Horton ER et al., 2015) adhesomes showed that 26 and 31 of the 73 regulated phosphoproteins were in common with these datasets, respectively. Together, these results suggest that EphA2, upon stimulation, regulates the phosphorylation of proteins involved in the regulation of cytoarchitecture, cell adhesion, and motilityTABLE 4Protein Classification of EphA2-Regulated PhosphoproteinsTABLE 5GO enrichment analysis of Cellular Compartment terms associated with the EphA2 -regulated proteinsTABLE 6GO enrichment analysis of Biological Process and Molecular Function terms associated with the EphA2 -regulated proteinsMotif and upstream kinase analyses confirm inhibition of the AKT / mT0R / GSK3 and MEK / ERK / RSK pathways as key mediators ofEphA2 downstream signaling.

[0364] To better understand the networks involved in mediating EphA2 downstream signalling, the inventors initially performed motif enrichment analysis of the regulated phosphorylation sites (Figure 10A-C; Figure 8B). This demonstrated an overrepresentation of the RXX[S / T*] and the extended RXRXX[S / T*] motifs, which are the preferred sites of AGC-kinase family members, including AKT, RSKs, S6K and SGKs. Also overrepresented in the dataset is the [S / T*]P proline-directed kinase motif, which is the preferred motif of CMGC kinases, including MAPKs, CDKs and GSK3. To further elucidate which kinases are the most likely intermediaries between activated EphA2 kinase and the phosphorylation of the identified downstream effector proteins, an upstream kinase analysis was performed (Figure 5D; Figure 8C). To model the EphA2 phosphorylation network, potential upstream kinases were assigned to the EphA2-regulated phosphorylation sites identified in the phosphoproteomic analysis based on known (black edges) and predicted (blue edges) kinase-substrate relationships by querying the PhosphoSitePlus (Hornbeck PV et aL, 2012), HRPD (Prasad TS et al., 2009), UniProt (UniProt C, 2023), and NetworKIN (binding R et al., 2008) databases. In addition, candidate AGC kinase and CMGC kinase substrates were assigned and organized according to the motifs of their regulated site. Upstream kinase node sizes are indicative of the number of connected substrates and hence the likelihood of a particular kinase to be a relevant kinase, mediating signals downstream of EphA2 and upstream of the identified EphA2-regulated effector phosphoproteins. Based on this analysis, AKT, ERK1 / 2, mTOR, GSK-3 and RSK2 are the most likely candidate kinases involved in the phosphorylation of effector proteins, which are regulated downstream of activated EphA2 kinase. The vast majority of the sites regulated by these kinases show reduced phosphorylation after EphA2 activation, suggesting that these kinases are inhibited in response to EphA2 activation. This notion was confirmed by Proteome Profiler Array and western blot analysis demonstrating strong inhibition of the mTOR / AKT / p70S6K / GSK3 and MEK / ERK / RSK pathways (Figure 10A; Figure 14).Adapter protein SHB is required to mediate EphA2-induced inhibition of the ERK / MAPK pathway.

[0365] To further validate the mass spectrometry findings and prioritise phosphoproteins for further studies, western blot analysis was performed for proteins with some of the largest fold-changes in phosphorylation. Reassuringly, western blot analysis of all the sites that were tested showed strong regulation and confirmed the phosphoproteomic data: The adapter proteins SHB (pY246) and PARD3 (pY - not site-specific) showed increased phosphorylation, whereas the adaptor protein GRB10 showed a strong reduction in phosphorylation at S476 (Figure 10B). The downstream effector proteins afadin (AFDN, S1799), NDRG1 (S330), stathmin (STMN1 , S16) and PRAS40 (T246), consistent with thephosphoproteomic data, all showed reduced phosphorylation (Figure 10B). The afadin and PRAS40 sites, for example, are known sites of AKT kinases. Consistently, the inventors observed concurrent near ablation of S473 phosphorylation of AKT isoforms 1 and 2 after stimulation, which is indicative of the inhibition of kinase activity (Figure 10B). To extend the findings beyond PC-3 prostate cancer cells, the inventors validated several of the phosphoprotein targets in a panel of prostate, breast, colorectal and brain cancer cell lines (Figure 10C). Inhibition of phosphorylation of Afadin (S1718 / 1799) and NDRG1 (S333) in response to ephrin-A1 stimulation was observed in some, but not all the cell lines. Inhibition, consistent with these sites conforming with the AKT consensus motif, was observed only in cell lines which responded to EphA2 stimulation with robust inhibition of Akt phosphorylation on S473 (PC-3, MDA-MB-231 , BT-519, HCT-116 and MN1). The adaptor protein SHB, on the other hand, showed consistent and strong upregulation of Y246 phosphorylation across all tested cell lines when stimulated with clustered ephrin-A1-Fc for 20 minutes. SHB is a ubiquitously expressed SH2 domaincontaining adaptor protein, which has been implicated in the signal transduction of several tyrosine kinase receptors). To test a possible role of SHB in EphA2 receptor tyrosine kinase signal transduction, the inventors transiently knocked down SHB using two distinct siRNA sequences before stimulating cells with ephrin-A1-Fc. Western blot analysis of AKT (S473) and Erk1 / 2 (T202 / 204) phosphorylation showed that SHB knockdown leads to an increase in basal ERK1 / 2 phosphorylation levels and diminishes inhibition of ERK1 / 2 phosphorylation in response to EphA2 receptor activation, while AKT signalling responses remained unchanged (Figure 12A). These data demonstrate a role for the adaptor protein SHB in the signal transduction of activated EphA2 via the Erk1 / 2 / MAPK pathway.The adherens junction protein afadin is an EphA2-requlated mediator of cell invasion.

[0366] Phosphorylation of the adherens junction protein Afadin on serine S1799 (the same site has been referred to as S1718 based on another isoform (Elloul S et al., 2014)) was the most strongly reduced phosphorylation site after EphA2 activation. The same site was described by Elloul et al. as an AKT substrate that, in its phosphorylated form, promotes cell migration / invasion in breast cancer (Elloul S et al., 2014). Thus, afadin was considered a strong candidate mediator of EphA2- regulated cell migration and invasion. Co-staining of afadin and EphA2 (Figure 12B) showed that afadin (red) in unstimulated cells localizes predominantly to the nucleus (blue) and to a lesser extent to distinct plasma membrane regions. Upon stimulation with ephrin-A1 , afadin is increasingly recruited to the plasma membrane, where it colocalizes with EphA2 (green). At later time-points (20 min), the inventors observed that afadin had been internalized together with EphA2 into punctate intracellular compartments, consistent with receptor-mediated endocytosis into endosomes. The interaction of EphA2 and afadin by co-immunoprecipitation of endogenous proteins was unable to be demonstrated (not shown). However, a preliminary proximity ligation assay showed increased signals indicative ofprotein-protein interactions / very close proximity (<40 nm) of EphA2 and afadin after 5 minutes of stimulation (Figure 15B). Regardless of the nature of the colocalization, these data demonstrate that EphA2 signalling is involved in regulating both the phosphorylation state and the cellular distribution of afadin. Furthermore, transient siRNA-mediated knockdown of afadin with two distinct siRNA sequences (Figure 12C) demonstrated a significant reduction in the invasion of PC-3 cells in Transwell invasion assays (Figure 12D), highlighting afadin as an important mediator of cell invasion in prostate cancer cells, similar to findings in breast cancer (Elloul S et al., 2014).

[0367] Taken together, these findings demonstrate that afadin is an EphA2-regulated phosphoprotein, which is involved in mediating invasion of prostate cancer cells.Discussion

[0368] In the first part of the study, the inventors characterized two EphA2 mAbs in vitro and in vivo in the context of prostate cancer: 1 F7, which actively blocks ephrin-induced kinase activation, and 4B3, which has activating properties. Notably, 1 F7 can bind to both human and mouse EphA2, while 4B3 only binds to human EphA2 on the tumour xenograft but does not interact with mouse host tissues. The inventors tested 1 F7 and 4B3 antibodies in both orthotopic and intravenous PC-3 xenograft models of human prostate cancer. In support of EphA2 receptor activation being tumour-suppressive, it was found that the 4B3 mAb slowed metastatic spread. In contrast, the 1 F7 mAb accelerated tumour progression at primary and metastatic sites. The pro-tumourigenic effect of 1 F7, which inhibits EphA2 kinase activation, is consistent with 1 F7 blocking any activating signals by ephrin ligands expressed on stromal or endothelial cells, thus countering any effect of EphA2 canonical signalling in reducing tumourigenesis and promoting kinase-independent pro-oncogenic functions of EphA2. In the intravenous PC-3 metastasis model, 1 F7-treated animals also displayed accelerated tumour progression and decreased survival.

[0369] The 4B3 mAb permits ephrin-induced EphA2 receptor activation and, if clustered, can activate human EphA2 receptors. This suggests that, in the presence of 4B3, PC-3 tumour cells, which themselves do not express significant levels of ephrin-A ligands, continue to receive activating, anti- oncogenic signals by ephrin ligands expressed on or shed by stromal and endothelial cells. In addition, the 4B3 lgG2a mAb, by binding to FcRII and FcRn receptors, may decorate the surface of myeloid and endothelial cells, raising the possibility that the antibody itself can activate human EphA2 receptors in vivo. In the present study, 4B3 showed efficacy in reducing metastatic spread and improved survival in the intravenous animal experiments. This effect was maintained when 4B3 was administered only in the days priorto and shortly after intravenous injection of PC-3 cells, suggesting an early anti-metastatic effect of EphA2 kinase activation. One possible explanation is that the 4B3 antibody functions in anagonistic capacity similar to other studies demonstrating anti-tumour effects of agonistic EphA2 mAbs in xenograft models. In these studies, EphA2 kinase activation, followed by receptor internalisation and degradation, implied that the underlying mechanisms involve induction of anti-oncogenic, canonical signalling and decreased EphA2 cell surface expression. Furthermore, EphA2 kinase activation diminishes the phosphorylation of EphA2 at S897, thereby countering the pro-invasive and pro- metastatic effects of non-canonical EphA2 signalling (Miao H et aL, 2009; Volz C et al., 2020; Li JY et al., 2019; Garcia-Monclus S et al., 2018; Zhang C et al., 2010). An alternative explanation for the anti- oncogenic effects of 4B3 in vivo, is the possibility that the interaction of 4B3 with the extracellular domain of EphA2 alters its ability to engage in multimeric complexes in the absence of ligand binding. Regardless of the mechanism, it is important to note that the 4B3 mAb is human-specific and highlights direct effects on tumour cells without any indirect effects on host stromal and vascular elements. This has clinical implications, as shown by a human-specific EphA2 antibody-drug conjugate MEDI-547, which was well tolerated and demonstrated anti-tumour efficacy in animal models but caused significant vascular toxicity in a phase I human trial that was halted (Jackson D et al., 2008; Annunziata CM et aL, 2013).

[0370] In seeking to better understand the observed anti-tumour effects of EphA2 receptor activation, a quantitative phosphoproteomics was performed to investigate downstream signalling in response to stimulation with ephrin-A1. The inventors show that PC-3 cells express high levels of EphA2 but negligible levels of other EphA receptors or ephrin-A ligands, making this a ‘clean’ model to specifically assess EphA2-mediated downstream kinase events. Previous studies have demonstrated that the activation of EphA2 induces inhibition of the PI3K / AKT, ERK / MAPK and FAK / SRC pathways (Miao H et al., 2009; Yang NY et aL, 2011 ; Miao H et aL, 2000; Miao H et aL, 2001), however downstream effector proteins remained largely understudied. It was found that EphA2 kinase activation led to changes in the phosphorylation of proteins predominantly involved in cell-cell and cell-matrix adhesion and cytoskeletal regulation. Consistently, these changes coincide with increased contractile forces, retraction of cell protrusions, and inhibition of locomotion. Motif and upstream kinase analyses of regulated phosphosites confirmed inhibition of AKT / mTOR and MEK / ERK pathways as key mediators of EphA2 signalling. Furthermore, the present study identified the adaptor protein SHB, which became phosphorylated on Y246 in response to EphA2 activation, as a necessary component in the signal transduction of ligand-activated EphA2 receptor. SHB siRNA knockdown demonstrated its role in mediating the inhibition of the ERK / MAPK pathway in response to EphA2 activation but did not impact on the PI3K / AKT pathway. SHB, a ubiquitously expressed adaptor protein, has previously been implicated in the signal transduction of several other receptor tyrosine kinases, including PDGFR, FGFR, VEGFR and EphB2. These studies demonstrated that receptor activation induces SHB tyrosine phosphorylation on several tyrosine residues, including Y246, a site that has been implicated in thebinding of RasGAP, a negative regulator of the Ras / ERK / MAPK signalling cascade. Thus, the results suggest a mechanism for EphA2-mediated inhibition of ERK / MAPK signalling. While the present study demonstrates inhibition of AKT, ERK1 / 2 and GSK-3B kinases, it is possible that EphA2 signalling also stimulates phosphatase activities that may contribute to the observed inhibition of protein phosphorylation of AKT and ERK1 / 2 substrates.

[0371] The AKT / mTOR and ERK / MAPK pathways relay signals by phosphorylating numerous substrates, that regulate a multitude of biological functions, including cell growth, survival, metabolism, and invasion. Intriguingly, the phosphoproteomic dataset indicated that EphA2 predominantly inhibits the phosphorylation of AKT and ERK substrates involved in cytoskeletal regulation, cell adhesion and invasion in prostate cancer cells. The selectivity of downstream substrates may, at least in part, be due to the subcellular localization of EphA2, which determines its protein interactions and the recruitment and translocation of substrates. A study by Perez White and colleagues identified EphA2-associated proteins in healthy keratinocytes using a proximity ligation assay in combination with mass spectrometry (Perez White BE et al., 2017). Of the present list of EphA2-regulated phosphoproteins, approximately one third (25 out of 73 proteins) were either identical (12 proteins) or paralogs (13 proteins) of proteins identified in this study. Consistently, the inventors observed that afadin, which is associated with EphA2 in keratinocytes (Perez White BE et al., 2017), is recruited to and colocalizes with EphA2 at the plasma membrane upon EphA2 activation. This coincided with afadin showing the strongest decrease in phosphorylation in the dataset of EphA2-regulated phosphoproteins. Afadin is a cell junction scaffolding protein. Early studies described two isoforms (Mandai K et al., 1997): a short splice variant, s-afadin, expressed in neural tissues, and a ubiquitously expressed, long splice variant, l-afadin, which contains an F-actin binding domain that connects the cell adhesion molecule nectin to the actin cytoskeleton (Mandai K et al., 1997; Takahashi K et al., 1999). But while the Uniprot database (UniProt C, 2023) lists six isoforms of human afadin (UniProt Accession No. P55196, the literature appears to refer to l-afadin more broadly as any of the larger / ubiquitously expressed isoforms. The inventors chose the canonical sequence (Uniprot isoform 4) for the numbering of the EphA2-regulated afadin phosphorylation site (S1799). The same phosphorylation site (KERQRLFS*QG) is also found in isoforms 3 (S1718) and 5 (S1809). Elloul et al. identified the S1718 / S1799 / S1809 site as an AKT substrate and demonstrated that the function of afadin can be switched from cell-cell adhesion to an invasion-promoting protein in breast cancer by regulating the phosphorylation of this site (Elloul S et al., 2014). Similarly, the present study highlights afadin as an important regulator of cell invasion in prostate cancer cells and further demonstrates that activated EphA2 alters the localization and leads to dephosphorylation of afadin at its AKT substrate site (S1718 / S1799 / S1809), suggesting a similar mechanism and EphA2 as an upstream regulator. Regulation of afadin by Eph receptors at cell junctions may indeed be a more common theme. Afadin has been shown to interact with EphB2 andEphB3 in a kinase activation-dependent manner at specialized sites of cell-cell contact in the brain, leading to increased EphB-mediated tyrosine phosphorylation of afadin (Fearnley GW et aL, 2019; Asano E et aL, 2011). More broadly, phosphorylation of cell junction proteins has been described as a common switch to regulate protein interactions and, consequently, function (Bertocchi C et aL, 2012). The present dataset of EphA2-regulated phosphoproteins was significantly enriched for adherence junction and cytoskeletal proteins, some of which are known to be able to switch between adhesive and invasion-promoting functions, including afadin, plakophilin, PARD3 and palladin among others (Elloul S et aL, 2014; Fearnley GW et aL, 2019; Asano E et aL, 2011).

[0372] In summary, these data demonstrate that EphA2-ephrin-A interactions induce downstream signalling on targets that promote cell adhesion and inhibit the formation of membrane protrusions and cell motility, thus acting in a tumour-suppressive manner and contributing to epithelial and endothelial integrity. Indeed, EphA2 has been shown to regulate adherens and tight junctions and vice versa in epithelial and endothelial cells (Fang WB et aL, 2008; Orsulic S et aL, 2000; Tanaka M et aL, 2005). Furthermore, the ephrin-blocking 1 F7 antibody interfered with the EphA2-ephrin linkage, which acts to maintain endothelial barrier function, and induced vascular leak, an effect that potentially contributes to metastasis. Tumour cells often show a concomitant loss of ephrin-A expression when EphA2 acts in a ligand-independent, pro-oncogenic manner (Miao H et aL, 2012; Wykosky J et aL, 2008). Thus, restoring kinase activation has been proposed as a therapeutic avenue for targeting EphA2-expressing human cancers (Barquilla A et aL, 2015). Consistent with the dichotomous role of this receptor, the EphA2-targeting mAbs with distinct activating and blocking functions demonstrated opposing tumour-suppressive and oncogenic properties. The in vivo antibody treatment studies indicate that the addition of an activating EphA2 mAb can restore EphA2 signalling in ephrin-A-negative aggressive epithelial cancers and inhibit pro-tumourigenic effects. Notably, the 1 F7 mAb, which blocks ephrin binding, thus preventing EphA2 activation, increased prostate cancer aggressiveness.Materials & MethodsCell Culture

[0373] PC-3 (ATCC #CRL-1435), LNCaP (ATCC #CRL-1740), DU145 (ATCC #HTB-81), MDA-MB-231 (ATCC #HTB-26), BT-519 (ATCC #HTB-122), MCF10A (ATCC CRL-10317), Lisp-1 (RRID:CVCL_9U26), HCT-116 (ATCC #CCL-247), U-87MG (ATCC #HTB-14) and U251 MG (ECACC #09063001) cell lines were maintained in RPMI 1640 and 2H-11 (ATCC #CRL-2163) in DMEM media, containing 10% fetal bovine serum (FBS), 2 mM L-Glutamine, 100 lU / mL penicillin and 100 pg / mL streptomycin. Human umbilical vein endothelial cells (HUVEC; Thermo Fisher Scientific #C0035C) were maintained in 200PRF medium containing low serum growth supplement. The primaryglioblastoma cell lines WK1 and MN1 were generated in house and were maintained as glioma neural stem cell (GNS) cultures using StemPro NSC SFM as per manufacturer’s guidelines. Cell line identity of human cell lines and mycoplasma-free status of cell cultures were confirmed by in-house short tandem repeat (STR) profiling and mycoplasma diagnostic testing services, respectively. PC-3 cells expressing luciferase (PC-3-luciferase) were generated using Firefly Luciferase Lentifect Purified Lentiviral Particles (GeneCopoeia). Briefly, 70-80% confluent cells in a 6-well were transduced with 2 pL of Lentifect particles in 2 ml of antibiotic-free media containing 8 pg / ml polybrene. After centrifugation (500 x g, 45 min), the cells were incubated for 24 h and medium replaced. Selection medium containing 1 pg / mL puromycin was added 72 h after transduction. For stimulation experiments, human ephrin-A1- Fc (recombinant soluble ephrin A1 fused to human IgGI Fc; custom production by CSL) or Fc control proteins (human IgG; Thermo Fisher Scientific #02-7102) were clustered with rabbit-anti-human secondary antibodies (Jackson ImmunoResearch #309005008) at a 2:1 ratio in serum-free RPMI media (1 h, 4°C) before stimulating serum-starved cells at a final concentration of 1 pg / mL. For stimulation experiments with clustered anti-EphA2 antibodies, mouse anti-EphA2 antibodies were clustered with Fc fragment-specific goat anti-mouse secondary antibody (Jackson ImmunoResearch #115-005-071) at a 2:1 ratio in serum-free RMPI media (1 h, 4°C) before stimulating serum-starved cells at a final concentration of 10 and 30 pg / mL, respectively. Cytochalasin D, was used at a final concentration of 1 pM.Development of Eph A2 monoclonal antibodies

[0374] The development of the EphA2 monoclonal antibodies (mAbs) used in this study has been reported previously (Herath Nl, Spanevello MD, Doecke JD, Smith FM, Pouponnot C, Boyd AW. Complex expression patterns of Eph receptor tyrosine kinases and their ephrin ligands in colorectal carcinogenesis. EurJ Cancer 2012; 48: 753-762). Briefly, the 4B3 hybridoma was derived from BALB / c mice and the 1 F7 hybridoma from EphA2 placental alkaline phosphatase (PLAP) reporter knockout mice immunized with EphA2-Fc protein, generated using an EphA2-Fc expression vector as the immunogen (Naruse-Nakajima C, Asano M, Iwakura Y. Involvement of EphA2 in the formation of the tail notochord via interaction with ephrinAI . Meeh Dev 2001 ; 102: 95-105).Bio-layer interferometry

[0375] The antibody binding kinetics of EphA2-specific mAbs to human EphA2-Fc-loaded biosensors were compared by bio-layer interferometry using the Octet RED system (Sartorius). Recombinant EphA2-Fc protein was conjugated to biotin (1 :1 molar ratio) using EZ-Link Sulfo NHS- LC-LC-Biotin (Thermo Fisher Scientific) following the manufacturer’s instructions. Excess biotin was removed using Zeba Spin desalting columns 7K MWCO. Biotinylated EphA2-Fc protein (50 pg / ml) wasloaded onto Octet Streptavidin Biosensors (Sartorius) according to the manufacturer’s instructions, and the binding of EphA2-specific antibodies and human ephrin-A1-Fc to individual sensors was recorded. Upon saturation, the probes were exchanged into 1 x Kinetics Buffer (1 mM Phosphate, 15 mM NaCI, 0.005% Tween 20 and 0.1 mg / mL BSA) to allow dissociation.Western blotting, immunoprecipitation and Proteome Profiler Array

[0376] Cells were lysed in lysis buffer A (20 mM Tris / HCI pH 7.4, 150 mM NaCI, 1 mM EDTA, 1% Triton X-100, PhosSTOP Phosphatase and complete, Mini, EDTA-free Protease Inhibitor Cocktails. Crude lysates were centrifuged (16,000 x g), supernatants were collected and protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories). Equal amounts of denatured protein samples were separated using SDS-PAGE and transferred onto PVDF membranes. Blocked membranes (5% BSA / PBS-Tween) were analyzed using the following primary antibodies: Anti-phospho-EphA2 / A3 / A4 (Y588+Y596) (#ab62256), anti-phospho-SHB (Y246) (#ab138388), anti-SHB (#ab129190) and anti-FAK (#ab40794) antibodies were purchased from Abeam. Phospho-EphA2 (S897) (#6347), anti-EphA2 (#6997), anti-phospho-AKT (S473) (#4060), anti- AKT (#2920), anti-phospho-ERK1 / 2 (T202 / T204) (#4370), anti-ERK1 / 2 (#4695), anti-phospho-MEK1 / 2 (S217 / 221) (#9154), anti-MEK1 / 2 (#9122), anti-phospho-FAK (Y397) (#8556), anti-phospho-SRC (Y416) (#2101), anti-SRC (#2110), anti-phospho-p70 S6 Kinase (T421 / S424) (#9204), anti-p70 S6 Kinase (#2708), anti-phospho-PRAS40 (T246) (#13175), anti-phospho-GSK-3a / p (S21 / S9) (#9327), anti-GSK-3a / p (#5676), anti-phospho-afadin (S1718) (#5485), anti-afadin (#D1Y3Z), anti-phospho- NDRG1 (S330) (#11899), anti-NDRG1 (#9485), anti-phospho-tyrosine (P-Tyr-100) (#9411), anti-p- actin (#4970) and anti-p-actin (#3700) antibodies were purchased from Cell Signaling Technology. The anti -PARD3 (#11085-1 -AP) antibody was purchased from Thermo Fisher Scientific, respectively. Blots were developed using goat anti-rabbit and goat anti-mouse IgG-HRP antibodies (both Agilent: #P044801-2, #P044701-2) and Clarity Western ECL Substrate (Bio-Rad Laboratories).

[0377] For immunoprecipitation experiments, equal amounts of cell lysates were pre-cleared with 20 pL of 50%-slurry Protein G Sepharose beads (Abeam #ab193259). Anti-PARD3 antibody (Thermo Fisher Scientific #11085-1 -AP) was bound to Protein G Sepharose beads (1 h, 4°C) and the unbound antibody was removed by washing with lysis buffer. 40 pL of antibody-coated beads (50%- slurry) were added to pre-cleared lysates and incubated for at least 2 h at 4°C. Beads were washed with lysis buffer and immunoprecipitated proteins were analyzed by western blotting. The Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems, #ARY003) was used according to the manufacturer’s instructions.Quantitative PCR

[0378] Total RNA was extracted and purified using the RNeasy mini kit according to the manufacturer’s instructions. First-strand cDNA was synthesized using oligo(dT) primers in conjunction with Superscript IV Reverse Transcriptase according to the manufacturer’s instructions. Quantitative real-time PCR was carried out using SYBR Green PCR Master Mix following the manufacturer’s instructions. The PCR primers used for SHB were F: 5’ GAT CCC TTT GAT GCC AAG AA and R: 5’ CTC TCC GAG TCC GAG TCA AC and have been reported previously in Schneider CA, Rasband WS, Eliceiri KW. NIH Image to Imaged: 25 years of image analysis. Nat Methods 2012; 9: 671-675. The following PCR conditions were used: 40 cycles of denaturation at 95°C for 30 s, followed by annealing and extension for 30 s at 63°C. All reactions were performed in technical triplicate for each of the biological triplicates on an ABI Viia 7 real-time PCR system. Cycle thresholds (Ct) were determined and exported using ABI Quantstudio 5 software. The fold change in SHB mRNA transcripts levels between groups was determined using the 2AACT- method with -actin as the reference house-keeping gene.Flow cytometry analysis

[0379] The cells were detached with 5 mM EDTA / PBS and washed with FACS buffer (2% FCS / PBS). 0.5-1 x10® cells were incubated in 5-10 pg / mL primary antibodies (1 F7 or 4B3 mAbs) or human EphA2-Fc (in-house), washed, then incubated with 5 pg / ml secondary antibodies donkey antimouse Alexa Fluor 647 (#A-31571) or goat anti-human Alexa Fluor 488 (#A-11013, both Thermo Fisher Scientific). Data were acquired on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (FlowJo, LLC).Immunofluorescence microscopy

[0380] For the stripe assay, glass coverslips were coated in a stripe-like pattern using silicone matrices by adapting the method of Knoll et al. Briefly, 10 pg / mL clustered ephrin-A1-Fc was used to fill the channels of the silicone matrices covered with a glass coverslip. To generate a binary substrate choice, alternating stripes were subsequently coated with 10 pg / ml clustered Fc control or 1 :100 Matrigel Basement Membrane Matrix (Corning, #354234). For immunofluorescence staining, PFA-fixed cells were permeabilized with 0.1 % Triton X-100 / PBS, blocked with 0.25% BSA / PBS and immunolabelled with the following primary antibodies: anti-EphA2 (1 F7 mAb), anti-afadin / AF-6 (Novus Biochemicals, #NBP1-90219), and anti-a-tubulin (Cell Signaling Technology, #2125). Rhodamine- phalloidin (#R415) and Alexa Fluor 488- and Alexa Fluor 647-conjugated secondary antibodies were purchased from Thermo Fisher Scientific. Ephrin-A1 -Fc-coated stripes were visualized using donkey anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific, #R37118). Coverslips were mounted usingProlong Gold Antifade Mountant with DAPI. Images were acquired with a Zeiss LSM780-NLO confocal microscope. The anisotropy of microtubules was analyzed using the FibrilTool plugin (Boudaoud et aL, 2014) in Imaged (Schneider et al., 2012).Proximity ligation assay

[0381] For the proximity ligation assay (PLA), cells were plated on coverslips, allowed to adhere overnight, serum-starved for 3 h, then stimulated with 1 pg / ml clustered ephrin-A1-Fc for 5 min or left untreated. PFA-fixed cells were permeabilized with 0.1% Triton X-100 / PBS prior to performing Duolink PLA. Duolink Fluorescent PLA was carried out according to the manufacturer’s instructions using the following reagents: Duolink In Situ PLA Probe Anti-Mouse PLUS (#DU092001), Duolink In Situ PLA Probe Anti-Rabbit MINUS (# DU092005), DUOlink In Situ Wash Buffers, Fluorescence (#DUO82049) and Duolink In Situ Detection Reagents Green (#DUO92014). Briefly, coverslips were blocked with Duolink Blocking Solution for 60 min, then incubated for 60 min with the anti-afadin antibody (Novus Biochemicals #NBP1-90219) and anti-EphA2 1 F7 mAb (in house), both diluted 1 :50 in Duolink Antibody Diluent. Coverslips incubated with anti-afadin antibody alone or anti-EphA2 antibody alone or in the absence of antibody serve as negative technical PLA controls. Coverslips were washed in Duolink Wash Buffer A, then incubated with the Duolink PLUS and MINUS PLA probes for 60 min. After washing in Duolink Wash Buffer A, coverslips were incubated with Duolink ligation solution for 90 min, again washed in Duolink Wash Buffer A and incubated with Duolink amplification solution for 100 min, then washed with Duolink Wash Buffer B. All incubation steps were carried out in a humidity chamber at 37°C. Slides were mounted in Prolong Gold Antifade Mountant with DAPI. Images were acquired with a Zeiss LSM780-NLO confocal microscope.Live cell brightfield imaging

[0382] PC-3 cells were plated in one well of a silicone culture insert (Ibidi, #IBI81176). Once confluent, the insert was removed, and cells were overlaid with Matrigel Basement Membrane Matrix (Corning #354234) diluted 1 :1.2 in CO2-independent medium, containing a final concentration of 1 pg / mL clustered ephrin-A1-Fc or Fc-controL Matrigel was allowed to gel before CO2-independent medium containing 1 pg / mL ephrin-A1-Fc or Fc control was overlaid. Time-lapse brightfield microscopy images were acquired on an 1X81 inverted microscope (Olympus) with a motorized stage and environmental chamber using xCellence Software (Olympus) and processed using Imaged (Schneider et al., 2012). siRNA Silencing and Transwell Invasion Assay

[0383] Lipofectamine2000 was used according to the manufacturer’s instructions. Ambion Silencer Select siRNA oligonucleotides were purchased from Thermo Fisher Scientific: Afadin siRNA sequence #1 : 5’-GGAUCACACUGGAUGCUCAtt-3’ and sequence #2: 5’-GCGUGUUACACGUUCCCAAtt-3’), SHB siRNA sequence #1 5’-GCAAAUAUGGUAUCACGGAtt-3’ and sequence #2: 5’-GGUAUCCAGUUAUAUGACAtt-3’, Grb10 siRNA sequence #1 : 5’- GAAUGCUCCUUUACCAGAAtt-3’ and sequence #2 5’-GGCUUUUUCUCCUCCGUGAtt-3’, and Negative Control #1 siRNA (#4390843). Per well of a 6-well plate 4 pl transfection reagent and 12.5 pmol siRNA were prepared in OptiMEM. The cells were cultured under antibiotic-free conditions. For transwell invasion experiments, cells were transfected, incubated for 30 h, serum-starved for 18 h, then set up using the QCM ECMatrix Cell Invasion Assay, 24-well (8 pm), fluorimetric (Merck #ECM 554) according to the manufacturer’s instructions. Briefly, cells were harvested using 5 mM EDTA / PBS. 2.5x105 cells in serum-free RPMI media were added to each transwell insert. FBS / RPMI medium (10%) was added to the lower chamber, and cells were incubated for 24 h.Animal studies

[0384] All experimental protocols for the animal studies were approved by the QIMR Berghofer Animal Ethics Committee (A0304-620M). No sample size calculations were performed prior to the study. The investigators were not blinded to the group allocation. However, intravital bioluminescence imaging was performed to objectively monitor and quantify tumour burden, supporting the validity of the survival data. At endpoint, animals were euthanised by cervical dislocation. Tissues samples were fixed in 10% neutral-buffered formalin, paraffin-embedded, and stained with hematoxylin and eosin (H&E).

[0385] Prostate orthotopic xenografts: PC-3-luciferase cells were orthotopically engrafted into the prostates of 6-8 week-old male NRG mice (NOD.Cg-Rag1tm1MOMIL2rgtm1wji / SzJ (Pearson T et al., 2008), The Jackson Laboratory, #007799). Mice received the analgesic Temgesic IP (0.03 mg / kg in PBS) prior to surgery and a top-up dose (0.06 mg / kg, SC) after surgery. Mice were anesthetized IP with 100 mg / kg ketamine and 10 mg / kg xylazine. The prostate was accessed through a lower midline abdominal incision by gently lifting the bladder through the incision. PC-3-luciferase cells (2.5x105) in 10 pl PBS were injected using a 27G micro-injection needle and Hamilton syringe. The needle was left in place for an additional minute. The bladder was placed back in the abdominal cavity, and muscle and skin layers were individually sutured using lyconate monofilament absorbable 5 / 0 sutures and sutures secured with Vetbond tissue adhesive. Mice were randomized into treatment groups before treatment commenced on day 10 after engraftment. The mice received thrice weekly IP injections of 8 mg / kg 1 F7 mAb, 4B3 mAb, or lgG1 (#BE0083) / lgG2a (#BE0085) InVivoMAb isotype control antibodies (1 :1 ratio, both BioXCell) in PBS or PBS vehicle control until endpoint.

[0386] Intravenous metastatic xenografts: Male NRG mice from multiple litters were randomized into treatment groups prior to the start of the experiment. PC-3-luciferase cells (5x105) in 200 pl PBS were injected via the lateral tail vein. Mice received 8 mg / kg 1 F7 mAb, 4B3 mAb, lgG1 / lgG2a isotype control mAbs or PBS vehicle control 2 days and 4 h prior to tumour cell engraftment. Mice either continued treatment thrice weekly until endpoint (“Protocol 1”) or received one final dose on day 2 after cell injection (“Protocol 2”). Mice were excluded from the experiment if humane endpoint was reached without mice demonstrating detectable tumour burden by intravital bioluminescence imaging.

[0387] Intravital and ex vivo bioluminescence imaging: Tumour burden was monitored using intravital bioluminescence imaging. Mice were anesthetized by isoflurane inhalation, injected IP with 100 pl of 5 mg / ml D-luciferin (Gold Biotechnology, #eLUCK-3G) in PBS and imaged using the MS Spectrum in vivo imaging system (PerkinElmer). For ex vivo imaging of lung metastatic burden, mice were euthanized 5 min after luciferin injection, and the lungs were dissected and imaged 5 min after euthanization. Signals were quantified using Living Image Software (PerkinElmer).

[0388] Vascular permeability studies: Vascular permeability was measured using the Miles Assay. Mice were injected with 200 pL of filter-sterilised 0.5% Evans Blue dye in PBS via the lateral tail vein. Mice were anesthetized with ketamine / xylazine as described above. Ten minutes after dye injection each mouse received 30 pl of 1 F7, 4B3 and isotype control mAbs (2 pg / mL each) and PBS control via intradermal injection into discrete areas of the flank. The mice were euthanized 20 min later. Evans Blue dye effusion into the dermis was photographed, and tissue biopsy cores were collected using 6 mm biopsy punches. Evans Blue dye was extracted in 400 pl formamide per core (48 h, 55°C) and quantified by spectrophotometry (A620nm).Sample preparation for phosphoproteomic mass spectrometry analysis

[0389] PC-3 cells were grown in RPM1 1640 for SILAC medium containing 10% dialyzed FBS, 100 U / mL Penicillin, 100 pg / mL Streptomycin. The medium was supplemented with 200 mg / L L-proline to prevent amino acid conversion (Bendall SC et al., 2008) and either 48 mg / L unlabelled “light” L-12Ce- arginine (Arg) and 200 mg / L L-12Ce-lysine (Lys) or equimolar amounts of the isotopic “heavy” L-13Ce- Arg and L-13Ce-Lys (all Cambridge Isotope Laboratories). After six passages cells showed >95% labelling efficiency, which did not increase further with subsequent passages. PC-3 cells grown under “heavy” and “light” conditions for 6 passages were serum-starved overnight, then incubated for 20 min with 1 pg / mL clustered ephrinA1-Fc and control Fc, respectively (“forwards experiment”). The experiment was done in biological quadruplet and labels were exchanged between conditions in replicates two and four (“reverse experiment”). Cells were washed thrice with ice-cold PBS and lysedon ice with RIPA Lysis and Extraction Buffer containing Roche PhosSTOP and complete, Mini, EDTA- free Protease Inhibitor Cocktails. Lysates were centrifuged (16,000 x g, 20 min, 4°C) and the supernatants were collected. Equal amounts (800 pg) of cell lysates from “heavy” and “light” conditions were pooled and proteins enriched using a 2D clean-up kit and quantitated using the 2D Quant kit. Samples (a total of 1.5 mg) were digested using a double digest with Lys-C and trypsin endoproteases as previously described (Dave KA et al., 2014). Briefly, proteins were precipitated using -20°C methanol. Protein pellets were washed thrice in 1 mL of -20°C 90% (v / v) methanol, centrifuged at 16,000 x g for 20 min at 4°C and pellets resuspended in 8 M urea / 100 mM NH4HCO3 and sonicated for 5 min. The urea concentration was adjusted to 6M using 50 mM NH4HCO3 before incubation with Lys-C at an enzyme-to-substrate ratio of 1 :100 at 37°C for 6 h. The samples were then diluted with 50 mM NH4HCO3 to adjust the urea concentration to 1.6 M before trypsin was added at an enzyme-to- substrate ratio of 1 :50, followed by incubation at 37°C for 18 h. Phosphopeptides were enriched with TiO2 beads. Briefly, 800pg of digested peptides were dried and resuspended in 1 M glycolic acid solution prepared in 5% (v / v) TFA, containing 80% (v / v) acetonitrile and 1 % (v / v) SDS. Subsequently, the solutions were diluted to 0.2% (v / v) SDS and loaded on to a TiO2 column (peptide: TiO2 beads ratio of 1 :4). The column was washed twice with 1% (v / v) and 0.1 % TFA containing 80% (v / v) and 20% (v / v) acetonitrile, respectively. Phosphopeptides were eluted with 25% ammonia and immediately acidified with formic acid. The eluates were dried and resuspended in 0.1% TFA containing 2% (v / v) acetonitrile.Mass spectrometry and data processing

[0390] All mass spectrometric analyses were performed using a Nano Ultra-High Performance Liquid Chromatograph (nUHPLC) (Waters NanoAcquity) interfaced with a high-resolution LTQ Orbitrap-VelosPro mass spectrometer (Thermo Fisher Scientific). Quantitative proteomic data (for normalization of phosphoproteomic data) were obtained by running a single shot 300 min LC-MS analysis on a 2 pg aliquot of the LysC / tryptic digest (four replicates). Four elutions from TiO2 column (phosphopeptide-enriched elutions) for each biological replicate were analysed by LC-MS using a 180 min LC gradient and the top 20 CID-MSA (multistage activation) method on the LTQ-Orbitrap-VelosPro. A Waters C18 BEH, 130A, 1.7 pm particle size, 75 pmx 200 pm analytical column and Waters 2G-V / M C18 Symmetry trap, 100A, 5 pm particle size, 180 pmx 20 mm trap were used for all analysis. A flow rate of 300 nL / min and a column temperature of 35°C were used.

[0391] RAW MS data files were analyzed using the MaxQuant computational platform (Cox J et al., 2008) (version 1.4.0.8). Proteins and peptides were identified using the Andromeda search engine (Cox J et al., 2011) by querying concatenated forward and reverse sequences from the complete human Uniprot database (UniProt C 2023) downloaded on 31 / 01 / 2013. To search for precursor and fragment ions, the mass tolerance was set to 6 ppm and 20 ppm, respectively. The enzyme specificitywas set to trypsin / P and Lys-C / P, allowing a maximum of two missed cleavages. The minimum peptide length was set to seven amino acids. Carboxamidomethyl-cysteine was defined as a fixed modification, whereas phosphorylation of serine / threonine / tyrosine, acetylation of protein N-termini, deamidation of asparagine / glutamine, and oxidation of methionine were defined as variable modifications. Peptides and proteins were identified with a maximum FDR of 1%. Reverse and contaminant hits were removed from the MaxQuant output file. Only phosphopeptides with class I phosphorylation sites, which had been assigned with a localization probability of >0.75 (Olsen JV et aL, 2006), were considered forfurther analysis. To assess the significance of outlier ratios from the bulk of the distribution ‘Significance B’ (calculated by MaxQuant version 1.4.0.8) was obtained by grouping protein subsets into intensity bins and correcting for multiple hypothesis testing with a Benjamini-Hochberg corrected p-value threshold of 0.05. Q-value estimation was computed using the ‘q value’ package in R (version 3.1.2). Phosphopeptides were considered to be regulated at a Benjamini-Hochberg corrected p-value threshold of <0.05. Phosphopeptides had to be significantly regulated in at least three out of four biological replicates to be included in the list of EphA2-regulated phosphopeptides for downstream bioinformatics analysis.Downstream bioinformatics analysis

[0392] Gene ontology (GO) and pathway enrichment analysis: Enrichment analyses of GO annotations (Ashburner M et al., 2000), KEGG (Kanehisa M et aL, 2016), and REACTOME (Fabregat A et aL, 2018) pathways were performed using the ClueGO plugin (Bindea G et aL, 2009) (version 2.3.4) in Cytoscape (Shannon P et aL, 2003) (version 3.2.1). Precompiled annotation files for QuickGO (Binns D et aL, 2009), KEGG and REACTOME Pathways were downloaded on 21 / 09 / 2017. All evidence codes except “Inferred from Electronic Annotation” were allowed. GO levels 2 to 8 were included in the analysis. The minimum number of genes for each term was set to 2 (GO Cellular Component) and 3 (other GO annotations, KEGG and REACTOME Pathways), and the minimum percent coverage of terms was specified as 7.5% (GO annotations) or 5% (KEGG and REACTOME Pathways). EphA2-regulated proteins were queried for enriched terms against our own background dataset (all detected phosphoproteins with class I phosphorylation sites; contaminant and reverse sequences removed). Enrichment was evaluated using a one-sided hypergeometric test. FDR was controlled using the Benjamini-Hochberg correction for multiple hypothesis testing. Significantly overrepresented terms (corrected p-value <0.05) were visualized, and related and redundant terms clustered with the ClueGO plugin in Cytoscape (kappa score threshold of 0.3 for GO enrichment and 0.4 for KEGG and REACTOME Pathway analyses). Clusters were manually annotated to summarize clusters’ themes.

[0393] Functional classification: Proteins were manually curated into protein classes based on information collated from the UniProt (UniProt C, 2023), GeneCards (Stelzer G et al., 2016), Human Protein Reference Database (HPRD) (Prasad TS et al., 2009), and Panther (Thomas PD et al., 2009) databases. Protein functions were assigned based on the protein class, GO analysis and information collated from the above databases. Proteins and their regulated phosphorylation sites were visualized using the Phosphopath App (Raaijmakers LM et al., 2015) in Cytoscape (Shannon P et aL, 2003) and imported into Adobe Illustrator for function overlay and layout editing.

[0394] Motif enrichment: The PhosphositePlus (Hornbeck PV et al., 2012) Motif Analysis Tool was used to query EphA2-regulated phosphosites for enriched linear phosphoserine and phosphothreonine motifs. 13-mer phosphosite-centered peptide sequences of regulated phosphosites were analyzed using the Motif-All algorithm (significance threshold of 1e05, support threshold of 0.05). A motif occurrence threshold of five was manually applied to significantly overrepresented motifs. Motif logos were generated using the PSP Production Algorithm of the PhosphositePlus Sequence Logo Tool.

[0395] Upstream kinase analysis: Known kinase-substrate relationships were extracted from PhosphositePlus (Hornbeck PV et al., 2012) (“putative in vivo kinases”), HPRD (Prasad TS et al., 2009), Uniprot (UniProt C, 2023) and KEA2 (http: / / www.maayanlab.net / KEA2 / index.html; library: “Literature Based Kinase-Substrate Library with Phosphosites”). Upstream kinase prediction analysis was performed using the NetworKIN database (Linding R et al., 2008). Kinases with a minimum score of three and a maximal score difference of four from the top scoring kinase were considered predicted upstream kinases. Kinase-substrate relationships were visualized in Cytoscape in conjunction with the Phosphopath App to visualize phosphosites.Statistical analysis

[0396] Statistical analyses, unless specified otherwise in the respective method sections, were performed using Prism 9 (GraphPad) and JMP (SAS Institute) software. Appropriate statistical tests for group comparisons were determined in consultation with the Statistics Unit at QIMR Berghofer Medical Research Institute. Applied tests include two-tailed unpaired Student’s t test, repeated measure ANOVA with post-hoc Tukey’s test, one-way ANOVA with post-hoc Sidak’s test, mixed model analysis and mixed-effect regression analysis. For survival analyses, the log-rank (Mantel-Cox) test was used to determine the significance between experimental groups. Applied statistical tests, sample sizes and p-values are indicated in the figure legends. QQ plots and homoscedasticity plots were examined to assess normal distribution and equal variances of the data, respectively. Where appropriate, data were log-transformed prior to statistical analysis. No sample size calculations were performed prior to thestudy. For in vitro experiment the sample size represents the number of biological replicates generated independently and on separate day. For in vivo experiments sample size represents the number of mice per treatment group from the same experiment or pooled across multiple identical experiments as indicated in the figure legends. The investigators were not blinded to the group allocations. A p-value of <0.05 was considered statistically significant. Unless specified otherwise in the figure legends, bar graphs represent the mean ± standard error (SE); box and whisker plots represent the median and interquartile range ± minimum / maximum values. Statistical significance is indicated by asterisks: *p < 0.05, **p < 0.01 , and *** p < 0.001 .

[0397] All publications, patents, patent applications and sequence accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0398] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.BIBLIOGRAPHYBarquilla A, Pasquale EB. Eph receptors and ephrins: therapeutic opportunities. Annu Rev Pharmacol Toxicol 2015; 55: 465-487Bartley TD, et al. B61 is a ligand for the ECK receptor protein-tyrosine kinase. Natur...

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An antigen-binding molecule that specifically binds EphA2, and upon binding activates EphA2 kinase activity.

2. The antigen-binding molecule of claim 1 , wherein the antigen-binding molecule does not bind to the ephrin binding site of EphA2.

3. The antigen-binding molecule of claim 1 or claim 2, wherein the EphA2 kinase activity comprises an increase or stimulation of phosphorylation of SHB via the Erk1 / 2 / MAPK pathway, at amino acid position Y246.

4. The antigen-binding molecule of any one of claims 1 to 3, wherein the binding to EphA2 increases or stimulates EphA2 internalisation.

5. An EphA2-binding molecule, comprising three heavy chain complementarity determining regions (CDRH1 , CDRH2, and CDRH3) and three light chain complementary determining regions (CDRL1 , CDRL2, and CDRL3) having an amino acid sequence set forth below or at least 85% identical thereto:

6. The EphA2-binding molecule of any one of claims 1 to 5, wherein the VH polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 71 or an amino acid sequence at least 85% identical thereto and / or the VL polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 72 or an amino acid sequence at least 85% identical thereto.

7. The EphA2-binding molecule of any one of claims 1 to 6, wherein the EphA2-binding molecule is an antibody (e.g. a monoclonal antibody) or antibody fragment.

8. The EphA2-binding molecule of any one of claims 1 to 7, wherein the VH polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 71 and the VL polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 72.

9. The EphA2-binding molecule of any one of claims 1 to 8, wherein the EphA2-binding molecule is a recombinant, human or humanized antibody or antibody fragment.

10. The EphA2-binding molecule of any one of claims 1 to 5, wherein the EphA2-binding molecule is a T cell receptor (TCR).

11. The EphA2-binding molecule of any one of the preceding claims, wherein the EphA2-binding molecule activates EphA2.

12. A chimeric antigen receptor (CAR) or BiTE comprising an EphA2-binding molecule according to any one of the preceding claims.

13. The EphA2-binding molecule of any one of claims 1 to 11 or the CAR of claim 12, for use in the treatment or prevention of a cancer in a subject.

14. An isolated nucleic acid encoding the EphA2-binding molecule according to any one of the preceding claims.

15. A genetic construct comprising an isolated nucleic acid of claim 14.

16. An expression vector comprising an isolated nucleic acid according to claim 14.

17. A cell comprising an isolated nucleic acid according to claim 14, or an expression vector according to claim 16.

18. A host cell comprising the isolated nucleic acid of claim 14, and / or the genetic construct of claim 12, and / or the expression vector of claim 16.

19. The host cell of claim 18, wherein the host cell is or comprises a T cell or NK cell.

20. A method of producing an isolated EphA2-binding molecule, said method comprising;(i) culturing the host cell of claim 18 or claim 19; and(ii) isolating said EphA2-binding molecule from said host cell cultured in step (i).21 . A method comprising culturing a cell comprising a nucleic acid according to claim 14, or an expression vector according to claim 16, under conditions suitable for expression of the antigen-binding molecule from the nucleic acid(s) or expression vector(s).

22. A composition comprising the EphA2-binding molecule according to any one of claim 1 to 11 or the CAR of claim 12 and a pharmaceutically acceptable carrier diluent or excipient.

23. The composition of claim 22, additionally comprising an agent (e.g., an immunotherapy agent, such as a checkpoint inhibitor).

24. A method of treating or preventing a proliferative disease (e.g., cancer) in a subject, said method including the step of administering a therapeutically effective amount of the EphA2- binding molecule according to any one of claim 1 to 11 or the CAR of claim 12 to the subject to thereby treat or prevent the proliferative disease (e.g., cancer) in the subject.

25. Use of the EphA2-binding molecule of any one of claims 1 to 11 or the CAR of claim 12 in the manufacture of a medicament for the prevention and / or treatment of a proliferative disease (e.g., cancer) in a subject.

26. The method of claim 24, or the use of claim 25, wherein the proliferative disease is cancer.

27. The method or use of any one of claims 24 to 26, wherein the cancer is a solid cancer or tumour.

28. The EphA2-binding molecule of claim 9, the method of claim 20 or the use of claim 21 , wherein the solid cancer or tumour is selected from prostate cancer, breast cancer, colorectal cancer, and brain cancer.

29. A method of detecting EphA2 or a cell expressing EphA2, said method including the step of forming a complex between the EphA2-binding molecule according to any one of claims 1 to 11 , and EphA2 to thereby detect EphA2 or the cell expressing EphA2.

30. The method of claim 29, wherein the cell is or comprises a cancer cell.31 . An isolated nucleic acid comprising, consisting or consisting essentially of a nucleic acid sequence set forth in any one of SEQ ID NOs: 73 to 78 or a nucleic acid sequence at least 85% identical thereto.

32. A composition comprising a plurality of antigen-binding molecules that specifically bind EphA2, and upon binding activates EphA2 kinase activity, wherein the plurality of antigenbinding molecules are present in a clustered form.

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