AU719910B2 - Novel gene encoding male-transplantation antigen - Google Patents

Description

i, WO 98/23739 PCT/AU97/00819

TITLE

"NOVEL GENE ENCODING MALE-TRANSPLANTATION

ANTIGEN"

FIELD OF THE INVENTION THIS INVENTION relates to a novel gene encoding malespecific transplantation antigen which has potential therapeutic value in treatment of transplantation-related diseases.

BACKGROUND OF THE INVENTION Rejection of male tissue grafts by genotypically identical female mice has been explained by the existence of a male-specific transplantation antigen, Histocompatibility Y (Eichwald et a., 1955, Transplant-Bull. 2 148-149), but the molecular nature of H-Y antigen has remained obscure. Hya, a murine minor histocompatibility locus controlling H-Y expression, has been localized to DSxr, a deletion interval of the short arm of the Y chromosome (McLaren et al., 1988, Proc. Nat/. Acad. Sci. U.S.A 85 6442-6445).

In mice, H-Y antigen comprises at least four distinct epitopes, each recognised by a specific T lymphocyte clone. It has recently been shown that one of these epitopes, H-YKk, is a peptide encoded by the Y-linked Smcy gene, presented at the cell surface with the H-2Kk major histocompatibility complex (MHC) molecule (Scott et al., 1995, Nature 376 695-698). However, deletion mapping and the analysis of variable inactivation of H-Y epitopes has suggested that the Hya locus may be genetically complex. This is supported by studies showing that Abelson-transformed cell lines bearing different Y chromosome mutations express different combinations of H-Y epitopes (King et al., 1994, Genomics 24 159-168). Reference also may be made to an article by Scott et a. (1991, Immunogenetics 33 54-61) in which the spread of X-inactivation into the Sxra portion of the XSxra chromosome in T16H/XSxra mice was shown to cause differential expression of H-Y epitopes. These observations are best explained by the existence of more than one H-Y antigen-producing locus. Thus, WOn/279n10 W 98/23739 rUII/AUY/ItU 2 while the H-YK k epitope encoded by Smcy has been identified, it is highly likely that epitopes encoded by other Y chromosome genes are also involved in accounting for the male-specific transplantation antigen.

Accordingly, a need exists for the identification and characterization of other H-Y epitopes encoded by the Y chromosome.

Identification and characterization of such genes and the H-Y epitopes encoded thereby would provide valuable insight into the complexity of responses caused by transplantation of tissue from a donor of one sex into a host of an opposite sex as well as therapies for treatment of transplantation-related diseases resulting therefrom.

OBJECT OF THE INVENTION Accordingly, it is an object of the present invention to provide novel nucleotide sequences and corresponding peptide and polypeptide sequences encoding a novel H-Y antigen or epitope thereof. It is also an object of the invention to provide methods for treatment of transplantation-related diseases, wherein a novel nucleotide sequence, and corresponding peptide and polypeptide sequences encoding the novel H-Y antigen or epitope thereof are used as therapeutic agents to treat such diseases.

SUMMARY OF THE INVENTION A novel gene designated "Uty" has been isolated from a male murine cDNA library. A subsequence of the Uty nucleotide sequence has been shown to encode a novel H-Y epitope designated H-YDb. Polypeptides comprising the Uty expression product and subsequences thereof are believed to encode a plurality of H-Y epitopes which may be useful for controlling rejection of male tissue grafts by female recipients and vice versa, for example, in the context of treating host versus graft disease and graft versus host disease. In addition, it is also believed that Uty is a candidate spermatogenesis gene and may therefore have utility in the treatment of male infertility.

819 WO 98/23739 PCT/AU97/00819 3 Accordingly, in a first aspect of the invention, there is provided an isolated nucleotide sequence selected from the group consisting of: an isolated nucleotide sequence comprising the entire sequence of nucleotides shown in FIG. la; (ii) an isolated nucleotide sequence comprising nucleotide 69 through nucleotide 3639 of FIG. la; and (iii) an isolated nucleotide sequence comprising nucleotide 804 through nucleotide 830 of FIG. 1 a.

It will be appreciated that the nucleotide sequences defined by above correspond respectively to the full-length sequence of Uty, the open reading frame sequence of Uty, and the nucleotide sequence encoding the novel H-Y epitope H-YDb.

The term "nucleotide sequence" as used herein designates mRNA, RNA, cRNA, cDNA or DNA. One of skill in the art will appreciate that cDNA is complementary DNA produced from a RNA template, usually by the action of RNA-dependent DNA polymerase (reverse transcriptase). If the RNA template has not been processed to remove the introns, the cDNA will not be identical to the gene from which the RNA was transcribed.

The invention also provides homologs of the Uty nucleotide sequences of the invention as described above. Such "Uty homologs", as used in this specification, include all nucleotide sequences encoding the UTY polypeptide of FIG. 1c; and all nucleotide sequences encoding sub-sequences of this polypeptide including but not limited to the sub-sequence encoding the H-YDb epitope. In this regard, it is well known to take advantage of codon sequence redundancy to incorporate changes in a nucleotide sequence without affecting the corresponding amino acid sequence.

The Uty homologs of the invention further include ^T 7/008 19 RECEIVED 2 Ju. 13 4 nucleotide sequences encoding polypeptides that have the same functional characteristics as the polypeptides of the invention or biological fragments thereof. For example, one of skill in the art will appreciate that conservative amino acid substitutions can be made in a polypeptide according to the invention ("parent polypeptide") and that such substituted polypeptides will retain the functional characteristics of the parent polypeptide.

The Uty homologs of the invention also cover alternately spliced variants which may be obtained by using a nucleotide sequence of the invention as a probe in relation to screening a male mouse fetal cDNA library.

The Uty homologs further comprise nucleotide sequences that hybridize with a nucleotide sequence in accordance with the invention under substantially stringent conditions. Suitable hybridization conditions are discussed below.

The Uty homologs of the invention may be prepared according to the following procedure: designing primers which are preferably degenerate which span at least a fragment of a nucleotide sequence of the invention; and (ii) using such oligonucleotide primers to amplify, via nucleotide sequence amplification techniques, said at least a fragment from a nucleic acid extract obtained from a suitable host. Suitable nucleotide sequence amplification techniques are well known to those of skill in the art and include, for example, the Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA) and Rolling Circle Replication (RCR). The suitable host from which a nucleic acid extract is obtained is preferably a mammal. Suitable mammals may be selected from the group consisting of human, mouse, rat, guinea pig and rabbit. By "obtained from" is meant that the nucleic acid extract R, is isolated from, or derived from, a particular source of the host. For W AMEINDED nHET XZ1. ^IPEA/AU h WO 98/23739 PCT/AU97/00819 example, the nucleic acid extract may be obtained from tissue isolated directly from a mammalian host.

Alternatively, a Uty homolog of the invention may be obtained from a nucleotide sequence library derived from mammalian tissue (such as male adult or fetal tissue). Such a library may be a male mouse cDNA library.

"Hybridization" is used here to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G.

Typically, nucleotide sequences to be compared by means of hybridization are analyzed using dot blotting, slot blotting, or Southern blotting. Southern blotting is used to determine the complementarity of DNA sequences. Northern blotting determines complementarity of DNA and RNA sequences or RNA and RNA sequences. Dot and Slot blotting can be used to analyze DNA/DNA, DNA/RNA or RNA/RNA complementarity. These techniques are well known by those of skill in the art. Typical procedures are described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, et al., eds.) (John Wiley Sons, Inc. 1995) at pages 2.9.1 through 2.9.20.

Briefly, for Southern blotting, DNA samples are separated by size using gel electrophoresis. The size-separated DNA samples are transferred to and immobilized on a membrane (typically, nitrocellulose) and the DNA samples are probed with a radioactively or enzymatically or fluorochromatically labeled complementary nucleic acid. In dot blotting and slot blotting, DNA samples are directly spotted onto a membrane (nitrocellulose or nylon). The membrane is then probed with a labeled complementary nucleic acid.

A probe is a biochemical labeled with a radioactive isotope or tagged in other ways for ease in identification. A probe is WO 98/23739 PCT/AU97/00819 6 used to identify a gene, a gene product or a protein. Thus a nucleotide sequence probe can be used to identify complementary nucleotide sequences. An mRNA probe will hybridize with its corresponding DNA gene.

Typically, the following general procedure can be used to determine hybridization under stringent conditions. A nucleotide according to the invention (such as Uty or a subsequence thereof) will be immobilized on a membrane using one of the above-described procedures for blotting. A sample nucleotide sequence will be labeled and used as a "probe." Using procedures well known to those skilled in the art for blotting described above, the ability of the probe to hybridize with a nucleotide sequence according to the invention can be analyzed.

One of skill in the art will recognize that various factors can influence the amount and detectability of the probe bound to the immobilized DNA. The specific activity of the probe must be sufficiently high to permit detection. Typically, a specific activity of at least 108 dpm/pg is necessary to avoid weak or undetectable hybridization signals when using a radioactive hybridization probe. A probe with a specific activity of 10 to 109 dpm/ug can detect approximately 0.5 pg of DNA. It is- well known in the art that sufficient DNA must be immobilized on the membrane to permit detection. It is desirable to have excess immobilized DNA and spotting 10 pg of DNA is generally an acceptable amount that will permit optimum detection in most circumstances. Adding an inert polymer such as 10% dextran sulfate (mol. wt. 500,000) or PEG 6000 to the hybridization solution can also increase the sensitivity of the hybridization. Adding these polymers has been known to increase the hybridization signal. See Ausubel, supra, at p 2.10.10.

To achieve meaningful results from hybridization between a first nucleotide sequence immobilized on a membrane and a second WO 98/23739 PCT/AU97/00819 7 nucleotide sequence to be used as a hybridization probe, sufficient probe must bind to the immobilized DNA to produce a detectable signal (sensitivity) and following the washing procedure, the probe must be attached only to those immobilized sequences with the desired degree of complementarity to the probe sequence (specificity).

"Stringency," as used in this specification, means the condition with regard to temperature, ionic strength and the presence of certain organic solvents, under which nucleic acid hybridizations are carried out. The higher the stringency used, the higher will be the degree of complementarity between the probe and the immobilized

DNA.

"Stringent conditions" designates those conditions under which only a nucleotide sequences that have a high frequency of complementary base sequences will hybridize with each other.

Exemplary stringent conditions are 0.75 M dibasic sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1% sarkosyl at about 42°C for at least about minutes, 6.0 M urea/0.4% sodium laurel sulfate/0.1% SSC at about 420 C for at least about 30 minutes, 0.1 X SSC/0.1% SDS at about 68 0 C for at least about 20 minutes, 1 X SSC/0.1% SDS at about 55°C for about one hour, 1 X SSC/0.1% SDS at about 62*C for about one hour, 1 X SSC/O.1% SDS at about 68 0 C for about one hour, 0.2 X SSC/0.1% SDS at about 55°C for about one hour, 0.2 X SSC/0.1% SDS at about 62 0 C for about one hour, and 0.2 X SSC/0.1% SDS at about 68°C for about one hour. See, e.g. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, et al., eds.) (John Wiley Sons, Inc. 1995), pages 2.10.1-2.10.16 of which are hereby incorporated by reference and Sambrook, et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989) at §§1.101-1.104.

Stringent washes are typically carried out for a total of WO 98/23739 PCT/AU97/00819 8 about 20 minutes to about 60 minutes. In certain instances, more than one stringent wash will be required to remove sequences that are not highly similar to Uty or a subsequence thereof. Typically, two washes of equal duration, such as two 15 or 30 minute washes, are used. One of skill in the art will appreciate that other longer or shorter times may be employed for stringent washes to ensure identification of sequences similar to Uty.

While stringent washes are typically carried out at temperatures from about 42°C to about 68°C, one of skill in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization typically occurs at about 20 to about 25°C below the Tm for DNA-DNA hybrids. It is well known in the art that Tm is the melting temperature, or temperature at which two nucleotide sequences dissociate. Methods for estimating Tm are well known in the art. See, e.g. Ausubel, supra, at page 2.10.8.

Maximum hybridization typically occurs at about 10 to about below the Tm for DNA-RNA hybrids.

Other typical stringent conditions are well-known in the art. One of skill in the art will recognize that various factors can be manipulated to optimize the specificity of the hybridization.

Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization between the Uty gene (or subsequence thereof) and other similar nucleotide sequences.

In a typical hybridization procedure, DNA is first immobilized on a membrane such as a nitrocellulose membrane or a nylon membrane. Procedures for DNA immobilization on such membranes are well known in the art. See, Ausubel, supra at pages 2.9.1-2.9.20. The membrane is prehybridized at 42°C for minutes in 0.75 M dibasic sodium phosphate/0.5 M monobasic -sodium phosphate/1 mM disodium EDTA/1% sarkosyl. Membranes are then hybridized at 420C in ACES hybridization solution (Life WO 98/23739 PCT/AU97/00819 9- Technologies, Inc., Gaithersburg, Md.) containing labeled probe for one hour. Next, membranes are subjected to two high stringency minute washes at 42 0 C in 0.75 M dibasic sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1% sarkosyl.

Following this, the membranes are washed with 2 X SSC at room temperature, to remove unbound probe.

In another typical hybridization procedure, DNA immobilized on -a membrane is hybridized overnight at 42 0 C in prehybridization solution. Following hybridization, blots are washed with two stringent washes, such as 6.0 M urea/0.4% sodium laurel sulfate/0.1% SSC at 420 C. Following this, the membranes are washed with 2 X SSC at room temperature.

Autoradiographic techniques for detecting radioactively labeled probes bound to membranes are well known in the art.

In another aspect of the invention, there is provided an isolated polypeptide comprising the entire sequence of amino acids depicted in FIG. 1c. This polypeptide corresponds to the UTY polypeptide.

The invention also includes within its scope a polypeptide encoded by the human Uty gene as well as mammalian homologs of the UTY polypeptide. As used in this specification, a "UTY polypeptide homolog" is a polypeptide encoded by a "Uty homolog," which is defined above. Thus, the invention contemplates polypeptides which are functionally similar to the UTY polypeptide.

Such polypeptides may contain conservative amino acid substitutions compared to the UTY polypeptide of FIG. 1c.

A UTY polypeptide of the invention may be prepared by a procedure including the steps of: ligating a DNA sequence encoding a recombinant UTY polypeptide or biological fragment thereof into a suitable expression vector to form an expression construct; *vrmr ww A ***Tvwvmfr f WO 98/23739 rL IAUY IIUU transfecting the expression construct into a suitable -host cell; expressing the recombinant protein; and isolating the recombinant protein.

As used in this specification, an expression construct is a nucleotide sequence comprising a first nucleotide sequence encoding a polypeptide, wherein said first sequence is operably linked to one or more regulatory nucleotide sequences (such as a promoter and a termination sequence) that will induce expression of said first sequence. Both constitutive and inducible promoters may be useful adjuncts for expression of a UTY polypeptide or UTY polypeptide homolog according to the invention. An expression construct according to the invention may be a vector, such as a plasmid cloning vector. A vector according to the invention may be a prokaryotic or a eukaryotic expression vector, which are well known to those of skill in the art.

Suitable host cells for expression may be prokaryotic or eukaryotic. One preferred host cell for expression of a polypeptide according to the invention is a bacterium. The bacterium used may be Escherichia coli. Alternatively, the host cell may be an insect cell such as, for example, SF9 cells which may be utilized with a baculovirus expression system.

The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al. (1989, supra, in particular Sections 16 -and 17).

In yet another aspect, the invention resides in a peptide corresponding to a sub-sequence of the UTY polypeptide or polypeptide homolog thereof. A peptide in accordance with the invention ("UTY peptide") includes but is not limited to a peptide having the sequence of amino acids corresponding to amino acid 246 1Y WO 98/23739 PCT/AU97/00819 11 through amino acid 254 of FIG. 1c. It will also be appreciated that changes to the sequence of such peptide may be made in accordance with the method of Joost van Neerren et al. (1996, Immunology Today 17 526-532), which is hereby incorporated by reference, to form substituted agonist/antagonist peptides which may enhance the efficacy of tolerance induction of a male-derived cell or tissue in a female recipient and vice versa.

UTY peptides in accordance with the invention may be prepared by chemical synthesis either manually or by utilizing an automated peptide synthesiser. Such peptides, for example, may be synthesized using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard which is included in a publication entitled "Synthetic _Vaccines" edited by Nicholson and published by Blackwell Scientific Publications.

In a further aspect of the invention, there is provided a method for inducing tolerance in a female recipient of a male-derived tissue, said method comprising the steps of transforming a cell from the female recipient with a vector comprising a Uty nucleotide sequence or Uty homolog operably linked to a constitutive promoter to form a transformed cell, and administrating said transformed cell to the female recipient prior to transplantation of the male-derived tissue into said female recipient.

SIn yet a further aspect of the invention, there is provided a method for inducing a female donor of a female-derived tissue to tolerate tissue of a male recipient before transplantation of the femalederived tissue into the male recipient, said method comprising the steps of transforming a cell from the female donor with a vector comprising a Uty nucleotide sequence or Uty homolog operably linked to a constitutive promoter to form a transformed cell, and administrating said transformed cell to the female donor prior to WO 98/23739 PCT/AU97/00819 12 transplantation of the female-derived tissue into said male recipient.

The terms "female recipient", "female donor", "male recipient" as used herein refer to mammalian recipients or donors of tissue.

The Uty nucleotide sequence or Uty homolog used to induce such tolerance may encode a plurality of distinct H-Y epitopes.

Preferably, the Uty nucleotide sequence or Uty homolog encodes one to three distinct H-Y epitopes. More preferably, the Uty nucleotide sequence or Uty homolog encodes one H-Y epitope such as, for example, the H-YDb epitope.

The cell to be transformed may be any suitable cell from the female recipient. Suitably, the cell to be transformed is a white blood cell. Preferably, the cell is an antigen presenting cell such as, for example, a CD341 while blood cell, a dendritic cell or a macrophage.

The invention also provides a method for inducing tolerance in a female recipient of a male-derived tissue, said method comprising the step of administrating a UTY polypeptide or polypeptide homolog to the female recipient prior to transplantation of the male-derived tissue into said female recipient.

The invention also contemplates a method for inducing a female donor of a female-derived tissue to tolerate tissue of a male recipient before transplantation of the female-derived tissue into the male recipient, said method comprising the step of administrating a UTY polypeptide or UTY polypeptide homolog to the female donor prior to transplantation of said female-derived tissue into said male recipient.

Preferably, the UTY polypeptide or UTY polypeptide homolog encodes one to three distinct H-Y epitopes. More preferably, the UTY polypeptide or polypeptide homolog encodes one H-Y epitope such as, for example, the H-YDb epitope.

WO 98/23739 PCT/AU97/00819 13 The UTY polypeptide or UTY polypeptide homolog may be administered directly. Alternatively, the UTY polypeptide or UTY polypeptide homolog may be administered by first combining such polypeptide with a cell from the female donor to form a mixture and subsequently administering the mixture to the female donor. In this regard, the cell which is combined with the UTY polypeptide or UTY polypeptide homolog may be an antigen presenting cell as described above.

In a still further aspect of the invention, there is provided a method for inducing tolerance in a female recipient of a male-derived tissue, said method comprising the step of administrating a peptide corresponding to a sub-sequence of the UTY polypeptide or polypeptide homolog thereof to the female recipient prior to transplantation of the male-derived tissue into said female recipient.

The invention also comprises a method for inducing a female donor of a female-derived tissue to tolerate tissue of a male recipient before transplantation of the female-derived tissue into the male recipient, said method comprising the step of administrating a UTY peptide to the female donor prior to transplantation of said female-derived tissue into said male recipient.

In the case of utilizing UTY peptides, if more than one distinct H-Y epitope is required to be utilized to increase the efficacy of tolerance induction, a corresponding number of UTY peptides encoding such epitopes may be linked directly or indirectly for administration to the female recipient/donor. Preferably, the UTY peptide used to -induce tolerance encodes the H-YDb epitope herein described.

The UTY peptide(s) may be administered to the female recipient/donor, for example, in soluble form or pre-bound to cells such as those referred to above.

According to a further aspect of the invention, there is provided a method for improving fertility of a mammalian sperm cell or WO 98/23739 PCT/AU97/00819 14 progenitor cell thereof, said method comprising the step of transforming said sperm cell or progenitor cell thereof with a vector comprising a Uty nucleotide sequence or Uty homolog operably linked to a constitutive promoter.

The invention further comprises a method for improving fertility of a mammalian sperm cell or progenitor cell thereof, said method comprising the step of delivering into said sperm cell or progenitor cell thereof a UTY polypeptide or UTY polypeptide homolog.

A sperm progenitor cell in accordance-with the present invention may be a primordial germ cell, a prospermatogonium, a spermatogonium or a spermatocyte.

The above methods directed to improving fertility of a mammalian sperm cell or progenitor cell thereof may be utilized to improve the propensity of mammalian sperm cells to fertilize an ovum.

It will be appreciated that such methods may also be used in concert with gene therapy techniques referred to herein to improve the fertility of future generations of a mammal having infertile sperm cells.

-The invention also comprises antibodies against the UTY polypeptide, UTY polypeptide homolog or UTY peptide. Such "anti- UTY antibodies" in accordance with the subject invention may include any suitable antibodies which bind to or conjugate with a UTY polypeptide, UTY polypeptide homolog or UTY peptide. For example, the anti-UTY antibodies may comprise polyclonal antibodies. Such antibodies may be prepared for example by injecting the UTY polypeptide, UTY polypeptide homolog or-UTY peptide of the invention into production species, which may include mice or rabbits, to obtain polyclonal antisera.

In lieu of the anti-UTY polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard method as for example, described in an article by Khler and Milstein (1975, Nature 256, 495-497) which is hereby incorporated by reference, or by more recent modifications thereof as for example, A U 7 008 1 RECEIVED 2 '3 JUN 198 described in "CURRENT PROTOCOLS IN IMMUNOLOGY" (1994, Ed.

J.E. Coligan, A.M. Kruisbeek, D.H. Marguiles, E.M. Shevach and W.

Strober, John Wiley and Son Inc. which is hereby incorporated by reference) by immortalising spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the peptides of the invention.

The invention also includes within its scope anti-UTY antibodies which comprise Fc or Fab fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the anti-UTY antibodies may comprise single chain Fv antibodies (scFvs) against the peptides of the invention. Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No 239,400 or the article by Winter and Milstein (1991, Nature, 349 293) which are hereby incorporated by reference.

The invention further contemplates a method for diagnosis of infertility in a male mammal, said method comprising the step of determining the presence or absence of a Uty nucleotide sequence or Uty homolog in a cell sample obtained from the male mammal wherein the absence of the Uty nucleotide sequence or Uty homolog is indicative of infertility in the male mammal.

The presence or absence of the Uty nucleotide sequence or Uty homolog in the cell sample may be determined using any suitable technique. For example, a labeled Uty nucleotide sequence or Uty homolog in accordance with the invention may be used as a probe in a Southern blot of a DNA extract obtained from the male mammal as described to above. Alternatively, a labeled Uty nucleotide sequence or Uty homolog may be utilized as a probe in a Northern blot of a total RNA extract or poly A RNA extract from the male mammal.

Preferably, a nucleic acid extract from the male mammal is utilized in concert with oligonucleotide primers corresponding to sense and 'E 1 'Lu C)

T'

WO 98/23739 PCT/AU97/00819 16 antisense sequences of a Uty nucleotide sequence or Uty homolog in a nucleotide sequence amplification reaction such as PCR. The above generic techniques are well known to persons skilled in the art.

The invention also comprises a method for diagnosis of _infertility in a male mammal, said method comprising the step of determining the presence or absence of mutations in a Uty nucleotide sequence or Uty homolog in a cell sample obtained from the male mammal wherein the presence of mutations in the Uty nucleotide sequence or Uty homolog is indicative of infertility in the male mammal.

Suitable methods for identifying mutations in a nucleic acid sequence which may be utilized in accordance with the invention include but are not limited to restriction fragment length polymorphism (RFLP) analysis, nucleotide sequencing, Ligase Chain Reaction (LCR) single-strand conformational polymorphism (SSCP) analysis and chemical cleavage methods. Alternatively, PCR may be employed for example using oligonucleotide primers specific for a mutant sequence.

In another aspect of the invention, there is provided a method for diagnosis of infertility in a male mammal, said method comprising the step of determining the presence or absence of a UTY polypeptide or UTY polypeptide homolog in a cell sample obtained from the male mammal wherein the absence of said UTY polypeptide or UTY polypeptide homolog is indicative of infertility in the male mammal.

Any suitable technique may be used. For example, an anti-UTY antibody in accordance with the invention having a label associated therewith may be utilized in immunoassays to determine the presence or absence of the UTY polypeptide or UTY polypeptide homolog in the cell sample. Such immunoassays may include, but are not limited to, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic WO 98/23739 PCT/AU97/00819 17 techniques (ICTs) which are well known those of skill in the art. For example, reference may be made to "CURRENT PROTOCOLS IN IMMUNOLOGY" (1994, supra) which discloses a variety of immunoassays which may be used in accordance with the present invention.

The anti-UTY antibody has a label associated therewith which includes the following: i. direct attachment of the label to the anti-UTY antibody; ii. indirect attachment of the label to the anti-UTY antibody; attachment of the label to another assay reagent which subsequently binds to the anti-UTY antibody; and iii. attachment to a subsequent reaction product of the anti-UTY antibody.

The label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu 34 a radioisotope and a direct visual label.

In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

A large number of enzymes suitable for use as labels is disclosed in United States Patent Specifications U.S. 4,366,241, U.S.

4,843,000, and U.S. 4,849,338, all of which are herein incorporated by reference. Suitable enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, Pgalactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme which is in solution.

Suitably, the fluorophore is selected from a group WO 98/23739 PCT/AU97/00819 18 including fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITL) or R-Phycoerythrin (RPE).

The therapeutic agents of the invention may also be utilized as part of a suitable drug delivery system to a particular tissue that may be targeted. In a broader sense, the potential uses for the Uty gene or its protein or peptide products or homologs respectively thereof conform to agents which may be used to induce tolerance in the context of host versus graft disease as well as graft versus host disease. As such, the gene or its protein or peptide products including homologs respectively thereof (or any part or combination of parts of either), can be described as a therapeutic agent. Thus, the therapeutic agent may be a Uty nucleotide sequence, including a subsequence of the full-length Uty gene, which may be used alone or in combination with any other molecule, UTY polypeptide, or polypeptide fragments thereof, or UTY peptide alone or in combination with any other molecule as well as antibodies to the UTY polypeptide, or polypeptide fragments, or UTY peptide alone or in combination with any other molecule.

Any suitable route of administration may be employed for providing a human or lower mammal the therapeutic agents of the invention. For example, oral, inhalational, rectal, parenteral, sublingual, buccal, intrathecal, epidural, intravenous, intra-articular, intramuscular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.

The method of administration of the therapeutic agent will differ depending on the intended use and or species, and may involve non-viral and viral vectors, cationic liposomes, retroviruses and adenoviruses such as, for example, described in Mulligan, (1993 Science 260 926-932) which is hereby incorporated by reference. Such methods may include: Local application of the therapeutic agent by injection (Wolff-et al., 1990, Science 247 1465-1468, which is hereby incorporated WO 98/23739 PCT/AU97/00819 19 by reference), surgical implantation, instillation or any other means. This method may be useful where effects are to be restricted to specific endothelial and/or smooth/striated muscles. This method may also be used in combination with local application by injection, surgical -implantation, instillation or any other means, of cells responsive to the therapeutic agent so as to increase the effectiveness of that treatment.

This method may also be used in combination with local application by injection, surgical implantation, instillation or any other means, of another factor or factors required for the activity of the therapeutic agent.

(ii) General systemic delivery by injection of DNA, oligonucleotides (Calabretta et al., 1993, Cancer Treat. Rev. 19 169-179, which is hereby incorporated by reference), RNA or protein, alone or in combination with liposomes (Zhu et al., 1993, Science 261 209-212, which is hereby incorporated by reference), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl. Biochem. 13 390-405, which is hereby incorporated by reference) or any other mediator of delivery. This method may be advantageous for all intended uses whether or not the effect is intended to be targeted to specific tissues or parts of the body, and regardless of whether the intended result is the induction of tolerance of a male-derived cell or tissue in a female recipient. Where specific targeting is required, this might be achieved by linking the agent to a targeting molecule (the so-called "magic bullet" approach employing for example, an antibody), or by local application by injection, surgical implantation or any other means, of another factor or factors required for the activity of the therapeutic agent, or of cells responsive to the therapeutic agent.

(iii) Injection or implantation or delivery by any means, of cells that have been modified ex vivo by transfection (for example, in the presence of calcium phosphate: Chen et al., 1987, Mole. Cell Biochem. 7 2745-2752, or of cationic lipids and polyamines: Rose et al., 1991, BioTech. 10 520-525, which articles are hereby incorporated by WO 98/23739 PCT/AU97/00819 reference), infection, injection, electroporation (Shigekawa et al., 1988, BioTech. 6 742-751, which is hereby incorporated by reference) or any other way so as to increase the expression or activity of Uty (gene or gene product or sub-sequence respectively thereof) in those cells. The modification may be mediated by plasmid, bacteriophage, cosmid, viral (such as adenoviral or retroviral; Mulligan, 1993, Science 260 926-932; Miller, 1992, Nature 357 455-460; Salmons et al., 1993, Hum. Gen. Ther.

4 129-141, which-articles are hereby incorporated by reference) or other vectors, or other agents of modification such as liposomes (Zhu et al., 1993, Science 261 209-212, which is hereby incorporated by reference), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl.

Biochem. 13 390-405, which is hereby incorporated by reference), or any other mediator of modification. The use of cells as a delivery vehicle for genes or gene products has been described by Barr et al., 1991, Science 254 1507-1512 and by Dhawan et al., 1991, Science 254 1509-1512, which articles are hereby incorporated by reference. Treated cells may be delivered in combination with any nutrient, growth factor, matrix or other agent that will promote their survival in the treated subject.

EXPERIMENTAL

MATERIALS AND METHODS cDNA cloning and seauencing An 11.5 dpc male fetal cDNA library was constructed in Agtl0 (Promega) by random-primed cDNA synthesis from polyA' RNA using an mRNA purification kit (Pharmacia) and a TimeSaver cDNA synthesis kit (Pharmacia). 3' RACE-PCR was performed essentially as described (Jeske et al., 1995, Nature Genet. 10 480-482). PolyA' RNA extracted from a whole male 11.5 dpc fetus was reverse transcribed with a modified oligo-dT primer AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGAT17 3' .and first-round PCR was performed with the adapter primer AAGGATCCGTCGACATCGATAAT 3' and the Uty primer WO 98/23739 PCT/AU97/00819 21 ATTAAATATTGTCTCCTCAAGATT A second-round of amplification was performed with adapter primer GACATCGATAATACGACTCACTAT 3' and the same Uty primer.

Sequencing was performed on both strands by dideoxy chain termination and products were analysed on a 373A DNA Sequencer (Applied Biosystems).

RT-PCR

Tissue dissection, RNA extraction, reverse transcription and PCR were performed as described previously (Jeske et al, 1995, supra; Koopman et al., 1990, Nature 348 450-452). RNA was derived from Swiss mice, excluding the embryonic stem cell sample derived from 129/Sv male D3 embryonic stem cells. The blastocyst sample represents half of the RT-PCR product yield after RNA extraction from 60 Swiss dpc embryos. Primers used for Uty RT-PCR in FIG. Ib) were AAATGCAGCTCGGACCAAATC 3' and 5' CTGAATGATGTGAAGCTGTC Primers used for amplification of Uty from genomic DNA ('gen' in FIG.

1b) were: 5' ATATCTGTGTTAATGCAAAGAAG 3' and ATGGTCCACATTGTCGATAGT 3'.

Southern blots DNA probes were labelled to high specific-activity with 32 P by random hexamer priming (Feinberg and Vogelstein, 1983, Anal. Biochem.

132 6-13). Southern analysis was performed at high stringency according to standard procedures (Sambrook et al., 1989, supra).

H-Y expression assays Uty expression constructs were made by insertion of the relevant cDNA subclones (FIG. 1b) into the vector pcDNAI (Invitrogen) downstream of the cytomegalovirus (CMV) immediate early gene promoter. EL4 cells were transfected with 10 pg of Uty construct and 1 pg of pHbAPr-1-neo (Gunning et al., 1987, Proc. Natl. Acad. Sci. U.S.A 84 4831-4835) by electroporation (Scott et al., 1992, Immunogenetics 36 86-94), Antibiotic-resistant stable transfectants were tested for H-YDb -1 WO 98/23739 PCT/AU97/00819 22 expression using a growth inhibition assay (Dudley et al., manuscript submitted). Triplicate aliquots of 100 pL of transfected cells (2x10 4 /mL in RPMI 1640 medium) in round-bottomed 96 well plates were incubated with either 100 pL of medium or 100 pL of medium containing 104 CTL clone (either CTL10 or 151.9). (The clones were used 4 or 5 days after the last stimulation). The plates were incubated at 37 0 C for 48 h and pulsed with 3 H-thymidine for 12 h. The cells were collected using a Tomtec 96-well plate harvester and counted using a Wallac 1205 Betaplate counter. The results are expressed as percent lysis, calculated from: ((100-(cpm cells with clone cpm clone alone)) x 100)/cpm with medium alone. The standard error of the triplicates was in the order of The assay is a sensitive indicator of the presence (positive lysis) or absence (no or negative lysis) of the epitope. Peptides were synthesised and HPLC-purified as described previously (Scott et al., 1995, supra) The peptides were made as 1 mM sterile stocks in PBS. Serial dilutions were made in triplicate. Irradiated female B10 -splenocytes (5x106) as antigen presenting cells and 104 CTL clone (CTL10 or 151.9) in 100 pL medium containing 5 units/mL recombinant IL-2 were added to the peptide dilutions. The cells were incubated for 72 h and pulsed and collected as described above. In this assay proliferation of the T cell clone is measured directly by 3 H thymidine incorporation. The results are expressed as dpm. The results for clone 151.9 were similar to those shown for Stabilisation of H-2Db molecules RMA-S cells (2x10 5 were incubated overnight at 26 0 C with serial 10-fold dilutions of peptide in a final volumeof 200 pL RPMI 1640 containing 0.5% bovine serum albumin (BSA). The cells were transferred to 370C and unstabilized class I molecules allowed to decay for 2 h. The cells were washed with PBS containing 0.5% BSA and 0.02% sodium azide. Cells were stained using FITC-conjugated B22.24 monoclonal anti-H-2Db antibody. The level of stabilisation of H-2Db was determined WO 98/23739 PCT/AU97/00819 23 by flow cytometry using a FACScan (Becton Dickinson, San Jose California, USA). Samples were gated according to forward and side scatter. The results are expressed as percent maximal binding. The maximal binding was determined from the mean fluorescence intensity of parental RMA cells. This value was 125.2 in experiment 1 (peptides UTY, UTX, 2, 5, 6 and 8) and 90.7 in experiment 2 (peptides UTY, UTX, 3, 4 and In each experiment the binding curves obtained for the UTY and UTX peptides, expressed as percent maximum binding, were virtually superimposable. The values from experiment 1 are shown in the figure.

RESULTS AND DISCUSSION The present invention arises from the discovery of a novel mouse Y chromosome gene designated Uty (ubiquitously-transcribed tetratricopeptide repeat gene on the Y chromosome) which encodes a novel H-Y epitope. A peptide WMHHNMDLI derived from the UTY protein was shown to encode an H-Y epitope designated H-YDb. Our data formally demonstrate that H-Y antigen is the product of more than one gene on the Y chromosome.

Uty was identified by a differential screen for transcripts expressed in a sex-specific fashion in mouse embryos (data not shown).

The UTY protein predicted from translation of the single major open reading frame of a composite Uty cDNA (FIG. 1 b c) exhibits homology to several members of the tetratricopeptide repeat (TPR) protein family.

TPR proteins contain multiple copies of a degenerate 34 amino acid motif first identified in genes controlling mitosis in lower eukaryotes (Goebl and Yanagida, 1991, Trends Biochem. Sci. 16 173- 177) and believed to mediate protein-protein interaction (Smith et al., 1995, Genes Development 9 2903-2910; Lamb et al., 1995, Trends Biochem. Sci. 20 257-259). UTY has eight TPR motifs at its N-terminus.

Althoughseveral TPR proteins involved in cell-cycle control were amongst the best matches detected by BLAST database searching, highest homology to a gene product of known function is to the TPR domain of WO 98/23739 PCT/AU97/00819 24 the yeast glucose repressor SSN6 (Schultz et al., 1990, Mol. Cell. Biol. 4744-4756) (FIG. Id). The nuclear phosphoprotein SSN6 contains TPR motifs at its N-terminus and is required for normal growth, sporulation and mating, acting as a transcriptional repressor when complexed with the WD40 repeat protein TUP1 (Keleher et al, 1992, Cell 68 709-719). The only gene product exhibiting homology to UTY both within and outside the TPR domain is that predicted from the sequence of the uncharacterized Caenorhabditis elegans gene, D2021.1 (accession no. U23513).

The sequence of UTY is rich in glutamine, serine and proline residues in certain regions (FIG. 1c). These amino acids are commonly -found in eukaryotic transcriptional activation domains (Tjian and Maniatis, 1994, Cell 77 Two potential nuclear localization signals are also present (FIG. 1c).

The expression of Uty in a range of fetal and adult tissues was- studied by reverse-transcriptase polymerase chain reaction (RT-PCR) (FIG. 2a). Transcripts were detected in all male tissues tested, including the gonads, male embryonic stem cells and blastocysts collected at 4 days post coitum (dpc). No transcripts were detected in a range of female tissues tested (data not shown). Northern blot analysis of poly A' RNA from male ES cells with a Uty probe indicated a transcript size of 5.5 kb (data not shown).

Hybridisation of a Uty cDNA probe (Probe A, FIG. 1b) to XY male and XX female mouse genomic DNA digests revealed a prominent male-specific fragment and two faint bands shared by males and females_ -(FIG. 2b). These shared fragments are derived from the X chromosome and correspond to a widely transcribed homologue of Uty which we term Utx and manuscript in preparation). To more precisely localise Uty on the Y chromosome, the same probe was also hybridised to genomic DNA from sex-reversed male mice of genotypes X/XSxra and X/XSxrb (McLaren et al., 1984, Nature 312 552-555; Burgoyne, 1993, WO 98/23739 PCT/AU97/00819 "Deletion mapping the functions of the mouse Y chromosome" In "Sex Chromosomes and Sex-Determining Genes" (eds. Reed, K.C. Graves, 353-368 (Harwood Academic Publishers, Chur)) and from two lymphoblastoid cell lines of genotype O/XSxra, one of which (13a-87) bears a small deletion within the translocated portion of the Y chromosome short arm (King et al., 1994, supra) (FIG. 2b). The pattern of hybridisation demonstrates that Uty maps to the Sxrb deleted region (tSxrb) of the mouse Y chromosome short arm in the vicinity of the Sxl D element (King et al., 1994, supra; Mitchell et al., 1992, Genomics 12 26-34; Bishop et al., 1988, "Molecular aspects of sex determination in mice: an alternative model for the origin of the Sxr region" In "Sex Determination in Mouse and Man" (eds. Maclaren, A. Ferguson-Smith, 119-124 (Royal Society, London); Capel et al., 1993, Nature Genet.

301-307), which is also deleted in cell line 13a-87 (King et al., 1994, supra). This localisation was confirmed by PCR amplification of Uty from the same genomic DNA samples (FIG. 2c).

Given its widespread transcription and chromosomal location, in addition to the absence of expression of the H-YAb and H-YDb epitopes from the Uty-negative cell line 13a-87 (King et al., 1994, supra), we investigated whether any H-Y epitopes might be encoded by Uty. Three constructs containing portions of the Uty cDNA cloned downstream of the cytomegalovirus immediate early gene promoter (FIG.

Sb) were transfected into EL4 cells. Transfectants were assayed for H-Y expression-using two anti-H-YDb-specific T cell clones, CTL10 and 151.9.

These clones can be used in a growth inhibition assay-which measures lysis of transfectants expressing H-YDb (as in FIG. 3a), or in a proliferation assay which measures -the ability of female antigen-presenting cells, pulsed with the appropriate peptide, to stimulate the clones (as in FIG. 3b). FIG. 3a shows that all three Uty constructs -conferred H-YDb expression.

To localise the epitope within the UTY protein, we utilised WO 98/23739 PCT/AU97/00819 26 the consensus sequence for peptides which are presented in association with the H-2Db MHC molecule (Rammensee et al., 1993, Ann. Rev.

Immunol. 11 213-244). This consensus (XXXXNXXXI) reflects the so-called anchor residues which are critical for binding of the peptide to H-2Db. Six peptides that correspond to this binding motif and are encoded by the smallest Uty construct were screened for their ability to stimulate proliferation of two anti-H-YDb T-cell clones (FIG. 3b). The UTY peptide WMHHNMDLI stimulated the anti-H-YDb T-cell clones CTL10 and 151.9, with maximal effect at a peptide concentration between 10 pM and 1 nM. The corresponding peptide encoded by Utx (WMHHTVDLL) caused no stimulation (FIG. 4a).

Whilst the UTY and UTX peptides differ at only three amino acid residues, the H-2Db anchor residues N and I of the UTY peptide are replaced by T and L in UTX. Substitution of T for N at position 5 (as in peptides 2 and 7) caused a 1,000-fold reduction in the ability of the peptide to bind to and stabilise expression of the H-2Db MHC molecule on RMA-S cells (Schumacher et al., 1990, Cell 62 563-567) (FIG. 4b) and to stimulate proliferation of the anti-H-YDb T cell clones (Fig 4a).

Substitution of L for I at position 9 had no effect on H-2Db stabilisation or T cell recognition. Although substitution for the M, D or L residues at positions 6, 7 and 8 did not affect H-2Db stabilisation, these residues were found to be critical for T cell recognition (FIG. 4).

Though it is commonly assumed that Hya is equivalent to a single gene, previous studies have suggested that this locus may be complex. Abelson-transformed cell lines bearing different Y chromosome mutations express different combinations of H-Y epitopes (King et al., 1994, supra). In addition, the spread of X-inactivation into the Sxra portion of the XSxra chromosome in T16H/XSxra mice causes differential expression of H-Y epitopes (Scott et 1991, supra). These observations are best explained by the existence of more than one H-Y antigen-producing locus. The identification of an H-YDb epitope in Uty WO 98/23739 PCT/AU97/00819 27 provides formal proof that H-Y antigen is encoded by more than one gene.

The H-YDb epitope resides within the atypical fifth TPR motif of UTY (FIG. Id), suggesting that this epitope may have arisen by neutral sequence drift, accelerated by the location of Uty on the non-recombining portion of the Y chromosome. However, evidence exists that Y-linked homologues of Uty are found in human and a marsupial and P.K., unpublished data), possibly reflecting a male-specific function for Uty which might also explain sequence divergence between the murine Xand Y-linked genes.

Minor histocompatibility antigens are peptides derived from intracellular proteins which have diverse roles, thus the cellular function of Uty remains unclear. The role played by many TPR proteins in eukaryotic cell-cycle control suggests that Uty may function in regulating cell division, or, like SSN6, some aspect of transcription. A role in general transcriptional regulation has also been proposed for Smcy. Like Uty, Smcy is a widely expressed Y-linked gene, encodes a male-specific transplantation antigen (Scott et al., 1995, supra; (Agulnik et al., 1994, Hum. Molec. Genet. 3 873-878; Kent-First et al., 1996, supra;) and has an X-linked homologue (Smcx) (Agulnik et al., 1994, Hum. Molec. Genet. 3 879-884).

Though Uty must be considered a candidate for the spermatogenesis gene Spy (Sutcliffe et al., 1989, Development 107 373-380), its ubiquitous expression suggests a role outside the germline.

A Y-linked locus has been described, termed Gdy (growth and development locus on the Y chromosome), which accelerates the growth of XY male preimplantation mouse embryos over their XX sibs (Burgoyne, 1993, Development 117 341-345). The expression of Uty in blastocysts and embryonic stem cells make it a candidate for Gdy. Determining the function of Uty will help to shed more light on the role of the mammalian Y chromosome in development.

WO 98/23739 PCT/AU97/0019 28 Induction of Tolerance Experiments performed by the applicants thus far have demonstrated the utility of peptides in accordance with the invention for inducing tolerance in female recipients of male-derived cells or tissues. In this regard, a UTY-derived peptide, WMHHNMDLI, pulsed onto female dendritic cells was shown to abrogate graft rejection responses in female mouse recipients of skin grafts derived from male mouse donors.

A logical extension of these results is to pretreat patients with the relevant UTY peptides for their HLA type, prior to grafting so that graft rejection responses (graft versus host and host versus graft) are abrogated or diminished in magnitude, so that lower amounts of nonspecific immunosuppressive drugs can be used.

In the case of host versus graft responses (as elicited by kidney and other organs grafts), the female recipient may be treated 7 to 28 days before transplantation of a partly or completely HLA matched organ by introduction of the relevant UTY peptide or a substituted agonist/antagonist peptide (Joost van Neerren et al., 1996, supra).

The selected peptide may be given by one or more of the following routes: intravenous subcutaneous intraperitoneal intramuscular intranasal or orally either in aqueous solution in quantities ranging from milligram to microgram, or prepulsed onto antigen presenting cells (APC). The APC may be either recipient white blood cells or specialized dendritic cells/macrophages generated therefrom by a period in culture, or in vitro grown cell lines selected for their peptide binding qualities (cf RMA-S). It will be appreciated that dendritic cells/macrophages may be grown in culture from their CD34* precursors in peripheral blood, for 7 to 10 days in the presence of differentiation and growth factors such as, for example, GMCSF and IL4. At the end of the culture period they may be harvested, washed and then pulsed with a saturating concentration of the selected UTY peptide, then washed again before being injected into the patient at doses from ten to one hundred WO 98/23739 PCT/AU97/00819 29 million cells by the selected route. Freshly isolated peripheral white blood cells, or in vitro cells lines may be alternative sources of APC, and these may be suitably peptide pulsed and washed before injection as for the cultured cells described above.

Strictly speaking, the above scenario constitutes a host versus graft response, but in principle tolerance induction utilizing the therapeutic agents of the invention may be extended to the treatment of the reverse situation, in which bone marrow transplant recipient patients, for example, are attacked by immunocompetent T lymphocytes from the donor. This is a difficult clinical problem in cases where donor and recipient are matched at the strongest transplantation antigens, HLA, (as in grafts exchanged between HLA matched siblings). If this protocol is to be transferred to humans, it would be necessary firstly to identify the relevant human UTY peptide homologs. In this regard, it is reasonably likely that these peptide homologs exist since another Y chromosome gene, Smcy, that encodes another H-Y peptide in mice also does so in humans (Scott et al., 1995, Nature 376 685-698; Wang et al., 1995, Science 269 1588-1590; Kent-First et al., 1996 Nature Genetics 14 128- 129).

Having identified the relevant UTY peptide or substituted peptide corresponding thereto for the human female donor in question, one would remove a sample of her blood, isolate from it CD34 white blood cells (dendritic cell precursors) and grow them in culture for seven days in the presence of the growth and differentiation factors, such as IL4 and GM-CSF (Sallusto et al., 1994 J. Exp. Med. 179 1109-1118 which is -hereby incorporated by reference). The differentiated dendritic cells would then be harvested and washed, and they would then be pulsed with the UTY peptide and administered to the female donor. Following a period of 7 to 28 days, when UTY peptide reactive donor T cells would be rendered anergic or tolerant, bone marrow removed for infusion into the HLA matched male recipient could be harvested from this donor and WO 98/23739 PCT/AU97/00819 transfused into the recipient. The peptide treatment is designed to inactivate the H-Y reactive T cells in the donor, and induce a population of regulatory cells which would interfere with the reactivity of other minor transplantation antigen reactive donor T cellspresent in the bone marrow inoculum (Davies et al., 1996, J. Immunol. 156 3602-3607 which is hereby incorporated by reference). This protocol is a modification of that tested in unpublished pilot experiments of the applicants in mice, for which female dendritic cell precursors have been cultured in vitro, harvested and pulsed with the appropriate UTY peptide, then administered to female mice subsequently grafted with male skin grafts. Reference also may be made to the utility of experimental protocols directed to the use of peptidepulsed dendritic cells in mice to immunize for a subsequently in vitro induced peptide response (Mayordom et al., 1995, Nature Medicine 1 1297-1302 which is hereby incorporated by reference).

In both host versus graft and graft versus host situations, the UTY peptide would render unresponsive those peptide specific T cells, and their presence would affect the activation of naive T cells with receptors specific for additional transplantation antigens. This type of bystander tolerance has been described for multiple minor histocompatibility antigens, but not analysed or induced to the single peptide level.

WO 98/23739 PCT/AU97/00819 31 FIGURE LEGENDS FIG. 1a. Nucleotide sequence of the UtycDNA.

FIG. lb. Structure of the Uty cDNA and predicted protein. Isolation of a composite Uty cDNA. Three overlapping cDNAs (15.1, m1.1 and L10.1) were isolated by screening of an 11.5 dpc male fetal library with probes derived from a screen for transcripts expressed differentially in developing male and female gonads. The region of the cDNA encoding the TPR repeats is shown as a solid box. 3' RACE-PCR was used to isolate an end-clone (R6) including an in-frame stop codon (marked with an asterisk). An in-frame stop codon is also present at the 5' end of clone 15.1. The open reading frame spans 3558 bp. The open box denotes the probe used for Southern analysis (FIG. 2b). The three constructs used in the CTL assay (FIG. 3) are depicted by the lines at the bottom of the figure (preceded by CMV). Constructs are: 1, Uty-BR1: nucleotides 1-1591; 2, Uty-15.1: nucleotides 1-2419; 3, Uty-BR2: nucleotides 1-2896.

The position of primer pairs used for RT-PCR and amplification of Uty from genomic DNA is indicated by arrows labelled 'ex' and 'gen', respectively. H= Hindlll; R EcoRI.

FIG. Ic. Predicted amino acid sequence of murine UTY protein.

Numbering for amino acids starts at the first in-frame methionine. The eight TPR motifs are underlined; the final four repeats are contiguous (see part The peptide encoding the H-YDb epitope is shown in bold.

Potential nuclear localisation signals conforming to the consensus K/R, K/R, X, K/R (Fenech et al., 1991, Nucleic Acids Res. 19 6737-6741) are shown in bold italics. The region defined by amino acids 393 to 525 is rich in glutamine residues and the region from amino acids 534 to 778 is rich in serine and proline FIG. Id. Alignment of the TPR domains of UTY and SSN6. The eight TPR motifs of UTY (amino acids 88 to 390) are shown aligned with TPR motifs three to ten (amino acids 114 to 398) of the yeast glucose repressor, SSN6 (accession number P14922). The TPR consensus (Con) WO 98/23739 PCT/AU97/00819 32 is shown above the firstrepeat. Each repeat is boxed, with consensus residues in bold. Identity is indicated by bars conservative matches by colons and semi-conservative pairings by dots. This alignment suggests that TPR motif number five of UTY and number seven of SSN6 are atypical in length. Conservation with respect to the TPR consensus at the ends of these motifs appears to be high, with divergence in sequence content and length in the middle. The H-YDb epitope described in this paper occurs in the central portion of this motif. The alignment was generated by the Wisconsin GCG program GAP.- FIG. 2a. Expression and chromosomal localisation of Uty. RT-PCR expression analysis of Uty in a sample of fetal (11.5 dpc) and adult male mouse tissues. Transcripts are observed in all male tissues tested. The upper 250 bp product is that predicted from the cDNA sequence. The lower 115 bp product is derived from a subset of transcripts lacking an intervening exon (encoding amino acids 394-438; FIG. Ib). Absence of this exon does not affect the translation reading frame. No product is observed with male genomic DNA due to the presence of two introns between the primer annealing sites. No expression was detected in any female samples (data not shown). Samples are: U, urogenital ridge; F, forelimb; N, lung; K, kidney; B, brain; S, spleen; G, gut; H, heart; ES, embryonic stem cell 6G, male genomic DNA; BI, negative control; L, liver; T, testis; Blasto, blastocyst. Data for several other positive male tissues are not shown.

FIG. 2b. Southern analysis of EcoRI-digested male and female genomic DNA with Uty probe A (FIG. la). A single, prominent male-specific fragment is observed, in addition to two fainter, shared fragments displaying approximately double intensity in females. These latter fragments are derived from an X-linked homologue of Uty (see text). The male-specific fragment is observed in the X/XSxra sample and cell line 13a (O/XSxra 13a) but not in X/XSxrb or line 13a-87 (O/XSxra 87).

FIG. 2c. PCR amplification of Uty using genomic DNA from cell lines 13a, WO 98/23739 PCT/AU97/00819 33 13a-87 and male, female and sex-reversed (X/XSxra and X/XSxrb) male mice. Uty primers were designed from within a single exon. A product of expected size (170 bp) is observed only in the XY, X/XSxra and 13a samples.

FIG. 3a. Uty expresses the H-YDb epitope. Identification of the smallest Uty construct that expresses H-YDb. EL4 cells transfected with each of three Uty constructs (FIG. la) are recognised by the anti-H-YDb-specific CTL clones. The lysis of negative control, untransfected EL4 cells is shown by the open bars and individual cloned EL4-Uty transfectants by grey bars. The standard errors calculated from the mean of triplicate samples are shown by the vertical bars. indicates samples not tested.

The results shown are: upper figure, using clone CTL10 (King et al., 1994, supra); lower figure, using clone, 151.9 (Simpson et al., 1995,7-Eur. J.

Immunol. 25 2813-2817). Apparent 'negative' lysis was observed in some instances indicating that these transfectant cells grew better in the presence of the T cell clone. Transfectants giving no or negative lysis are those which either fail to take upUty construct DNA or to express the epitope encoded by it. These were not investigated further, but the positive results observed were confirned by using the complementary approach of syntheticpeptides shown in part FIG. 3b. Identification of the peptide from Uty-BR1 that expresses the H-YDb epitope. Six peptides conforming to the H-2Db-binding motif (Rammensee et al., 1993, supra) were tested for their ability to stimulate the H-YDb-specific T cell clones. Grey circles show control stimulation by B10 male and female cells. Closed triangles show the stimulation by peptide 1 (LIDCNVCTL); closed circles, peptide 2 (WMHHNMDLI); open squares, peptide 3 (KSCNNTSAL); closed squares, peptides 4, 5 and 6 (SSLQNKTKL, NQNGNHTL, GDAQNHFLRI).

FIG. 4a. The effect of amino acid substitution of the H-YDb peptide epitope on T cell recognition and binding to the H-2Db class I molecule. T cell recognition: the grey circles show stimulation by male and female WO 98/23739 PCT/AU97/00819 34 control cells; closed circles, solid line, the unsubstituted UTY peptide, WMHHNMDLI; open circles, dotted line, the UTX peptide, WMHHTVDLL, peptide 2, WMHHTMDLL, and peptide 3, WMHHNVDLL; closed boxes, dotted line, peptide 4, WMHHNVDLI; open boxes, peptide WMHHNMALI; closed triangles, peptide 6, WMHHNMDAI; grey triangles, peptide 7, WMHHTMDLI; and grey boxes, solid line, peptide 8, WMHHNMDLL. (Substituted residues are underlined).

FIG. 4b. Stabilisation of H-2Db class I molecules on RMA-S cells by substituted H-YDb peptides. Stabilisation by the UTY and UTX peptides and substituted peptides 2-8 are shown using the same symbols as in FIG. 4a.

Claims (38)

1. An isolated nucleotide sequence selected from the group consisting of: an isolated nucleotide sequence comprising the entire sequence of nucleotides shown in FIG. la; (ii) an isolated nucleotide sequence comprising nucleotide 69 through nucleotide 3639 of FIG. la; and (iii) an isolated nucleotide sequence comprising nucleotide 804 through nucleotide 830 of FIG. la.

2. A Uty homolog.

3. A homolog according to claim 2, wherein said homolog is obtained from a mammal selected from the group consisting of human, mouse, rat, guinea pig and rabbit.

4. A homolog according to claim 2, wherein said homolog is obtained from male adult tissue. A homolog according to claim 2, wherein said homolog is obtained from male fetal tissue.

6. A homolog according to claim 2, wherein said homolog encodes the entire amino acid sequence of FIG. 1c or sub-sequence thereof.

7. A homolog according to claim 6, wherein said sub- sequence consists essentially of: Trp-Met-His-His-Asn-Met-Asp-Leu-lle.

8. A homolog according to any one of claims 2 to 7, wherein y AMSNODE SHEET CAU 7 0 0 8 1 RECEIVED 2 3 JUN 1398 36 said homolog hybridizes under substantially stringent conditions to a nucleotide sequence according to claim 1.

9. An isolated polypeptide comprising the entire sequence of amino acids depicted in FIG. 1c. A UTY polypeptide homolog.

11. A homolog according to claim 10, wherein said homolog is obtained from a mammal selected from the group consisting of human, mouse, rat, guinea pig and rabbit.

12. A homolog according to claim 11, wherein said homolog is obtained from male adult tissue.

13. A homolog according to claim 11, wherein said homolog is obtained from male fetal tissue,

14. A peptide corresponding to a sub-sequence of a polypeptide according to claim 9. A peptide according to claim 14, wherein said peptide consists essentially of the sequence: Trp-Met-His-His-Asn-Met-Asp- Leu-Ile.

16. A homolog of a peptide according to claim 14 or claim

17. A method for inducing tolerance in a female recipient of a male-derived tissue, comprising transforming a cell from the female recipient with a vector comprising a nucleotide sequence according to u1 w'bGD r C RECEIVED 2 3 JUN 1938 37 claim 1 or Uty homolog operably linked to a constitutive promoter to form a transformed cell, and administrating said transformed cell to the female recipient prior to transplantation of the male-derived tissue into said female recipient.

18. A method for inducing a female donor of a female-derived tissue to tolerate tissue of a male recipient before transplantation of the female-derived tissue into the male recipient, said method comprising transforming a cell from the female donor with a vector comprising a nucleotide sequence according to claim 1 or Uty homolog operably linked to a constitutive promoter to form a transformed cell, and administrating said transformed cell to the female donor prior to transplantation of the female-derived tissue into said male recipient.

19. A method according to claim 17 or claim 18, wherein the nucleotide sequence or Uty homolog encodes one to three distinct H-Y epitopes. A method according to claim 17 or claim 18, wherein the nucleotide sequence or Uty homolog encodes a single H-Y epitope.

21. A method according to claim 17 or claim 18, wherein the cell to be transformed is a white blood cell.

22. A method according to claim 21, wherein the white blood cell is an antigen presenting.

23. A method according to claim 22, wherein the antigen presenting cell is selected from the group consisting of a CD34 white blood cell, a dendritic cell and a macrophage. i-i A* AU 00- 1 9 RECEIVED 2 3 JUN 1998 38

24. A method for inducing tolerance in a female recipient of a male-derived tissue, comprising administrating a polypeptide according to claim 9 or UTY polypeptide homolog to the female recipient prior to transplantation of the male-derived tissue into said female recipient. A method for inducing a female donor of a female-derived tissue to tolerate tissue of a male recipient before transplantation of the female-derived tissue into the male recipient, said method comprising administrating a polypeptide according to claim 9 or UTY polypeptide homolog to the female donor prior to transplantation of said female-derived tissue into said male recipient.

26. A method according to claim 24 or claim 25, wherein the polypeptide or UTY polypeptide homolog encodes one to three distinct H-Y epitopes.

27. A method according to claim 24 or claim 25, wherein the polypeptide or UTY polypeptide homolog encodes a single H-Y epitope.

28. A method according to claim 24 or claim 25, wherein the polypeptide or UTY polypeptide homolog is administered directly.

29. A method according to claim 24 or claim 25, wherein the polypeptide or UTY polypeptide homolog is administered by first combining the polypeptide or UTY polypeptide homolog with a cell from the female donor to form a mixture and subsequently administering the mixture to the female donor. A method according to claim 29, wherein the cell is an antigen presenting cell. 39

31. A method according to claim 30, wherein the antigen presenting cell is selected from the group consisting of a CD34 white blood cell, a dendritic cell and a macrophage.

32. A method for inducing tolerance in a female recipient of a male-derived tissue, comprising administrating a peptide corresponding to a sub-sequence of a polypeptide according to claim 9 or UTY polypeptide homolog to the female recipient prior to transplantation of the male-derived tissue into said female recipient.

33. A method for inducing a female donor of a female-derived tissue to tolerate tissue of a male recipient before transplantation of the female-derived tissue into the male recipient, said method comprising the step of administrating a peptide corresponding to a sub-sequence of a polypeptide according to claim 9 or UTY polypeptide homolog to the female donor prior to transplantation of said female-derived tissue into said male recipient.

34. A method according to claim 32 or claim 33, wherein the peptide encodes one to three distinct H-Y epitopes. A method according to claim 32 or claim 33, wherein the peptide encodes a single H-Y epitope.

36. A method for improving fertility of a mammalian sperm cell or progenitor cell thereof, comprising transforming said sperm cell or progenitor cell thereof with a vector comprising a nucleotide sequence according to claim 1 or Uty homolog operably linked to a constitutive promoter.

37. A method for improving fertility of a mammalian sperm )4 ?A9 7 0 08 1 9 RECEIVED 2 3 JUN 1998 cell or progenitor cell thereof, comprising the step of delivering into said sperm cell or progenitor cell thereof a polypeptide according to claim 9 or UTY polypeptide homolog.

38. An anti-UTY antibody.

39. A method for diagnosis of infertility in a male mammal, comprising determining the presence or absence of a nucleotide sequence according to claim 1 or Uty homolog in a cell sample obtained from the male mammal wherein the absence of said sequence or homolog is indicative of infertility in the male mammal. A method for diagnosis of infertility in a male mammal, comprising determining the presence or absence of mutations in a nucleotide sequence according to claim 1 or Uty homolog in a cell sample obtained from the male mammal wherein the presence of mutations in said sequence or homolog is indicative of infertility in the male mammal.

41. A method for diagnosis of infertility in a male mammal, comprising determining the presence or absence of a polypeptide according to claim 9 or UTY polypeptide homolog in a cell sample obtained from the male mammal wherein the absence of said polypeptide or polypeptide homolog is indicative of infertility in the male mammal.

42. A pharmaceutical composition comprising a nucleotide sequence according to claim 1 or a Uty homolog.

43. A method for treatment of infertility,, comprising administering to a male patient in need of such treatment a I.SA/A phamaceutically effective amount of the composition of claim 42.

44. A pharmaceutical composition comprising a polypeptide according to claim 9 or a UTY polypeptide homolog. A pharmaceutical composition comprising a peptide corresponding to a sub-sequence of the polypeptide of claim 9 or UTY polypeptide homolog.

46. A method for inducing tolerance in a female recipient of a male-derived tissue, comprising administrating to a female recipient in need of said induction a phamaceutically effective amount of a composition according claim 44 or claim

47. A method for inducing a female donor of a female-derived tissue to tolerate tissue of a male recipient before transplantation of the female-derived tissue into the male recipient, comprising administrating to a female donor in need of said induction a phamaceutically effective amount of a composition according claim 44 or claim UjI