DDX3y specific binding domains

Abstract

Disclosed herein are antigen-specific binding domains that specifically bind to DDX3Y. Examples herein include antibodies that bind to DDX3Y and do not cross-react with DDX3X and compositions comprising the same.

Description

DDX3Y SPECIFIC BINDING DOMAINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore provisional application no. 10202303462Q, filed 08 December 2023, the contents of it being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of molecular biology. In particular, the present invention relates to immunology and biotechnology.STATEMENT OF DEPOSIT

[0003] The following hybridoma cell lines were deposited in CellBank Australia with the following accession numbers: CBA20230043 (hDDX3Y-44E3); CBA20230044 (hDDX3Y- 43D1); CBA20230045 (hDDX3Y-41H3E9); CBA20230046 (hDDX3Y-43E5A6); CBA20230047 (hDDX3Y-56A9A5); and CBA20230048 (hDDX3Y-72E8A4).BACKGROUND OF THE INVENTION

[0004] DEAD box proteins, characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), are putative helicases. They axe implicated in a number of cellular' processes involving alteration of nucleic acid secondary structure such as transcription and translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their gene distribution, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division. The human genome codes for more than 30 DEAD box proteins, among which include DEAD-box helicase 3 Y-linked (DDX3Y) and its homolog, DEAD-box helicase 3 X-linked (DDX3X).

[0005] DDX3Y is encoded by the DDX3Y gene found on the Y chromosome. Its homolog, DDX3X, is encoded by the DDX3X gene found on the X chromosome. Although DDX3Y and DDX3X are homologous, they are believed to have very different physiological roles. DDX3Y is only expressed in significant amounts in the male testes and is believed to have a limited physiological role in sperm maturation, whereas DDX3X is ubiquitously expressed in cells of both male and female tissues and is thought to have multiple physiological roles.

[0006] At present, there were no commercially available antibodies that bind specifically to DDX3Y. Due to the homologous nature of the DDX3Y and DDX3X protein sequences, havingabout 92% homology, all commercially available antibodies that bind DDX3Y can also recognize and cross-react with DDX3X.[ 00071 Thus, there is an unmet need for an antibody that binds to DDX3Y, and which does not substantially bind to or cross-react with DDX3X.SUMMARY

[0008] In one aspect, the present disclosure refers to an antigen-specific binding domain comprising a heavy chain variable domain and / or a light chain variable domain, wherein the heavy chain variable domain comprises three heavy chain CDRs of CDRH1, CDRH2, and CDRH3 and wherein the light chain variable domain comprises three light chain CDRs of CDRL1 , CDRL2, and CDRL3, wherein: a. CDRH1 is selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102; or a modified CDRH1 sequence thereof; b. CDRH2 is selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104; or a modified CDRH2 sequence thereof; c. CDRH3 is selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106; or a modified CDRH3 sequence thereof; d. CDRL1 is selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110; or a modified CDRL1 sequence thereof; e. CDRL2 is selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112; or a modified CDRL2 sequence thereof; and f. CDRL3 is selected from the group consisting of SEQ ID NO: 18, 34, 50, 66, 82, 98, 114; or a modified CDRL3 sequence thereof. .

[0009] In another aspect, the present disclosure refers to a hybridoma cell line with deposition number CBA20230043.

[0010] In yet another aspect, the present disclosure refers to a hybridoma cell line with deposition number CBA20230044.

[0011] In a further aspect, the present disclosure refers to a hybridoma cell line with deposition number CBA20230045.

[0012] In another further aspect, the present disclosure refers to a hybridoma cell line with deposition number CBA20230046.

[0013] In another aspect, the present disclosure refers to a hybridoma cell line with deposition number CBA20230047.

[0014] In yet another aspect, the present disclosure refers to a hybridoma cell line with deposition number CBA20230048.

[0015] In a further aspect, the present disclosure refers to an antibody isolated from the hybridoma line as disclosed herein.

[0016] In another aspect, the present disclosure refers to a kit comprising the antigen-specific binding domain as disclosed herein, the hybridoma cell line as disclosed herein, and / or the antibody as disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0018] Fig, 1 shows western blot analysis demonstrating that commercially available anti- DDX3Y monoclonal antibodies (purchased from MyBioSource and Sigma) cross-react with DDX3X and are thus non-specific.

[0019] Figs. 2A to 2G shows western blot analysis demonstrating binding to DDX3Y. HEK 293T cells were transduced with adenoviral vectors to overexpress DDX3X, DDX3Y, or empty vector (EV). Supernatants from the clones indicated above the blots were used to perform western blots on lysates from transduced cells. Where shown, lines indicate bands at -73ED which is the molecular weight of DDX3X and DDX3Y. Supernatants from clones 34G11, 43D1, 44E3, 74E4, and 74G1 selectively stained DDX3Y. Clone 75B 1 was non-selective, and no appreciable staining was noted from other clones.

[0020] Fig. 3 shows western blot analysis of antibodies purified from the supernatants of clones 34G11 and 44E3, used to stain lysates from HEK293T cells overexpressing DDX3X or DDX3Y. Actin was used as a loading control. Both clones showed selective staining for DDX3Y.

[0021] Figs. 4A and 4B shows western blot analysis of antibodies purified from the supernatants of clones 34G11, 43D1, 44E3, 74E4, and 74G1. A) The antibodies were used to stain lysates from HEK293T cells overexpressing DDX3X or DDX3Y. Lines indicate bands at ~73kDa which is the molecular weight of DDX3X and DDX3Y. All purified antibodies showed selective staining for DDX3Y although 74G1 showed weaker staining. B) The antibodies were used to stain lysates from aggressive B-cell lymphoma cell lines Raji (male), U2932 (female), and HEK293T cells overexpressing DDX3X and DDX3Y. Arrows and asterisks indicate exogenous and endogenous DDX3Y, respectively. Staining from clones 74E4 and 74G1 was not observed in these experiments.

[0022] Fig. 5 shows western blot analysis demonstrating that all three hybridoma clones derived from DDX3Y-1 (34G11, 43D1, 44E3) and four hybridoma clones derived from DDX3Y- 2 (41H3E9, 43E5A6, 56A9A5, 72E8A4) have strong reactivity to DDX3Y and no reactivity to DDX3X.

[0023] Fig. 6 shows detection and binding of endogenously expressed DDX3Y and / or DDX3X by anti-DDX3Y antibodies in tissue samples. Fig. 6A shows immunohistochemistry (IHC) staining results as determined by 34G11, 44E3-1 and 43D1-1. Clones 44E3-1 and 43D1-1 are suitable for IHC, showing low background in ovary and specific staining in testis. Clone 34G11 has high background staining in ovary, similar to high background signals in female cell line as determined by immunoblot analysis. Fig. 6B shows immunohistochemistry (IHC) staining results as determined by 44E3-1. The 44E3-1 purified antibodies stained stronger for prostate carcinoma (CA) than normal prostate.

[0024] Fig. 7 shows immunohistochemistry (IHC) results as determined by purified antibody from clone 44E3, showing negative stain on ovary (negative control), positive stain on testis (positive control), as well as various staining intensities in tumour tissues.

[0025] Fig. 8 shows immunohistochemistry (IHC) results as determined by clones derived from DDX3Y-2. Fig. 8A shows IHC staining of negative (ovary) control, utilizing the indicated monoclonal antibodies at pH 6 and pH 9. Antibody 41H3E9 showed the least background staining and was selected for further optimization for IHC. Fig. 8B shows IHC staining of negative (ovary) and positive (testis) controls, utilizing the monoclonal antibody 41H3E9 using the indicated dilutions at pH 6 and pH 9. Mild background staining is noted at higher antibody concentrations in the negative control, however this is clearly distinguishable from the specific staining seen in the positive control at all concentrations.DEFINITIONS

[0026] As used herein, the term “antigen-specific binding domain” or “antigen-binding molecule” refers to a molecule capable of binding to a given target (also referred to as an antigen), and includes within its scope molecules such as, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multi-specific antibodies (for example, including but not limited to, bispecific or tri-specific antibodies), and fragments and derivatives thereof (for example, including but not limited to a variable fragment (Fv), a single chain variable fragment (scFv), an antigen- binding fragment (Fab), an antigen-binding fragment arm 1 (Fabl), a monovalent antigen-binding fragment containing one Fab fragment and a portion of the hinge region (F(ab’)), a single chain antigen-binding fragment (scFab), a bivalent antigen-binding fragment including two Fab fragments linked by a disulfide bridge at the hinge region (F(ab’)2), an antigen-binding fragment arm 2 (Fab2), diabodies, triabodies, single-chain antibody molecules (for example, including but not limited to scFv), tctrabodics, linear antibodies, a single chain variable fragment conjugated to a constant fragment (scFv-Fc). minibodies, and single domainantibodies (for example, including but not limited to, VHH)). Antigen- specific binding domains as disclosed herein can specifically bind to the relevant targets disclosed herein such as DDX3Y (for example, including but not limited to, human DDX3Y).

[0027] As used herein, the term “anti-DDX3Y antibody” refers to an antibody that binds DDX3Y. By the same understanding, the term “anti-DDX3 Y binding domain” refers to an antigenspecific binding domain that binds DDX3Y.

[0028] As used herein, the term “antibody” (Ab) or “antibodies”, also referred to as “immunoglobulin”, refers to a molecule or fragments of a molecule that bind to a target antigen. Such molecules can be, but are not limited to, an immunoglobulin (Ig) molecule and immunologically active portions of an 1g molecule.

[0029] As used herein, the term “immunoglobulin (Ig)” includes molecules that are naturally produced in that they are generated by an organism reacting to a target antigen, and also glycoproteins that are synthetically produced or engineered molecules (for example, including but not limited to, rccombinantly produced antibodies, chimeric antibodies, humanised antibodies etc.). Immunoglobulin molecules can be of any class (also referred to as isotypes), for example, including but not limited to, IgG, IgE, IgM, IgD, IgA, and IgY, or subclass (for example, including but not limited to, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2).

[0030] The immunoglobulin molecules referred to herein include, but is not limited to, full length functional mutants, variants, or derivatives thereof, which retain the essential antigenbinding features of the immunoglobulin molecule, and can include dual specific, bispecific, multispecific, and dual variable domain antibodies.

[0031] Structures of immunoglobulin molecules have been well characterized in the art. Briefly, each Ig molecule comprises four polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof. Heavy chains typically comprise a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein as Cn or CH). The heavy chain constant region is typically comprised of three domains, CHI, CH2 and CH3. The heavy chains are generally inter-connected via disulfide bonds in a so- called “hinge region”. Light chains typically comprise a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region is typically comprised of one domain. As used herein, the term “domain” and “region” are used interchangeably.

[0032] Heavy chain variable region and light chain variable region refer to the portion of the heavy chain or light chain, respectively, that is involved in binding the Ig molecule to a target antigen. The variable regions of the heavy chain and light chain of a native Ig molecule may befurther subdivided into regions of hypervariability, also called “complementarity-determining regions”, “CDRs”, “hypervariability regions” or “HVRs”, which are interspersed within regions that are more conserved, referred to as “framework regions” or “FR”. In general, there are four FRs in each full-length heavy chain variable region and four FRs in each full-length light chain variable region. Three CDRs are interspersed within the four FRs in the heavy chain variable region and three CDRs are interspersed within the four FRs in the light chain variable region. The three CDRs in a heavy chain variable region may be referred to as CDRH1, CDRH2 and CDRH3, and the three CDRs in a light chain variable region may be referred to as CDRL1, CDRL2 and CDRL3, numbered sequentially starting from the N-terminus. CDRs are primarily responsible for binding to an epitope of an antigen.

[0033] The amino acid residues that encompass the CDRs as defined by each of Kabat, Chotia, and MacCallum (which are different naming conventions in the art) are set forth below in Table 1 as a comparison.0034] As used herein, the term “specific” in reference to the term “antigen- specific binding domain”, or the term “specifically bind(s)”, refers to the situation in which an antigen- binding molecule will not show any statistically significant binding to molecules other than its target antigen. For example, in the context of the present disclosure, antigen-specific binding domains that bind DDX3Y, as well as anti-DDX3Y specific antibodies, as disclosed herein can bind to DDX3Y and do not substantially bind to molecules other than DDX3Y (for example, including but not limited to, DDX3X). Tn other words, the antigen-specific binding domains or antibodies disclosed herein selectively bind to DDX3Y.

[0035] As used herein, “do not substantially bind”, in reference to binding of an antibody or fragment thereof as disclosed herein to a target antigen refers to the presence or absence of off- site binding or unintended binding of the antibody to targets other than the intended target. The term “substantially” refers to the fact that an antibody will bind mainly to its target, but that some degreeof off-site binding would be acceptable. In other words, in the context of the present disclosure, the antigen-specific binding domains or antibodies disclosed herein does not substantially bind to DDX3X or targets other than DDX3 Y. In another example, in the context of the present disclosure, the antigen-specific binding domains or antibodies disclosed herein selectively bind to DDX3Y.

[0036] As used herein, the term “monoclonal antibody” or “monoclonal antibodies” refer to antibodies that are produced from a single B cell. For example, monoclonal antibodies described herein may be made by fusing a short-lived, antibody-producing B cell to a fast-growing cell such as a cancer cell (sometimes referred to as an “immortalised cell”), to arrive at a hybrid cell, also referred to as a hybridoma (cell). A hybridoma cell can multiply rapidly, thereby creating B cell clones that produce the antibody produced by the B cell. Monoclonal antibodies are target-specific in that they are produced against a particular epitope on a target antigen.L0037J As used herein, the term “polyclonal antibody” or “polyclonal antibodies” refer to antibodies that are produced from multiple different B cells. Polyclonal antibodies are produced against different epitopes on a target antigen and may also cross-rcact with other antigens.

[0038] As used herein, an “isolated” or “purified” protein (for example, including but not limited to, an antibody or fragment thereof) is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived.

[0039] As used herein, the term “human antibody” refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human gcrmlinc immunoglobulin sequences.

[0040] As used herein, the term “humanized antibody” refers to a genetically engineered nonhuman antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody CDRs, onto a homologous human acceptor framework region. The human framework region may comprise one or more amino acid mutations.

[0041] As used herein, the term “modified”, in reference to a nucleic acid sequence, refers to the inclusion of one or more mutations such as, but not limited to, substitutions, additions, deletions and translocations. In reference to an amino acid sequence, the term “modified” refers to amino acid variations present in the amino acid sequence, compared to an unmodified version of the same amino acid (for example, a wild-type sequence). Modifications as used herein include conservative mutations, wherein the mutations do not substantially change the structure or reduce the ability of the antibody to bind the antigen. As used herein, a “modified nucleic acid sequence”or “modified amino acid sequence” is still capable of specifically binding to DDX3Y. In other words, a “modified nucleic acid sequence” or “modified amino acid sequence” does not lose its ability to bind specifically to DDX3Y Additionally, as used herein, the “modified nucleic acid sequence” or “modified amino acid sequence” does not result in cross-reactivity of the antibody or antigen- specific binding domain disclosed herein to DDX3X.

[0042] As used herein, a “modified nucleic acid sequence” refers to a nucleic acid sequence that deviates from the corresponding unmodified nucleic acid sequence by inclusion of one or more mutations. In one example, the unmodified nucleic acid sequence can be either a wildtype sequence, or a reference sequence. In some examples, the modified nucleic acid sequence as disclosed herein includes one, two, or three, or more nucleic acids different from the unmodified nucleic acid sequence. In some examples, the modified nucleic acid sequence as disclosed herein has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the corresponding unmodified nucleic acid sequence.

[0043] As used herein, a “modified amino acid sequence” refers to an amino acid sequence that deviates from the corresponding unmodified amino acid sequence by inclusion of one or more substitutions, additions or deletions. In some examples, the modified amino acid sequence as disclosed herein includes one or more amino acids different from the unmodified amino acid sequence. In some examples, the modified nucleic acid sequence as disclosed herein encodes a protein having an amino acid sequence that has at least 80% e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the amino acid sequence of a protein encoded by the corresponding unmodified nucleic acid sequence.

[0044] As used herein, the term “percent (%) identity” with respect to a reference amino acid sequence or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotide residues in a candidate sequence that are identical with the amino acid residues or nucleotide residues in the reference sequence, after aligning the sequences and introducing gaps.

[0045] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B”; “A or B”; “A” (alone) and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following examples: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone) and C (alone).DETAILED DESCRIPTION

[0046] DDX3Y and DDX3X are DNA / RNA helicases with high protein sequence homology, showing about 92% homology to each other. In spite of their homology, DDX3Y and DDX3X have different physiological roles and different expression profiles. However, studying their physiological roles and expression profiles individually has been difficult because hitherto, no commercially available antibodies have been shown to be specific for DDX3Y. Due to the high protein sequence homologies between DDX3Y and DDX3X, all commercially available antibodies that bind DDX3Y cross-react with DDX3X. Accordingly, it had not been possible previously to comprehensively and reliably study the physiological role and expression profile of DDX3Y independently of DDX3X.

[0047] In developing the anti-DDX3Y specific antibodies, three peptide sequences were identified, showing the most differences between the DDX3X and DDX3Y protein. These sequences were identified in the N-terminal domain by aligning the amino acid (aa) sequences of DDX3X and DDX3Y. The three peptides were shown to be specific for DDX3Y to varying degrees. The three peptides are: amino acids 2 to 13 (SHVVVKNDPELD, denoted as DDX3Y-1; SEQ ID NO: 1), amino acids 22 to 33 (NSEKQSGGASTA, denoted as DDX3Y-3; SEQ ID NO: 2), and amino acids 106 to 118 (IGNRERPGFGRFER, denoted as DDX3Y-2; SEQ ID NO: 3). The amino acid numbering as disclosed here is based on the protein sequence of Human DDX3Y with NCB1 Reference Sequence: NP_001116137.1.Alignment of Human DDX3X and DDX3Y[ 00481 The identical matches are marked by letter code in a line between the two queries (top query in black, bottom query in grey); ‘homologous’ substitutions (determined by the scoring matrix used) are marked by ‘+’ symbol in a line between the two queries; gaps are marked by either blank spaces (when occurring outside of the identified peptide sequences) or black spaces (when occurring within the identified peptide sequences) in a line between the two queries; and the three identified peptide sequences from DDX3Y (denoted herein as SEQ ID NOs: 1, 2, and 3, respectively) arc marked by underline:DDX3X 1 MSHVAVENALGLDQQFAGLDLNS SDNQSGG- STASKGRYI PPHLRNREATKGFYDKDS SGW 60DDX3X 121 RGGNS RWCDKSDEDDWSKPLPPSERLEQELFSGGNTGINFEKYDDI PVEATGNNCPPHI E 180DDX3X 661 GN 662

[0049] The three peptides, DDX3Y-1 (SEQ ID NO: 1), DDX3Y-2 (SEQ ID NO: 3) and DDX3Y-3 (SEQ ID NO: 2), were selected and used to develop anti-DDX3Y antibodies (that is, antibodies that bind to or target DDX3Y) disclosed herein. Cysteine was added to either the N’- terminal or C’-terminal of each of the three peptides in order to enable keyhole limpet hemocyanin (KLH) conjugation. No significant alignment was detected between DDX3Y-1 peptide and its DDX3X counterpart. Even though the DDX3Y-2 and DDX3Y-3 peptide sequences are about 70% similar to their DDX3X counterparts, sequence interruptions by gaps or mismatched amino acid with distinct side chain properties allowed generation of independent DDX3Y-specific antibodies without cross reactivity with DDX3X.Alignment of DDX3Y-3 and corresponding DDX3X peptide

[0050] NSEKQSGGASTAC (amino acids 22 to 33 of DDX3Y with C’-terminal cysteine; denoted as SEQ ID NO: 230)Query SEKQSGGASTA (DDX3Y)S+ QSGG STASubj ect SDNQSGG-STA (DDX3X )Alignment of DDX3Y-2 and corresponding DDX3X peptide

[0051] IGNRERPGFGRFERC (amino acids 106 to 119 of DDX3Y with C’-terminal cysteine, denoted as SEQ ID NO: 231)Query IGNR-ERPGFGRFER ( DDX3Y)IG R +R GFG FERSubj ect IGSRGDRSGFGKFER ( DDX3X)

[0052] Specificity of the anti-DDX3Y antibodies was achieved using stringent selection criteria applied to hybridoma clones. Hybridoma clones which showed specificity to DDX3Y by ELISA, and showed no cross-reaction with DDX3X by western blot and immunohistochemistry (IHC) were selected for analysis.

[0053] For the clones derived from DDX3Y-1 peptide, western blot analysis was performed using 53 clones (16 clones from Balb / c mouse #M0168; 4 clones from C57BL / 6 mouse #M0208; 26 clones from C57BL / 6 mouse #M0209; and 7 clones from C57BL / 6 mouse #M0210) on protein lysates from cells forcibly overexpressing DDX3X. The results showed that among the 53 clones that were screened, 5 clones specifically bound to DDX3Y with little or no cross-reaction with DDX3X. The 5 clones are 34G11 from #M0210; 43D1 from#M0168; 44E3 from #M0168; 74E4 from#M0209; and 74G1 from#M0209 (Figs. 2A, 2B, 2F and 4A). Moreover, the 3 clones 34G11, 43D1 and 44E3, demonstrated strong reactivity to DDX3Y and no reactivity to DDX3X (Fig. 5).

[0054] A further western blot analysis was performed using the 5 clones on Raji (a male cell line) and U2932 (a female cell line) cell lines to confirm the binding activity and specificity of the antibodies to DDX3Y. The results showed that clones 44E3 and 43D1 had no signals in U2932, and had strong signals in Raji, indicating the specificity of these clones to endogenous DDX3Y without cross-reaction with endogenous DDX3X (Fig. 4B).

[0055] Similarly, when the three hybridoma clones 34G11, 43D1 and 44E3, were used for immunohistochemistry staining, the results demonstrate that subclones 44E3-1 and 43D1-1,derived from parental clones 44E3 and 43D1, respectively, show low background staining in the ovary sample and specific staining in the testis sample (Fig. 6A). However, clone 34G11 showed high background staining in the ovary sample (Fig. 6A), similar to the high background signal in U2932 female cell line as determined by the western blot (Fig. 4B). Based on the immunohistochemistry studies, antibody 44E3-1 appeared to show the least non-specific staining with 1:1,000 dilution and appeared to stain stronger for prostate cancer (CA) than normal prostate (Fig. 6B).

[0056] For the clones derived from peptide DDX3Y-2, a western blot analysis was performed using 4 clones (41H3E9, 43E5A6, 56A9A5 and 72E8A4) on protein lysates from cells forcibly overexpressing DDX3X. The results showed that all 4 clones specifically bound to DDX3Y without cross-reacting with DDX3X (Figs. 5 and 8). Moreover, the immunohistochemistry staining showed the strongest and most specific signal amongst the 4 clones was obtained using the antibody 41H3E9 (Figs. 8A and 8B).

[0057] Also described herein arc antigen peptides, for example obtained from mouse DDX3Y, that can be used to generate anti-DDX3Y antibodies. In particular, it was found that when aligning the amino acid (aa) sequences of human DDX3Y and mouse DDX3Y, the amino acid sequences of peptides DDX3Y-1 and DDX3Y-2 described herein do not share a high degree of similarity to the corresponding amino acid positions on mouse DDX3Y. However, the amino acid sequences of DDX3 Y-3 described herein are about 70% similar to the corresponding amino acid positions of the mouse counterpart. Thus, contemplated herein are anti-DDX3Y specific antibodies or antigenspecific binding domains that bind human DDX3Y and do not substantially bind to mouse DDX3Y.Alignment of Human DDX3Y and Mouse DDX3Y

[0058] The identical matches are marked by letter code in a line between the two queries (top query in black, bottom query in grey); ‘homologous’ substitutions (determined by the scoring matrix used) are marked by ‘+’ symbol in a line between the two queries; gaps are marked by cither blank spaces (when occurring outside of the identified peptide sequences) or black spaces (when occurring within the identified peptide sequences) in a line between the two queries; and the three identified peptide sequences from human DDX3Y (denoted herein as SEQ ID NOs: 1, 2, and 3, respectively) are marked by underline:SEQ ID NO: 1 SEQ ID NO: 2Hs DDX3Y 1 XSHVWKNDPELDQQLANLDL-NSEKQSGGASTASKGRYI PPHLRNREASKGFHDKDSSG 59

[0059] Also contemplated herein are anti-DDX3Y specific antibodies or antigen-specific binding domains that are generated by immunizing an animal with DDX3Y-1, DDX3Y-2 and / or DDX3Y-3. In one example, the anti-DDX3Y specific antibodies or antigen- specific binding domains that are generated by immunizing an animal with a peptide having an amino acid sequence SHVVVKNDPELDC (amino acids 2 to 13 of human DDX3Y and a C-tcrminal cysteine; SEQ ID NO: 229). In one example, the anti-DDX3Y specific antibodies or antigen-specific binding domains that are generated by immunizing an animal with a peptide having an amino acid sequence IGNRERPGFGRFERC (amino acids 106 to 119 of human DDX3Y and a C-terminal cysteine; SEQ ID NO: 231). In some examples, the animal is a mammal. The mammal can be, but is not limited to, a rabbit, rat, guinea pig, hamster, goat, sheep, horse, cows, or mouse. In one example, the mammal is a mouse or a rabbit.Antigen-Specific Binding Domains

[0060] The present disclosure refers to antigen- specific binding domains that bind to DDX3Y. In one example, there is described an antigen-specific binding domain that is specific for DDX3Y and does not substantially bind to DDX3X. In other words, the antigen- specific binding domains claimed herein may have little to no binding with DDX3X. In some cases, while binding to DDX3X may be present, such binding is not considered to be statistically relevant.

[0061] In some examples, the anti-DDX3Y antibody (also referred to as the antibody that binds DDX3Y or antigen- specific binding domain that binds to DDX3Y) is a full-length antibody, which comprises two heavy chains and two light chains, each comprising a variable domain and a constant domain. In another example, the anti-DDX3Y antibody can be an antigen-binding fragment of a full-length antibody or a fragment thereof. In some examples, the antigen- specific binding domain is a mutant of a full-length antibody or fragment thereof, a fusion protein comprising an antibody portion that binds DDX3Y, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity. In some examples, the fragment is an antigen-binding fragment (Fab), including but not limited to Fabl , Fab2, F(ab') and F(ab')2- In some examples, the fragment is a variable fragment (Fv) consisting of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and Vn regions pair to form monovalent molecules known as single chain Fv (scFv). A person of skill in the art would be able to derive any one of the fragments described herein from a full antibody using methods known in the art. In one example, the antigen-binding fragment can be, but is not limited to, a Fv, a scFv, a Fab, a scFab, a F(ab’), a F(ab’)2, a diabody, a triabody, a scFv-Fc, a minibody, or a single domain antibody.

[0062] In some examples, the anti-DDX3Y antibody is an antibody of a clone such as, but not limited to, 44E3, 43D1, 41H3E9, 43E5A6, 56A9A5 or 72E8A4.

[0063] As used herein, the term “reference antibody of clone 44E3” refers to an antibody that specifically binds DDX3 Y and comprises a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 117 to 123 and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 125 to 131. As used herein, the term “reference antibody of clone 43D1” refers to an antibody that specifically binds DDX3Y and comprises a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 133 to 139 and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 141 to 147. As used herein, the term “reference antibody of clone 41H3E9” refers to an antibody that specifically binds DDX3Y and comprises a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 165 to 171 and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 173 to 179. As used herein, the term “reference antibody of clone 43E5A6” refers to an antibody that specifically binds DDX3Y and comprises a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 181 to 187 and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 189 to 195. As used herein, the term “reference antibody of clone 56A9A5” refers to an antibody that specifically binds DDX3Y and comprises a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 197 to 203 and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 205 to 211. As used herein, the term “reference antibody of clone 72E8A4” refers to an antibody that specifically binds DDX3Y and comprises a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 213 to 219 and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 221 to 227.

[0064] Disclosed herein is, as an example, an antigen-specific binding domain comprising heavy chain CDRs of CDRH1, CDRH2, and CDRH3 and / or light chain CDRs of CDRL1, CDRL2, and CDRL3.

[0065] In some examples, the CDRH1 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86 or 102, or a modified CDRH1 sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 6, 22, 38, 54, 70, 86 or 102. In some examples, the CDRH2 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88 or 104, or a modified CDRH2 sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 8, 24, 40, 56, 72, 88 or 104. In some examples, the CDRH3 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90 or 106, or a modified CDRH3 sequence thereof having at least 80% e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 10, 26, 42, 58, 74, 90 or 106.

[0066] In some examples, the CDRL1 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94 or 110, or a modified CDRL1 sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 14, 30, 46, 62, 78, 94 or 110. In some examples, the CDRL2 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96 or 112, or a modified CDRL2 sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 16, 32, 48, 64, 80, 96 or 112. In some examples, the CDRL3 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 18, 34, 50, 66, 82, 98 or 114, or a modified CDRL3 sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 18, 34, 50, 66, 82, 98 or 1 14.

[0067] Thus, in one example, there is disclosed an antigen-specific binding domain comprising a heavy chain variable domain and / or a light chain variable domain, wherein the heavy chain variable domain comprises heavy chain CDRs of CDRH1, CDRH2, and CDRH3 and wherein the light chain variable domain comprises light chain CDRs of CDRL1, CDRL2, and CDRL3, wherein: a. CDRH1 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102; or a modified CDRH1 sequence thereof; b. CDRH2 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104; or a modified CDRH2 sequence thereof; c. CDRH3 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106; or a modified CDRH3 sequence thereof; d. CDRL1 is encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110; or a modified CDRL1 sequence thereof; e. CDRL2 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112; or a modified CDRL2 sequence thereof; or f. CDRL3 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 18, 34, 50, 66, 82, 98, 114; or a modified CDRL3 sequence thereof, and wherein the modified CDRs have nucleic acid sequences that have at least 90% identity to the corresponding nucleic acid sequences.

[0068] In another example, the antigen-specific binding domain described herein comprises the heavy chain CDRs encoded by nucleic acid sequences wherein the CDRH1 is SEQ ID NO: 6, the CRDH2 is SEQ ID NO: 8, the CRDH3 is SEQ ID NO: 10, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In another example, the heavy chain CDRs are encoded by nucleic acid sequences wherein the CDRH1 is SEQ ID NO:22, the CRDH2 is SEQ ID NO: 24, the CRDH3 is SEQ ID NO: 26, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In another example, the heavy chain CDRs are encoded by nucleic acid sequences wherein the CDRH1 is SEQ ID NO: 38, the CRDH2 is SEQ ID NO: 40, the CRDH3 is SEQ ID NO: 42, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In another example, the heavy chain CDRs are encoded by nucleic acid sequences wherein the CDRH1 is SEQ ID NO:54, the CRDH2 is SEQ ID NO: 56, the CRDH3 is SEQ ID NO: 58, or modified amino acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In another example, the heavy chain CDRs are encoded by nucleic acid sequences wherein the CDRH1 is SEQ ID NO: 70, the CRDH2 is SEQ ID NO: 72, the CRDH3 is SEQ ID NO: 74, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In another example, the heavy chain CDRs are encoded by nucleic acid sequences wherein the CDRH1 is SEQ ID NO: 86, the CRDH2 is SEQ ID NO: 88, the CRDH3 is SEQ ID NO: 90, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In another example, the heavychain CDRs are encoded by nucleic acid sequences wherein the CDRH1 is SEQ ID NO: 102, the CRDH2 is SEQ ID NO: 104, the CRDH3 is SEQ ID NO: 106, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences.[0069 J Further described herein, as an example, the heavy chain CDRs of the antigen- specific binding domains disclosed herein include but are not limited to g. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 6, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 8, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 10, or modified nucleic acid sequences thereof; h. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO:22, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 24, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 26, or modified nucleic acid sequences thereof; i. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO:54, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 56, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 58, or modified nucleic acid sequences thereof; j. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 70, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 72, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 74, or modified nucleic acid sequences thereof; k. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 86, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 88, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 90, or modified nucleic acid sequences thereof; or 1. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 102, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 104, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 106, or modified nucleic acid sequences thereof.

[0070] In another example, there is disclosed an antigen-specific binding domain comprising the light chain CDRs encoded by nucleic acid sequences wherein the CDRL1 is SEQ ID NO: 14, the CDRL2 is SEQ ID NO: 16, the CDRL3 is SEQ ID NO: 18, or modified nucleic acid sequences thereof having at least 80% e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In some examples, the light chain CDRs are encoded by nucleic acid sequences wherein the CDRL1 is SEQ ID NO: 30, the CDRL2 is SEQ ID NO: 32, the CDRL3 is SEQ ID NO: 34, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In some examples, the light chain CDRs arc encoded by nucleic acid sequences wherein the CDRL1 is SEQ ID NO: 46, the CDRL2 is SEQ ID NO: 48, the CDRL3 isSEQ ID NO: 50, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In some examples, the light chain CDRs are encoded by nucleic acid sequences wherein the CDRL1 is SEQ ID NO: 62, the CDRL2 is SEQ ID NO: 64, the CDRL3 is SEQ ID NO: 66, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In some examples, the light chain CDRs are encoded by nucleic acid sequences wherein the CDRL1 is SEQ ID NO: 78, the CDRL2 is SEQ ID NO: 80, the CDRL3 is SEQ ID NO: 82, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In some examples, the light chain CDRs are encoded by nucleic acid sequences wherein the CDRL1 is SEQ ID NO: 94, the CDRL2 is SEQ ID NO: 96, the CDRL3 is SEQ ID NO: 98, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences. In some examples, the light chain CDRs are encoded by nucleic acid sequences wherein the CDRL1 is SEQ ID NO: 110, the CDRL2 is SEQ ID NO: 112, the CDRL3 is SEQ ID NO: 114, or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the respective nucleic acid sequences.

[0071] Also disclosed herein is, as an example, the light chain CDRs of the antigen- specific binding domains disclosed herein include but are not limited to: m. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 14, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 16, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 18, or modified nucleic acid sequences thereof; n. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 30, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 32, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 34, or modified nucleic acid sequences thereof; o. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 62, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 64, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 66, or modified nucleic acid sequences thereof; p. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 78, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 80, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 82, or modified nucleic acid sequences thereof; q. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 94, a CDRL2 encoded by a nucleic acid sequenceset forth in SEQ ID NO: 96, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 98, or modified nucleic acid sequences thereof; or r. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 110, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 112, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 114, or modified nucleic acid sequences thereof.

[0072] In another example, the present disclosure describes an antigen- specific binding domain comprising a heavy chain variable domain, wherein the heavy chain variable domain has a nucleic acid sequence comprising sequences as set forth in SEQ ID NOs: 5 to 11 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 5 to 1 1 . In another example, the heavy chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 21 to 27 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 21 to 27. In another example, the heavy chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 37 to 43 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 37 to 43. In another example, the heavy chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 53 to 59 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 53 to 59. In another example, the heavy chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 69 to 75 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 69 to 75. In another example, the heavy chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 85 to 91 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 85 to 91. In another example, the heavy chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 101 to 107 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 101 to 107.

[0073] In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises heavy chain variable domain, wherein the framework regions of the heavy chain variabledomain FR1, FR2, FR3 and FR4 comprise a nucleic acid sequence set forth in SEQ ID NOs: 5, 7, 9 and 11, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%>, 97%>, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 5, 7, 9 and 11, respectively; SEQ ID NOs: 21, 23, 25 and 27, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g. , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 21, 23, 25 and 27, respectively; SEQ ID NOs: 37, 39, 41 and 43, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 37, 39, 41 and 43, respectively; SEQ ID NOs: 53, 55, 57 and 59, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 53, 55, 57 and 59, respectively; SEQ ID NOs: 69, 71, 73 and 75, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 69, 71, 73 and 75, respectively; and SEQ ID NOs: 85, 87, 89 and 91, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 85, 87, 89 and 91, respectively; SEQ ID NOs: 101, 103, 105 and 107, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 101, 103, 105 and 107, respectively.

[0074] Also disclosed herein, as an example, the heavy chain variable domain of the antigenspecific binding domains disclosed herein include but are not limited to: s. SEQ ID NOs: 5 to 11 or modified nucleic acid sequences thereof; t. SEQ ID NOs: 21 to 27 or modified nucleic acid sequence thereof; u. SEQ ID NOs: 53 to 59 or modified nucleic acid sequence thereof; v. SEQ ID NOs: 69 to 75 or modified nucleic acid sequence thereof; w. SEQ ID NOs: 85 to 91 or modified nucleic acid sequence thereof; and x. SEQ ID NOs: 101 to 107 or modified nucleic acid sequence thereof.

[0075] In another example, the present disclosure relates to an antigen- specific binding domain comprising a light chain variable domain comprising the nucleic acid sequences as set forth in SEQ ID NOs: 13 to 19 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 13 to 19. In another example, the light chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 29 to 35 or modifiednucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 29 to 35. In another example, the light chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 45 to 51 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 45 to 51. In another example, the light chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 61 to 67 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 61 to 67. In another example, the light chain variable domain comprising the nucleic acid sequences as set forth in SEQ ID NOs: 77 to 83 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 77 to 83. In another example, the light chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 93 to 99 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 93 to 99. In another example, the light chain variable domain comprises the nucleic acid sequences as set forth in SEQ ID NOs: 109 to 115 or modified nucleic acid sequences thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 109 to 115.

[0076] In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises light chain variable domain, wherein the framework regions of the light chain variable domain FR1 , FR2, FR3 and FR4 comprise a nucleic acid sequence set forth in SEQ ID NOs: 13, 15, 17 and 19, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 13, 15, 17 and 19, respectively; SEQ ID NOs: 29, 31, 33 and 35, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g. , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 29, 31, 33 and 35, respectively; SEQ ID NOs: 45, 47, 49 and 51, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 45, 47, 49 and 51, respectively; SEQ ID NOs: 61, 63, 65 and 67, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 61, 63, 65 and 67, respectively; SEQ ID NOs: 77, 79, 81 and 83, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 77, 79, 81 and 83, respectively; and SEQ ID NOs: 93, 95, 97 and 99, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 93, 95, 97 and 99, respectively; SEQ ID NOs: 109, 111, 113 and 115, respectively, or a modified nucleic acid sequence thereof having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 109, 1 1 1 , 1 13 and 1 15, respectively.

[0077] Also disclosed herein, as an example, the light chain variable domain of the antigenspecific binding domains disclosed herein include but are not limited to: y. SEQ ID NOs: 13 to 19, or modified nucleic acid sequence thereof; z. SEQ ID NOs: 29 to 35, or modified nucleic acid sequence thereof; aa. SEQ ID NOs: 61 to 67 or modified nucleic acid sequence thereof; bb. SEQ ID NOs: 77 to 83 or modified nucleic acid sequence thereof; cc. SEQ ID NOs: 93 to 99 or modified nucleic acid sequence thereof; and dd. SEQ ID NOs: 109 to 115 or modified nucleic acid sequence thereof.

[0078] In some examples, the modified nucleic acid sequence encodes a modified amino acid sequence, wherein the modified amino acid sequence differs from the unmodified amino acid sequences disclosed herein by one or more amino acids. In some examples, the one or more modifications in the amino acid sequence can be present in the CDR sequences. In some examples, the CDR sequence comprises 1 to 3, 1 or 2, or 1 modification(s). In one example, the modified amino acid sequences disclosed herein may differ by 1 or 2 amino acids from the unmodified amino acid sequences disclosed herein.

[0079] In some examples, the one or more modifications is one or more substitutions, and the one or more substitutions include conservative amino acid substitutions which do not materially or significantly affect the specificity and / or activity of the antibodies or antigen-specific binding domains disclosed herein. Both conservative and non-conscrvativc amino acid substitutions arc contemplated herein for the CDR region so long as the binding activity of the antibodies or antigenspecific binding domain is not substantially affected.

[0080] Amino acid modifications may be made in the variable region sequence outside of the CDRs so as to retain the CDR sequences. Thus, changes in the variable region sequence or alternative non-homologous variable region sequences may be introduced or utilized, such that the CDR sequences are maintained and the remainder of the variable region sequence maybesubstituted. In some examples, the modifications are in the framework sequence. It is contemplated that the variable region sequence comprises only a few modifications, preferably from 1 to 10, preferably from 1 to 9, preferably from 1 to 8, preferably from 1 to 7, preferably from 1 to 6, preferably from 1 to 5, preferably from 1 to 4, preferably from 1 to 3, preferably 1 or 2, or preferably 1 modification(s). In some examples, the modification is a substitution, wherein the substitution includes particularly conservative amino acid substitutions which do not materially or significantly affect the binding activity of the antibodies or antigen-specific binding domains disclosed herein. Conservative and non-conservative amino acid substitutions are contemplated herein for the variable region.

[0081] In some examples, the anti-DDX3Y antibody or antigen-specific binding domain disclosed herein comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises CDRH1, CDRH2 and CDRH3 having modified nucleic acid sequences, wherein the modified nucleic acid sequences encode CDRH1, CDRH2 and CDRH3, respectively, each having an amino acid sequence with at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, as disclosed herein. In some examples, the modified nucleic acid sequence encodes a CDRH1 having an amino acid sequence with at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 118, 134, 150, 166, 182, 198 or 214. In some examples, the modified nucleic acid sequence encodes a CDRH2 having an amino acid sequence with at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 120, 136, 152, 168, 184, 200 or 216. In some examples, the modified nucleic acid sequence encodes a CDRH3 having an amino acid sequence with at least 80% (e.g., 80%, 85%, 90%, 91 %>, 92%>, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 122, 138, 154, 170, 186, 202 or 218.1n some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a light chain variable domain, wherein the light chain variable domain comprises CDRL1, CDRL2 and CDRL3 having modified nucleic acid sequences, wherein the modified nucleic acid sequences encode CDRL1, CDRL2 and CDRL3, respectively, each having an amino acid sequence with at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, as disclosed herein. In some examples, the modified nucleic acid sequence encodes a CDRL1 having an amino acid sequence with at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and anyincrement therein) sequence identity to SEQ ID NOs: 126, 142, 158, 174, 190, 206 or 222. In some examples, the modified nucleic acid sequence encodes a CDRL1 having an amino acid sequence with at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 128, 144, 160, 176, 192, 208 or 224. In some examples, the modified nucleic acid sequence encodes a CDRL3 having an amino acid sequence with at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 130, 146, 162, 178, 194, 210 or 226.

[0082] Also described herein, as an example, the antigen-specific binding domains disclosed herein comprise a modified nucleic acid sequence that includes but is not limited to: ee. the modified nucleic acid sequence encodes a CDRH1 having an amino acid sequence with at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 134, 150, 166, 182, 198 and 214; ff. the modified nucleic acid sequence encodes a CDRH2 having an amino acid sequence with at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 120, 136, 152, 168, 184, 200 and 216; gg. the modified nucleic acid sequence encodes a CDRH3 having an amino acid sequence with at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 122, 138, 154, 170, 186, 202 and 218; hh. the modified nucleic acid sequence encodes a CDRL1 having an amino acid sequence with at least 80% identity to an amino acid sequence is selected from the group consisting of SEQ ID NOs: 126, 142, 158, 174, 190, 206 and 222; ii. the modified nucleic acid sequence encodes a CDRL2 having an amino acid sequence with at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 128, 144, 160, 176, 192, 208 and 224; and / or jj. the modified nucleic acid sequence encodes a CDRL3 having an amino acid sequence with at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 146, 162, 178, 194, 210 and 226.

[0083] In some examples, the heavy chain variable domain CDRH1, CDRH2, and CDRH3 amino acid sequences have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 118, 120 and 122, respectively; SEQ ID NOs: 134, 136 and 138, respectively; SEQ ID NOs: 150, 152 and 154, respectively; SEQ ID NOs: 166, 168 and 170, respectively; SEQ ID NOs: 182, 184 and 186, respectively; SEQ ID NOs: 198, 200 and 202, respectively; or SEQ ID NOs: 214, 216 and 218, respectively. In some examples, the light chain variable domain CDRL1, CDRL2, and CDRL3 amino acid sequences have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NOs: 126, 128 and130, respectively; SEQ ID NOs: 142, 144 and 146, respectively; SEQ ID NOs: 158, 160 and 162, respectively; SEQ ID NOs: 174, 176 and 178, respectively; SEQ ID NOs: 190, 192 and 194, respectively; SEQ ID NOs: 206, 208 and 210, respectively; and SEQ ID NOs: 222, 224 and 226, respectively.[0084 J Also described herein, as an example, the modified nucleic acid sequences of the heavy chain CDRs encode heavy chain CDRs that includes but is not limited to: kk. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 118, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 120, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 122; 11. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 134, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 136, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 138; mm. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 166, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 168, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 170; nn. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 182, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 184, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 186; oo. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 198, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 200, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 202; or pp. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 214, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 216, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 218.

[0085] Also described herein, as an example, the modified nucleic acid sequences of the light chain CDRs encode light chain CDRs that includes but is not limited to: qq. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 126, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 128, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 130; rr. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 142, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 144, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 146; ss. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 174, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 176, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 178; tt. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 190, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 192, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 194; uu. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 206, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 208, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 210; or vv. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 222, a CRDL2 having anamino acid sequence set forth in SEQ ID NO: 224, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 226.

[0086] In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 117 to 123. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 133 to 139. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 149 to 155. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 165 to 171. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 181 to 187. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 197 to 203. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%>, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 213 to 219.

[0087] In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a heavy chain variable domain, wherein the framework regions of the heavy chain variable domain FR1, FR2, FR3 and FR4 comprise an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain variable region FR1, FR2, FR3 and FR4 amino acid sequence set forth in SEQ ID NOs: 117, 119, 121 and 123, respectively; SEQ ID NOs: 133, 135, 137 and 139, respectively; SEQ ID NOs: 149, 151, 153 and 155, respectively; SEQ ID NOs: 165, 167, 169 and 171, respectively; SEQ ID NOs: 181, 183, 185 and 187, respectively; SEQ ID NOs: 197, 199, 201 and 203, respectively; or SEQ ID NOs: 213, 215, 217 and 219, respectively.

[0088] Also disclosed herein, as an example, die modified nucleic acid sequences of die heavy chain variable domain encode a heavy chain variable domain having amino acid sequences including but not limited to: ww. SEQ ID NOs: 117 to 123; xx. SEQ ID NOs: 133 to 139; yy. SEQ ID NOs: 165 to 171; zz. SEQ ID NOs: 181 to 187; aaa. SEQ ID NOs: 197 to 203; or bbb. SEQ ID NOs: 213 to 219.

[0089] In some examples, the modified nucleic acid sequences encode a light chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 125 to 131. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 141 to 147. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 157 to 163. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 173 to 179. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 189 to 195. In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 205 to 21 1 . In some examples, the modified nucleic acid sequences encode a heavy chain variable domain comprising amino acid sequences having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to each of SEQ ID NOs: 221 to 227.

[0090] In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises light chain variable domain, wherein the framework regions of the light chain variable domain FR1, FR2, FR3 and FR4 comprise an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain variable region FR1, FR2, FR3 and FR4 amino acid sequence set forth in SEQ ID NOs: 125, 127, 129 and 131, respectively; SEQ ID NOs: 141, 143, 145 and147, respectively; SEQ ID NOs: 157, 159, 161 and 163, respectively; SEQ ID NOs: 173, 175, 177 and 179, respectively; SEQ ID NOs: 189, 191, 193 and 195, respectively; SEQ ID NOs: 205, 207, 209 and 211, respectively; and SEQ ID NOs: 221, 223, 225 and 227, respectively.

[0091] Also disclosed herein, as an example, the modified nucleic acid sequences of the light chain variable domain encode a light chain variable domain having amino acid sequences including but not limited to: ccc. SEQ ID NOs: 125 to 131; ddd. SEQ ID NOs: 141 to 147; eee. SEQ ID NOs: 173 to 179; fff. SEQ ID NOs: 189 to 195; ggg. SEQ ID NOs: 205 to 211; or hhh. SEQ ID NOs: 221 to 227.

[0092] Also disclosed herein are hybridoma cell lines that produce anti-DDX3Y specific antibodies. In one example, the hybridoma cell line is a hybridoma cell line with deposition number CBA20230043. In one example, the hybridoma cell line is a hybridoma cell line with deposition number CBA20230044. In one example, the hybridoma cell line is a hybridoma cell line with deposition number CBA20230045. In one example, the hybridoma cell line is a hybridoma cell line with deposition number CBA20230046. In one example, the hybridoma cell line is a hybridoma cell line with deposition number CBA20230047. hi one example, the hybridoma cell line is a hybridoma cell line with deposition number CBA20230048.

[0093] In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a heavy chain variable domain comprising heavy chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230043. In some examples, the anti-DDX3Y antibody or antigenspecific binding domain comprises a heavy chain variable domain comprising heavy chain CDRs having at least 80% e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain CDRs of an antibody from the hybridoma cell hne with deposition number CBA20230044. In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises a heavy chain variable domain comprising heavy chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230045. In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises a heavy chain variable domain comprising heavy chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230046. In some examples, the anti-DDX3Y antibody or antigen- specific binding domaincomprises a heavy chain variable domain comprising heavy chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230047. In some examples, the anti-DDX3Y antibody or antigenspecific binding domain comprises a heavy chain variable domain comprising heavy chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230048.

[0094] In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a light chain variable domain comprising light chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230043. In some examples, the anti-DDX3Y antibody or antigenspecific binding domain comprises a light chain variable domain comprising light chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230044. In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a light chain variable domain comprising light chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230045. In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises a light chain variable domain comprising light chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230046. In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises a light chain variable domain comprising light chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230047. In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a light chain variable domain comprising light chain CDRs having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain CDRs of an antibody from the hybridoma cell line with deposition number CBA20230048.

[0095] In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230043. In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230044. In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230045. In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain variable domain of an antibody from the hybridoma cell hne with deposition number CBA20230046. In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230047. In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230048.

[0096] In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a light chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230043. In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a light chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% and any increment therein) sequence identity to the light chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230044. In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises a light chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230045. In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a light chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230046. In some examples, the anti-DDX3Y antibody or antigen- specific binding domain comprises a light chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230047. In some examples, the anti-DDX3Y antibody or antigen-specific binding domain comprises a light chain variable domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain variable domain of an antibody from the hybridoma cell line with deposition number CBA20230048.

[0097] In some examples, the antibody or antigen-specific binding domain comprises a heavy chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230043. In some examples, the antibody or antigen- specific binding domain comprises a heavy chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230044. In some examples, the antibody or antigen- specific binding domain comprises a heavy chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230045. In some examples, the antibody or antigen- specific binding domain comprises a heavy chain constantdomain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230046. In some examples, the antibody or antigen-specific binding domain comprises a heavy chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230047. In some examples, the antibody or antigen- specific binding domain comprises a heavy chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the heavy chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230048.

[0098] In some examples, the antibody or antigen-specific binding domain comprises a light chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230043. In some examples, the antibody or antigen- specific binding domain comprises a light chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230044. In some examples, the antibody or antigen- specific binding domain comprises a light chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230045. In some examples, the antibody or antigen-specific binding domain comprises a light chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230046. In some examples, the antibody or antigen-specific binding domain comprises a light chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230047. In some examples, the antibody or antigen- specificbinding domain comprises a light chain constant domain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the light chain constant domain of an antibody from the hybridoma cell line with deposition number CBA20230048.L0099J In certain examples, the anti-DDX3Y antibody or antigen-specific binding domain can comprise nucleic acid sequences from, but not limited to, human, murine (for example, including but not limited to, mouse and rat), cricctinc (for example, including but not limited to, hamster), leporine (for example, including but not limited to, rabbit and hare), cavine (for example, including but not limited to, guinea pig), caprine (for example, including but not limited to, goat), ovine, bovine, equine antibodies or antigen-specific binding domains.

[0100] In one example, the antibody or antigen-specific binding domain as described herein is a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (for example, but not limited to, bispecific or trispecific antibodies), an antigen-binding fragments and derivatives thereof.

[0101] Monoclonal antibodies derived by hybridoma technique from another species than human, such as mouse or rabbit, can be humanized, which means that a non-human antibody is genetically engineered to be more human. The methods of humanization of antibodies are well known within the art, among the more common methods are CDR grafting.

[0102] Portions or fragments of the anti-DDX3Y antibodies or antigen- specific binding domains disclosed herein are contemplated and incorporated, including portions or fragments that include those that arc modified or fused to reagents, labels, other domains or fragments, therapeutic drugs, or payloads.

[0103] In one example, the anti-DDX3Y antibody or antigen-specific binding domain is conjugated to a payload. In some examples, the payload is selected from a group consisting of toxin, a polymer, biologically active proteins, nucleic acids and fragments thereof radionuclides chelated metals, nanoparticles and reporter groups. In yet another example, the antibody is conjugated to a payload. In some examples, the payload is selected from, but not limited to, a group consisting of toxin, a polymer, biologically active proteins, cytotoxic drugs and agents, nucleic acids and fragments thereof radionuclides chelated metals, nanoparticles and reporter groups. In one example, the antibody or antigen-specific binding domain is conjugated to a label. In some examples, the label is a radiolabel. In another example, the label is a radiolabel compatible for use in in vilro diagnostic techniques and in vivo radio-imaging techniques. In the instance of in vivo imaging, the antibody or antigen-specific binding domain as disclosed herein may be conjugated to an imaging agent rather than a radioisotope, including but not limited to a magneticresonance image enhancing agent, wherein for instance an antibody molecule is loaded with a large number of paramagnetic ions through chelating groups. In another example, the antibody comprises an antibody-drug conjugate.

[0104] An antigen-binding fragment of an antibody can be produced for example, enzymatically or chemically by fragmentation of an intact antibody and / or recombinantly from a gene encoding the partial antibody sequence. In another example, the antigen-binding fragment of an antibody can be wholly or partially synthetically produced.

[0105] Also disclosed herein are compositions comprising at least one of the anti-DDX3Y specific monoclonal antibodies, or nucleic acids encoding the same. The anti-DDX3Y antibodies or antigen-specific binding domains as disclosed herein may be provided as isolated and / or purified molecules, for example from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes other than the sequence encoding a polypeptide with the required function.

[0106] In one example, the present disclosure refers to an isolated nucleic acid molecule encoding an anti-DDX3Y antibody or antigen- specific binding domain as disclosed herein. Nucleic acid molecules include DNA and RNA and may be wholly or partially synthetic. The present disclosure also refers to constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide encoding an antibody or antigenspecific binding domain as disclosed herein. The present disclosure further refers to a recombinant host cell which comprises one or more of the constructs as disclosed herein.

[0107] In one example, the present disclosure refers to anti-DDX3Y antibodies or antigenspecific binding domains isolated from the one or more hybridoma cell lines disclosed herein.

[0108] In another example, the present disclosure refers to a kit comprising one or more of the antibodies or antigen-specific binding domains as disclosed herein, a nucleic acid molecule encoding the anti-DDX3 Y antibody or antigen-specific binding domain as disclosed herein, and / or one or more of the hybridoma cell lines as disclosed herein. In some examples, the kit can further comprise instructions for use of the anti-DDX3Y antibody or antigen-specific binding domain. In some examples, the kits disclosed herein arc in suitable packing. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g. , sealed Mylar or plastic bags), and the like. In some examples, the kit can further comprise additional components such as buffers and reagents.

[0109] The anti-DDX3Y antibodies and antigen-specific binding domains disclosed herein are specific to DDX3Y and do not cross-rcact with DDX3X as confirmed by ELISA, Western Blot and Immunohistochemistry. Moreover, the anti-DDX3Y antibodies and antigen- specific bindingdomains disclosed herein will enable researchers to confidently study DDX3Y expression in cancer and normal tissues in a much more robust fashion than has been hitherto possible with commercially available antibodies.

[0110] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred examples and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the ait, and that such modifications and variations arc considered to be within the scope of this invention.

[0111] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.

[0112] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0113] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0114] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso ornegative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0115] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0116] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.EXPERIMENTAL SECTION

[0117] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), cell biology, and immunology, which are within the skill of the art. Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0118] Hybridomas were developed by Genscript, under commercial arrangement and with the guidance of the co-invcn tors. Gcnscript then shipped the final antibodies and hybridomas to the co-inventors.Commercially Available Anti-DDX3Y Antibodies

[0119] Commercially available anti-DDX3Y antibodies were purchased and analysed for specificity to DDX3Y and cross reactivity with DDX3X, to be used as a basis for comparison. In particular, anti-DDX3Y antibodies, MBS6152109 from My Biosource and WH0008653M1 from Sigma Aldrich were purchased and analysed via western blot. Western blots were performed on lysates from HEK293T cells overexpressing DDX3X and DDX3Y. The two antibodies showed strong cross-reactivity with DDX3X (Fig 3). Additionally, manufacturers of commercially available anti-DDX3Y antibodies were contacted to discuss specificity of the antibodies (Table 2). Antibodies that stained HeLa cells (a female cell line) or human ovary were defined as nonspecific, since DDX3Y is Y-chromosomc linked and therefore only found in males.Table 2.Production of Hybridoma Clones Derived from DDX3Y-1Hybridoma Generation

[0120] The DDX3Y-l peptide (SHVWKNDPELD; SEQ ID NO: 1) was modified to add a N- terminal cysteine to facilitate keyhole limpet hemocyanin (KLH) conjugation for immunization.This peptide CSHVVVKNDPELD (SEQ ID NO: 228) was synthesized and injected into 3 Balb / c mice (#M0168, #M0169, #M0170) and 3 C57BL / 6 mice (#M0208, #M0209, #M0210). Following 4 to 5 rounds of immunization, 4 mice with higher titers of antiserum (1:128k to 1:512k) were selected for cell fusion and hybridoma generation.Hybridoma Selection and Clone Characterisation

[0121] Hybridoma cells were cultured in DMEM with 10% FBS, and the supernatants were tested for presence of anti-DDX3Y antibodies by ELISA with DDX3Y-1 peptide. The supernatants which tested positive for the DDX3Y-1 peptide by ELISA were further confirmed for their specificity against DDX3Y without cross-reactivity with DDX3X via western blotting.

[0122] To characterize hybridoma-derived monoclonal antibodies for their specific reactivity to DDX3X and DDX3Y, a forced overexpression platform on HEK293T cells was used in conjunction with Western blot analysis. Briefly, HEK293T cells were transiently transfected with pCDNA3-mChcrry as empty vector control (EV), or pCDNA3 carrying Flag tagged DDX3X or DDX3Y. Total lysates harvested by direct lysis with 2x Laemmli buffer from the post-transfected cells were measured for protein amount using RC DC protein assay kit (Bio-Rad). 5pg of protein lysates derived from HEK293T cells overexpressing DDX3X and DDX3Y, as well as EV as background control were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane using TransBlot TurboTM transfer system RTA transfer kit (Bio-Rad). The membranes were blocked with 5% BSA for 1 hour at room temperature, followed by overnight incubation at 4°C in hybridoma supernatant diluted 5 times in 5% BSA. After rinsing with TBST buffer, membranes were incubated with goat-anti-mouse secondary antibodies conjugated with horseradish peroxidase in 5% BSA for 1 hour at room temperature. Immobilon Western Chemiluminescent HRP substrate (Merck Millipore) was added to the membrane and immunoreactivity presented by the chemiluminescence signals were detected using UV Illuminescent Alliance MINI HD4 chemiluminescence imaging system (UVItec).Table 3.[00123 J A total of 53 clones (16, 4, 26, and 7 clones from 4 mice) were screened (Table 3), and clones specific for DDX3Y without cross-reaction with DDX3X were identified (Figs. 2 and 4A). The five clones identified to be specific for DDX3Y without cross-reaction with DDX3X were then subcloned and analysed for binding activity in Raji (a male cell line) and U2932 (a female cell line) (Fig. 4B). Three hybridoma clones were found to have specific binding in Raji and were then selected for further analysis and sequencing. The three hybridoma clones derived from DDX3Y-1 are 34G11 (mouse IgG2b, K), 43D1 (mouse IgGl, K; deposition number CBA20230044) and 44E3 (mouse IgGl, K; deposition number CBA20230043), the CDRs of which were sequenced.Production of Hybridoma Clones Derived from DDX3Y-2Hybridoma Generation

[0124] The DDX3Y-2 peptide (IGNRERPGFGRFER; SEQ ID NO: 3) was modified to add a C-terminal cysteine to facilitate KLH conjugation for immunization. This peptide 1GNRERPGFGRFERC (SEQ ID NO: 231) was synthesized and injected into 5 Balb / c mice (#2369, #2370, #2371, #2372 and #2373). Following 4 to 5 rounds of immunization, ELISA testing using DDX3Y-2 and a counter-screen using a corresponding DDX3X peptide (SRGDRSGFGKFERGC, SEQ ID NO: 232) revealed that mice #2370 and #2371 had high antiserum titers against DDX3Y-2 (1:512k) and low cross-reactivity with the corresponding DDX3X peptide (1 :64k), and were therefore selected for cell fusion and hybridoma generation.Hybridoma Selection and Clone Characterisation

[0125] The same counter screening approach described in the “Production of Hybridoma Clones Derived from DDX3Y-1” section above was implemented for the hybridoma clones screening. Four hybridoma clones derived from DDX3Y-2 were obtained, namely 41H3E9 (mouse IgGl, K; deposition number CBA20230045), 43E5A6 (mouse IgGl, K; deposition number CBA20230046), 56A9A5 (mouse IgGl, K; deposition number CBA20230047) and 72E8A4 (mouse IgG2a, K; deposition number CBA20230048). The four hybridoma clones demonstrated strong reactivity to DDX3Y and no reactivity to DDX3X (Fig. 5).Production of Hybridoma Clones Derived from DDX3Y-3

[0126] The DDX3Y-3 peptide (NSEKQSGGASTA, SEQ ID NO: 2) was modified to add a C- terminal cysteine to facilitate KLH conjugation for immunization. This peptide NSEKQSGGASTAC (SEQ ID NO: 230) was synthesized and injected into 5 Balb / c mice.Sequencing Monoclonal Antibodies Derived from Hybridoma Cells

[0127] To purify antibodies form the selected hybridomas, total RNA was isolated from the hybridoma cells following the technical manual of RNA-easy Isolation Reagent. Total RNA was then reverse transcribed into cDNA using isotype- specific anti-sense primers or universal primers following the technical manual of SMARTScribe Reverse Transcriptase. The antibody fragments of VH and VL were amplified according to the standard operating procedure of rapid amplification of cDNA ends (RACE) of GcnScript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. No less than five colonies with inserts of correct sizes were sequenced for each fragment. The sequences of different clones were aligned and the consensus sequence of these clones was provided.Characteristics of Monoclonal Antibodies Derived from Hybridoma Cells

[0128] Purified antibodies from the selected hybridomas were obtained from culture supernatants using a standard industrial protocol and subjected to immunohistochemistry staining. In particular, clones which demonstrated strong reactivity to DDX3Y and no reactivity to DDX3X were used to stain human ovary (negative control) and testis (positive control) formalin-fixed paraffin-embedded tissue sections to confirm their staining specificity (Figs. 6 and 7). Results from these IHC studies showed the strongest and most specific signal was obtained using the antibodies 44E3, 43D1 and 41H3E9 (Figs. 6A and 8B). IHC staining was done using the BOND EpitopeRetrieval 2 kit with antibody concentration 1:1,000 incubated at 25°C for 25 minutes. Further experiments had been performed to study the expression of DDX3Y in human male colon, liver, and lung tissue specimens, representative images of which showed that various staining intensities were detected in corresponding tumour tissues (Fig. 7).SEQUENCE LISTING* Amino acids 2 to 13 of Human DDX3Y with NCBI Reference Sequence: NP_001116137.1.**Amino acids 22 to 33 of Human DDX3Y with NCBI Reference Sequence: NP_001116137.1.***Amino acids 106 to 119 of Human DDX3Y with NCBI Reference Sequence: NP_001116137.1. **** Amino acids 109 to 122 of Human DDX3X, corresponding DDX3X peptide of DDX3Y-2***** Amino acids 1 to 660 of Human DDX3Y with NCBI Reference Sequence: NP_001116137.1. Amino acid positions 2, 55, 118: acetylation sites. Amino acid positions 101, 103, 129, 181, 454, 612 : phosphorylation sites. Amino acid positions 178-206: Q motif. Amino acid positions 180-402: Dead-box helicase. Amino acid positions 225-229, 504, 525, 529, 532: ATP binding site. Amino acid positions 345-348: DEAD box motif. Amino acid positions 345- 348: Mg-H- binding site. Amino acid positions 380-382: motif III. Amino acid positions 424-534: helicase C. Amino acid positions 447-450, 470-471, 496-498: nucleotide binding region.

Claims

CLAIMS1. An antigen- specific binding domain comprising a heavy chain variable domain and / or a light chain variable domain, wherein the heavy chain variable domain comprises heavy chain CDRs of CDRH1, CDRH2, and CDRH3 and / or wherein the light chain variable domain comprises light chain CDRs of CDRL1, CDRL2, and CDRL3, wherein: a. CDRH1 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102; or a modified CDRH1 sequence thereof; b. CDRH2 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104; or a modified CDRH2 sequence thereof; c. CDRH3 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106; or a modified CDRH3 sequence thereof; d. CDRL1 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110; or a modified CDRL1 sequence thereof; e. CDRL2 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112; or a modified CDRL2 sequence thereof; and f. CDRL3 is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 18, 34, 50, 66, 82, 98, 114; or a modified CDRL3 sequence thereof, wherein the modified CDRs have nucleic acid sequences that have at least 90% identity to the corresponding nucleic acid sequences.

2. The antigen-specific binding domain according to claim 1, wherein the heavy chain CDRs are selected from the group consisting of: g. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 6, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 8, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 10, or modified nucleic acid sequences thereof; h. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO:22, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 24, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 26, or modified nucleic acid sequences thereof; i. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO:54, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 56, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 58, or modified nucleic acid sequences thereof;j. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 70, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 72, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 74, or modified nucleic acid sequences thereof; k. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 86, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 88, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 90, or modified nucleic acid sequences thereof; and l. a CDRH1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 102, a CDRH2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 104, a CDRH3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 106, or modified nucleic acid sequences thereof.

3. The antigen- specific binding domain according to any one of the preceding claims, wherein the light chain CDRs are selected from the group consisting of: m. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 14, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 16, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 18, or modified nucleic acid sequences thereof; n. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 30, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 32, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 34, or modified nucleic acid sequences thereof; o. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 62, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 64, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 66, or modified nucleic acid sequences thereof; p. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 78, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 80, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 82, or modified nucleic acid sequences thereof; q. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 94, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 96, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 98, or modified nucleic acid sequences thereof; and r. a CDRL1 encoded by a nucleic acid sequence set forth in SEQ ID NO: 110, a CDRL2 encoded by a nucleic acid sequence set forth in SEQ ID NO: 112, a CDRL3 encoded by a nucleic acid sequence set forth in SEQ ID NO: 114, or modified nucleic acid sequences thereof.

4. The antigen- specific binding domain according to any one of the preceding claims, wherein the heavy chain variable domain comprises a sequence encoded by sequences selected from the group consisting of:s. SEQ ID NOs: 5 to 11 or modified nucleic acid sequences thereof; t. SEQ ID NOs: 21 to 27 or modified nucleic acid sequence thereof; u. SEQ ID NOs: 53 to 59 or modified nucleic acid sequence thereof; v. SEQ ID NOs: 69 to 75 or modified nucleic acid sequence thereof; w. SEQ ID NOs: 85 to 91 or modified nucleic acid sequence thereof; and x. SEQ ID NOs: 101 to 107 or modified nucleic acid sequence thereof.

5. The antigen- specific binding domain according to any one of the preceding claims, wherein the light chain variable domain comprises a sequence encoded by sequences selected from the group consisting of: y. SEQ ID NOs: 13 to 19, or modified nucleic acid sequence thereof; z. SEQ ID NOs: 29 to 35, or modified nucleic acid sequence thereof; aa. SEQ ID NOs: 61 to 67 or modified nucleic acid sequence thereof; bb. SEQ ID NOs: 77 to 83 or modified nucleic acid sequence thereof; cc. SEQ ID NOs: 93 to 99 or modified nucleic acid sequence thereof; and dd. SEQ ID NOs: 109 to 115 or modified nucleic acid sequence thereof.

6. The antigen- specific binding domain according to any one of the preceding claims, wherein: ee. the modified nucleic acid sequence encodes a CDRH1 having an amino acid sequence with at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 134, 150, 166, 182, 198 and 214; ff. the modified nucleic acid sequence encodes a CDRH2 having an amino acid sequence with at least 80%> identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 120, 136, 152, 168, 184, 200 and 216; gg. the modified nucleic acid sequence encodes a CDRH3 having an amino acid sequence with at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 122, 138, 154, 170, 186, 202 and 218; hh. the modified nucleic acid sequence encodes a CDRL1 having an amino acid sequence with at least 80% identity to an amino acid sequence is selected from the group consisting of SEQ ID NOs: 126, 142, 158, 174, 190, 206 and 222; ii. the modified nucleic acid sequence encodes a CDRL2 having an amino acid sequence with at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 128, 144, 160, 176, 192, 208 and 224; and / orjj. the modified nucleic acid sequence encodes a CDRL3 having an amino acid sequence with at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 146, 162, 178, 194, 210 and 226.

7. The antigen- specific binding domain of claim 2, wherein the modified nucleic acid sequences of the heavy chain CDRs encode: kk. a CDRH1 having an amino acid sequence set forth in SEQ ID NO:118, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 120, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 122;11. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 134, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 136, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 138; mm. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 166, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 168, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 170; nn. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 182, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 184, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 186; oo. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 198, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 200, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 202; or pp. a CDRH1 having an amino acid sequence set forth in SEQ ID NO: 214, a CRDH2 having an amino acid sequence set forth in SEQ ID NO: 216, a CRDH3 having an amino acid sequence set forth in SEQ ID NO: 218.

8. The antigen- specific binding domain of claim 3, wherein the modified nucleic acid sequences of the light chain CDRs encode: qq. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 126, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 128, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 130; rr. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 142, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 144, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 146;ss. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 174, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 176, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 178; tt. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 190, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 192, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 194; uu. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 206, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 208, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 210; or vv. a CDRL1 having an amino acid sequence set forth in SEQ ID NO: 222, a CRDL2 having an amino acid sequence set forth in SEQ ID NO: 224, a CRDL3 having an amino acid sequence set forth in SEQ ID NO: 226.

9. The antigen- specific binding domain of claim 4, wherein the modified nucleic acid sequences of the heavy chain variable domain encode a heavy chain variable domain having amino acid sequences set forth in: ww. SEQ ID NOs: 117 to 123; xx. SEQ ID NOs: 133 to 139; yy. SEQ ID NOs: 165 to 171; zz. SEQ ID NOs: 181 to 187; aaa. SEQ ID NOs: 197 to 203; or bbb. SEQ ID NOs: 213 to 219.

10. The antigen-specific binding domain of claim 5, wherein the modified nucleic acid sequences of the light chain variable domain encode a light chain variable domain having amino acid sequences set forth in: ccc. SEQ ID NOs: 125 to 131; ddd. SEQ ID NOs: 141 to 147; eee. SEQ ID NOs: 173 to 179; fff. SEQ ID NOs: 189 to 195; ggg. SEQ ID NOs: 205 to 211; or hhh. SEQ ID NOs: 221 to 227.

11. The antigen- specific binding domain according to any one of the preceding claims, wherein the antigen- specific binding domain is a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody, or an antigen-binding fragment thereof.

12. The antigen- specific binding domain of claim 11, wherein the antigen-binding fragment is selected from the group consisting of Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, and single domain antibodies.

13. A hybridoma cell line with deposition number CBA20230043.

14. A hybridoma cell line with deposition number CBA20230044.

15. A hybridoma cell line with deposition number CBA20230045.

16. A hybridoma cell line with deposition number CBA20230046.

17. A hybridoma cell line with deposition number CBA20230047.

18. A hybridoma cell line with deposition number CBA20230048.

19. An antibody isolated from the hybridoma cell line of any one of claim 13 to 18.

20. A kit comprising the antigen- specific binding domain as defined in any one of claims 1 to 12, the hybridoma cell line as defined in any one of claims 13 to 18, and / or the antibody of claim 19.

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