Cancer diagnosis and treatment

Abstract

The present invention relates to a method for detecting or predicting cancer, particularly paediatric cancer, in a subject, such as a foetal or paediatric subject. The method involves determining, from a biological sample obtained from the subject or the subject's mother during the peripartum period or during pregnancy, the presence of autoantibodies against a plurality of selected antigens or proteins which have been found to be associated with cancer (i.e. paediatric cancer-associated autoantigens). The invention also provides devices which may be used for diagnosis or prediction of cancers, particularly paediatric cancers.

Description

[0001] CANCER DIAGNOSIS AND TREATMENT

[0002] The present invention relates to a method for detecting or predicting cancer, particularly paediatric cancer, in a subject, such as a foetal or paediatric subject. The method involves determining, from a biological sample obtained from the subject or the subject’s mother during the peripartum period or during pregnancy, the presence of autoantibodies against a plurality of selected antigens or proteins which have been found to be associated with cancer (i.e. paediatric cancer-associated autoantigens). The invention also provides devices which may be used for diagnosis or prediction of cancers, particularly paediatric cancers.

[0003] Autoantibodies are common in healthy people but also involved in autoimmune disorders1 2. They can appear shortly after birth and increase towards older age3-6. Circulating autoantibodies at low concentrations can be detected in up to 40% of healthy individuals7It has been estimated that up to 15% of healthy children are positive for antinuclear autoantibodies, and in 3% of these the concentrations are regarded as clinically significant8. Although the repertoires are highly heterogenous, many autoantibodies are shared by a large fraction of healthy people1.

[0004] The pathogenic effects of autoantibodies are not limited to classical autoimmune diseases. In cancer patients, mutated proteins, so-called neoantigens, can trigger the generation of autoantibodies that cross-react with the wild type (wt) proteins, and wt proteins aberrantly expressed or post-translationally modified9in the tumour can also induce autoantibodies. Such anti-tumour antibodies indicate a blunted immune reaction to the tumour cells that might be activated by new immunotherapies. Tumour- associated autoimmunity may give rise to paraneoplastic syndromes in distant tissues10’11. Both positive and negative association between autoimmune disease and cancer have been reported.

[0005] Paediatric cancers arise at ages when their tissues-of-origin go through critical developmental processes and waves of proliferation (Figure 1a). These cancers are known to differ fundamentally from those occurring in adult subjects. Despite the higher risk of mutations due to the large number of cell divisions in growing tissues, paediatric cancers are rare and characterized by paucity of somatic mutations13’15. Although genetic predisposition has been thought to play an important role in early cancers, only 6-10% of cases have so far been shown to have likely pathogenic gene variants14’16.

[0006] The understanding of the aetiology of paediatric cancer remains unsatisfactory and, as a consequence, there is a distinct lack of known biomarkers that can be used in the diagnosis or prediction of the likelihood of development of paediatric cancers. The lack of biomarkers and heterogeneity of paediatric cancers, but also the caution required when treating small, sensitive patients with experimental drugs, has hindered the development of improved treatments for these patients in great need. Thus, most current treatments for children were developed more than 30 years ago, and more than 95% of cancer survivors develop serious health-related issues, including second cancers. Indeed, without a means to enable early diagnosis or prediction of paediatric cancer development, early detection and treatment of such cancers remains a challenge, with a huge impact on lost life quality and years.

[0007] There is a need therefore for biomarkers to be identified which can be used to diagnose and / or predict the development of paediatric cancers in subjects. In particular, biomarkers that work across cancer types would be valuable. Early detection or prediction of these cancers are essential in improving the survival chances of patients at risk of developing, or those with, paediatric cancers. Novel, non-toxic treatments would also be an important achievement.

[0008] A group of paediatric cancer-associated autoantibodies have now been identified as being enriched in samples from patients with different types of paediatric cancer. In particular, the present inventors have found that serum samples from paediatric cancer patients were surprisingly enriched in autoantibodies against proteins involved in DNA damage repair and genome integrity maintenance pathways, inter alia. Such proteins are generally cancer-protecting, so-called tumour suppressor proteins, and heritable mutations in similar proteins are known to predispose to cancer. Using indirect immunofluorescence and confocal microscopy, the inventors performed a systematic analysis of the capability of autoantibodies from patients and controls to penetrate various human cell types and assessed the co-localization of autoantibodies with some of the autoantigens identified by protein arrays. They found that autoantibodies from paediatric patients efficiently penetrated live cells with specific patterns of subcellular localisation, and co-localized with some of the enriched autoantigens or caused their dislocation from their usual localisation in the nuclei. On the contrary, healthy controls or even adult cancer patients’ sera did not show these patterns and levels of penetrating capacity. The inventors conclude that these autoantibodies compromise genome integrity by interfering with critical nuclear proteins and thus may predispose to cancer.

[0009] Such autoantibody biomarkers may therefore be used in the diagnosis and / or prediction of paediatric cancers in patients either before the cancer has developed or during the early stages of the disease. Furthermore, peptides based on the sequence of the antigen epitopes might be used as a treatment to block the action of the antibodies (e.g. Havasi, A., W. Lu, H. T. Cohen, L. Beck, Z. Wang, C. Igwebuike and S. C. Borkan (2017). "Blocking peptides and molecular mimicry as treatment for kidney disease." Am J Physiol Renal Physiol 312(6): F1016-F1025.) or even to target and ablate memory B cells producing these autoantibodies (Stepanov, A. V., A. A. Belogurov, Jr., N. A. Ponomarenko, 0. A. Stremovskiy, L. V. Kozlov, A. M. Bichucher, S. E. Dmitriev, I. V. Smirnov, 0. G. Shamborant, D. S. Balabashin, L. P. Sashchenko, A. G. Tonevitsky, A. Friboulet, A. G. Gabibov and S. M. Deyev (2011). "Design of targeted B cell killing agents." PLoS One 6(6): e20991.), thus preventing disease.

[0010] It is therefore an object of the invention to provide methods and devices for detecting these paediatric cancer-associated autoantibody biomarkers in subjects. Such information is useful in the diagnosis or prediction of paediatric and other cancers.

[0011] In one embodiment, the invention provides a method of detecting or predicting cancer in a subject, the method comprising the step: (a) determining, from a biological sample obtained from:

[0012] (i) the subject;

[0013] (ii) the subject’s mother during the peripartum period; or

[0014] (iii) the subject’s mother whilst pregnant with the subject; the presence of autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen, Sm Antigen, ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CPNE6, CRAT, PLCD4, DHX58, DMD, HSPA13, SRP19, and ELOB / TCEB2 and optionally also PRDX2, wherein the presence of autoantibodies against the selected antigens or proteins within the biological sample is indicative of the presence of a cancer in the subject or is predictive of the future development of a cancer in the subject.

[0015] CGGBP1 and CCT4 may also be included in the group.

[0016] The subject is preferably a mammalian subject. The mammal may be human or nonhuman. For example, the subject may be a farm mammal (e.g. sheep, horse, pig, cow or goat), a companion mammal (e.g. cat, dog or rabbit) or a laboratory test mammal (e.g. mouse, rat or monkey). In particularly-preferred embodiments, the subject is a human subject.

[0017] The subject is preferably a foetal, neonatal, paediatric, infant, adolescent or adult subject.

[0018] As used therein, the term “foetal” subject or “foetus” refers to an unborn subject which has all of its major organ systems. In human subjects, a foetus is defined as an unborn subject from week 11 (gestational age) until birth.

[0019] As used therein, the term “neonatal” subject or “neonate” refers to a subject from birth to a period shortly after birth. In human subjects, the term “neonatal” subject or “neonate” refers to subjects from 0 to 4 weeks old. The neonate may be premature, full term or post-mature.

[0020] As used therein, the term “paediatric” subject refers to a subject from birth to adulthood. In human subjects, the term “paediatric” refers to subjects from 0 to 20 years old.

[0021] In some embodiments, the term subject is a human who is 0-1 , 1-5, 5-12, 12-16 or 16- 20 years old.

[0022] The subject may be an infant. In human subjects, the term “infant” refers to subjects from 1 month to 1 year.

[0023] As used therein, the term “adolescent” subject refers to a subject during the period from puberty to adulthood, e.g. between 10 and 20 years old in humans.

[0024] In some embodiments, the subject is an adult. As used therein, in human subjects, the term “adult” refers to a subject from 20 years old and older. The term “young adult” may be used to define a human subject between 20 and 30 years old.

[0025] The subject may be (biologically) male or female.

[0026] The human may be one who is at risk from a particular disease or disorder, e.g. a cancer. The human may be one who has previously suffered from a particular disease or disorder, e.g. a cancer. The human may be one who is genetically-related to another human who is at risk from a particular disease or disorder, e.g. a cancer. For example, the human subject may be an adult mother of a neonate who is at risk from a particular disease or disorder, e.g. a cancer. The subject may be one who is at risk of developing a cancer.

[0027] Whereas the IgG from older subjects are generated by their own immune system, the IgG in subjects at a few months of age would be expected to be largely maternal. It is therefore likely that very early cancers initiate during pregnancy, upon the possible influence of maternal autoantibodies which would circulate in the foetus, and would disappear from the subject’s blood within the first year of life.

[0028] As used herein, the term “subject’s mother during the peripartum period” refers to the subject’s biological mother, i.e. the person who is pregnant with the subject, is giving birth to the subject or who has previously given birth to the subject. The peripartum period is the period immediately preceding and immediately following the birth of the subject, e.g. from one month before the birth of the subject to 6 months following the birth of the subject.

[0029] The biological sample may also be obtained from the subject’s mother whilst pregnant with the subject, preferably wherein the biological sample is or was obtained during or after week 17, after week 20 or after week 25 (gestational age) of the pregnancy.

[0030] As used herein, a control subject may be defined as a non-diseased subject, i.e. a subject without a present or previous cancer, or a healthy subject. The control subject may have other health issues not related to the cancer.

[0031] The biological sample is preferably a blood sample, i.e. whole blood, blood serum or blood plasma. In other embodiments, the biological sample is cerebrospinal fluid (CSF), lymph or saliva.

[0032] In most embodiments, the method of the invention is performed on a biological sample which has previously been obtained from the subject or from the subject’s mother during pregnancy or in the peripartum period, i.e. the method does not include step of obtaining the biological sample from the subject or subject’s mother. In some embodiments, however, the method may additionally comprise the step of obtaining one or more biological samples from the subject or from the subject’s mother during pregnancy or in the peripartum period. In some embodiments, the biological sample is obtained from the subject during the peripartum period. In some embodiments, the autoantibodies are detected directly in the biological sample, e.g. from within a biological sample of the subject or subject mother’s blood, preferably the subject or subject mother’s blood serum.

[0033] The biological sample may be processed before the presence of autoantibodies is determined, e.g. to separate serum or plasma from other blood components such as cells, or by enriching for the presence of antibodies (e.g. IgG) in the biological sample.

[0034] In some embodiments, the biological sample is one which was obtained from the subject’s mother prior to the birth of the subject, e.g. 1 -8 weeks, preferably 1 -4 weeks prior to birth (or expected birth), or within 0-1 , 0-6 or 0-12 months after the birth of the subject.

[0035] In some embodiments, the biological sample is one which was obtained from the umbilical cord between the subject and the subject’s mother, preferably umbilical cord blood.

[0036] In the case of blood collected from the umbilical cord at birth, the biological samples are from the subject, whereas the IgG in that biological sample are maternal IgG, and the IgM are from the subject.

[0037] In other embodiments, the biological sample is one which was obtained from the subject when the subject was 0-1 , 1-2, 2-5, 5-12, 12-16 or 16-20 years old.

[0038] In general, the detecting or predicting methods of the invention are carried out in vitro or ex vivo.

[0039] The Applicant has identified the autoantigens of the invention as being paediatric cancer-associated autoantigens. As used herein, the term “autoantibodies” refers to antibodies that recognise the subject’s own (i.e. self) antigens or proteins, i.e. autoantigens. In other words, the autoantibodies recognise (auto)antigens that are on or are derived from the subject’s own (i.e. self) antigens or proteins. As used herein, the term “autoantigen” refers to one of the subject’s (self) antigens or proteins that is recognized by the immune system of the subject. Under normal conditions, these self-antigens or proteins should not be the target of the immune system, but in certain diseases, the subject’s associated B and T cells are not deleted and instead attack these auto-antigens.

[0040] As used herein, the term “subject’s own (i.e. self) antigens or proteins” includes the subject’s own (self) antigen and protein complexes.

[0041] In most embodiments of the invention, the autoantibodies are against the subject’s wildtype antigens or proteins. In other words, the autoantibodies are not against neoantigens. As used herein, the term “neoantigens” refers to mutated, i.e. non-wild- type proteins, that are entirely absent from the normal human genome.

[0042] In some embodiments, the plurality of the subject’s (self) antigens or proteins is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27 of the subject’s (self) antigens or proteins, preferably at least 5, 10, 15, 20, 25 or 27, more preferably at least 26 or 27 of the subject’s (self) antigens or proteins. In other embodiments, the plurality of the subject’s (self) antigens or proteins is preferably 2-3, 3-5, 5-10, 10-15, 15-20, 20-25 or 26-27 or all of the antigens or proteins. In some embodiments, the term “plurality” refers to at least four of the antigens or proteins.

[0043] In some embodiments, the term “plurality” refers to at least ten of the antigens or proteins. In some embodiments, the term “plurality” refers to at least 15 of the antigens or proteins. In some embodiments, the term “plurality” refers to at least 20 of the antigens or proteins. In some embodiments, the term “plurality” refers to at least 25, at least 26 or at least 27 of the antigens or proteins.

[0044] The presence or absence of an autoantibody in the biological sample may be determined by any suitable means known to the skilled person. For example, the presence or absence of an autoantibody in the biological sample may be determined by protein array, Western blot, a lateral flow device, or an ELISA assay (e.g. a multiplex flow-based ELISA). The detection of the autoantibodies may be done using antibodies at an appropriate dilution, e.g. 1 / 20, 1 / 40 or 1 / 100 dilution.

[0045] The cancer is preferably a paediatric cancer. Paediatric cancers, also known as childhood cancers, are cancers that occur in foetuses, neonates, paediatric subjects, infants, adolescents or young adults. These cancers can vary in type and often arise at ages when their tissues-of-origin go through critical development processes and waves of cellular proliferation. In some embodiments, the paediatric cancer is a cancer selected from the group consisting of blood, brain, spinal cord, lymphatic system, muscle, bone, nerve cells, kidney and eye cancers.

[0046] In some embodiments, the paediatric cancer is neuroblastoma. In other embodiments, the paediatric cancer is glioma. In other embodiments, the paediatric cancer is ganglioglioma, pontine glioma, high grade glioma or supratentorial glioma. In other embodiments, the paediatric cancer is Wilms’ tumour. In other embodiments, the paediatric cancer is a leukaemia, e.g. acute lymphoblastic leukaemia (ALL). In other embodiments, the paediatric cancer is a sarcoma, e.g. osteosarcoma, rhabdomyosarcoma or Ewing’s sarcoma, preferably, osteosarcoma or rhabdomyosarcoma.

[0047] In some embodiments, the antigens or proteins are tumour suppressors. A tumour suppressor protein is a protein that controls cell growth and regulates cell division and replication. The binding of autoantibodies to such proteins prevents the tumoursuppressing function of those antigens or proteins, thus leading to uncontrolled cell division and cell replication. The following antigens and proteins have been identified as tumour suppressor antigens or proteins: APBB1 , TP53I3, ACOT12, CRAT, PLCD4 and DMD.

[0048] In some embodiments, the plurality of the subject’s (self) antigens or proteins are at least 5 antigens or proteins, preferably wherein the selected antigens or proteins are or comprise RAD52, UBE2N, CRAT, PLCD4 and HSPA13. In some embodiments, the plurality of the subject’s (self) antigens or proteins are at least 10 antigens or proteins, preferably wherein the selected antigens or proteins are or comprise APBB1 , NUMA1 , RAD52, UBE2N, ACOT12, CRAT, PLCD4, DHX58, HSPA13 and SRP19.

[0049] In some embodiments, the plurality of the subject’s (self) antigens or proteins are at least 15 antigens or proteins, preferably wherein the selected antigens or proteins are or comprise ALKBH7, APBB1 , DMC1 , MSH5, NUMA1 , RAD52, TP53I3, UBE2N, ACOT12, CRAT, PLCD4, DMD, DHX58, HSPA13 and SRP19, and optionally also PRDX2.

[0050] In some embodiments, the plurality of the subject’s (self) antigens or proteins are at least 20 antigens or proteins, preferably wherein the selected antigens or proteins are or comprise ALKBH7, APBB1 , DMC1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CRAT, PLCD4, DMD, DHX58, HSPA13, SRP19, TCEB2 and ADRM1 , and optionally also PRDX2.

[0051] In some embodiments, the autoantibodies are antibodies against proteins which are involved in DNA damage detection or repair detection or genome integrity maintenance.

[0052] In some preferred embodiments, the autoantibodies are against a plurality of antigens or proteins selected from the group consisting of the proteins in the following table.

[0053] Table 1 : Proteins which are involved in DNA damage detection or repair or in genome integrity maintenance

[0054] The proteins referred to herein are named according to GeneCards and UniProt.

[0055] In some embodiments of the invention, the plurality of the subject’s (self) antigens or proteins are selected from the group which includes PRDX2.

[0056] In some embodiments of the invention, the plurality of the subject’s (self) antigens or proteins are selected from the group which does not include PRDX2. In some embodiments, the autoantibodies are antibodies against proteins which are involved in lipid metabolism.

[0057] In some preferred embodiments, the autoantibodies are against a plurality of antigens or proteins selected from the group consisting of the proteins in the following table.

[0058] Table 2: Proteins which are involved in lipid metabolism

[0059] In some embodiments, the autoantibodies are autoantibodies against biomarkers for autoimmunity. Such proteins are involved in cancer development and progression (Fernandez, L. P., M. Gomez de Cedron and A. Ramirez de Molina (2020). "Alterations of Lipid Metabolism in Cancer: Implications in Prognosis and Treatment." Front Oncol 10: 577420).

[0060] In some embodiments, the autoantibodies are autoantibodies against a plurality of antigens or proteins selected from the group consisting of the antigens and proteins in the following table.

[0061] Table 3: Antigens and proteins which are biomarkers for autoimmunity.

[0062] The antigens and complexes listed in the above table are not single proteins. In each case, the main proteins are listed.

[0063] In some embodiments of the invention, the subject’s (self) antigens or proteins are selected from a group which does not include the above antigens and complexes.

[0064] In some preferred embodiments, the autoantibodies are autoantibodies against a plurality of antigens or proteins selected from the group consisting of the proteins in the following table.

[0065] Table 4: Proteins

[0066] In some embodiments, the autoantibodies are autoantibodies against CGGBP1 or CCT4.

[0067] In some embodiments, the plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of CRAT, RD52 and DHX58. These antigens / proteins were found by confocal microscopy to be colocalized with patients’ autoantibodies.

[0068] In some embodiments, the plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of Scl-70 Antigen, RAD52, PLCD4, HSPA13 and SPR19, and the cancer is neuroblastoma.

[0069] In some embodiments, the plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of Scl-70 Antigen, RAD52, CRAT, PLCD4 and HSPA13, and the cancer is glioma.

[0070] In some embodiments, the plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of Scl-70 Antigen, MSH5, UBE2N, CRAT, DMD and HSPA13, and the cancer is Wilms’ tumour.

[0071] In some embodiments, the plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of CRAT, DHX58 and DMD, and the cancer is leukaemia, preferably ALL.

[0072] In some embodiments, the two plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of Scl-70 Antigen, TP53I3, AC0T12 and CRAT, and the cancer is osteosarcoma / rhabdomyosarcoma / Ewing’s sarcoma, preferably osteosarcoma or rhabdomyosarcoma. In yet another embodiment, the invention provides a method of detecting or predicting leukaemia in an adult subject, wherein the plurality of the subject’s (self) proteins are or are selected from the group consisting of ALKHB7, APBB1 , MSH5, NUMA1 , POLR2H, RAD52, RCHY1 , CRAT, DMD and HSPA13.

[0073] More specifically, the invention provides a method of detecting or predicting leukaemia in an adult subject, the method comprising the step:

[0074] (a) determining, from a biological sample obtained from the subject, the presence of autoantibodies against a plurality of the subject’s (self) proteins selected from the group consisting of ALKHB7, APBB1 , MSH5, NUMA1 , POLR2H, RAD52, RCHY1 , CRAT, DMD and HSPA13, wherein the presence of autoantibodies against the selected proteins within the biological sample is indicative of the presence of leukaemia in the subject or predictive of the future development of leukaemia in the subject.

[0075] In some preferred embodiments, the selected proteins are all of ALKHB7, APBB1 , MSH5, NUMA1 , POLR2H, RAD52, RCHY1 , CRAT, DMD and HSPA13.

[0076] As used herein, the term "is indicative of the presence of a cancer in the subject or predictive of the future development of a cancer in the subject” means that there is a positive correlation between the presence of autoantibodies against a plurality of the selected antigens or proteins as defined herein and the presence of a cancer in that subject or an increased likelihood that the subject will develop a cancer.

[0077] Thus, the presence of autoantibodies against a plurality of the selected antigens or proteins in a biological sample from the subject means an increased likelihood or statistically-significant chance of the subject having or developing a cancer. Significance may be measured by any suitable statistical technique known to the skilled person, e.g. Student’s t-test.

[0078] In another embodiment, the invention provides a method of obtaining information relevant to the diagnosis of cancer in a subject, the method comprising the step: (a) determining, from a biological sample obtained from:

[0079] (i) the subject;

[0080] (ii) the subject’s mother during the peripartum period; or

[0081] (iii) the subject’s mother whilst pregnant with the subject; the presence of autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen, Sm Antigen, ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CPNE6, CRAT, PLCD4, DHX58, DMD, HSPA13, SRP19, and ELOB / TCEB2 and optionally also PRDX2.

[0082] As used herein, the term "obtaining information relevant to the diagnosis of cancer” in the subject means that the information obtained from the method can provide information contributing towards or necessary for the diagnosis of a cancer in the subject.

[0083] In another embodiment, the invention provides a method of preventing cancer in a subject, the method comprising the steps:

[0084] (a) determining, from a biological sample obtained from:

[0085] (i) the subject;

[0086] (ii) the subject’s mother during the peripartum period; or

[0087] (iii) the subject’s mother whilst pregnant with the subject; the presence of autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen, Sm Antigen, ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CPNE6, CRAT, PLCD4, DHX58, DMD, HSPA13, SRP19, and ELOB / TCEB2 and optionally also PRDX2, wherein the presence of autoantibodies against the selected antigens or protein within the biological sample is predictive of the future development of a cancer in the subject; and (b) if a prediction of the future development of a cancer in the subject has been obtained, administering to the subject a prophylactic treatment appropriate for preventing the cancer.

[0088] As used herein, the term “prophylactic treatment” is a treatment that, once administered, reduces the risk or removes the possibility of a subject at risk of developing a cancer from eventually being diagnosed with a cancer.

[0089] In another embodiment, the invention provides a method of preventing the future development of a cancer in a subject, the method comprising the steps of:

[0090] (a) administering a prophylactic treatment appropriate for preventing the future development of a cancer to the subject, wherein, prior to administration, an indication of the future development of a cancer in the subject has been obtained by a method comprising the steps of:

[0091] (b) determining, from a biological sample obtained from:

[0092] (i) the subject;

[0093] (ii) the subject’s mother during the peripartum period; or

[0094] (iii) the subject’s mother whilst pregnant with the subject; the presence of autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen, Sm Antigen, ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CPNE6, CRAT, PLCD4, DHX58, DMD, HSPA13, SRP19, and ELOB / TCEB2 and optionally also PRDX2, wherein the presence of autoantibodies against selected antigens or protein within the biological sample is predictive of the future development of a cancer in the subject.

[0095] As used herein, the term “preventing the future development of a cancer” also means reducing the risk of (future) development of a cancer. Similarly, the term “preventing” also includes reducing the risk. Preferably, the subject is one who has not previously been diagnosed with or is showing any indication of a cancer. Numerous prophylactic treatments for cancers are known in the art.

[0096] Suitable treatments include ablation of the subject’s B-cell clones and the administration of blocking peptides (mimotopes), e.g. as disclosed herein.

[0097] In accordance with another embodiment, the invention provides a method of preventing a cancer in a subject, the method comprising administering effective amounts of one or more blocking peptides to a subject in need thereof, wherein the blocking peptides are peptides which bind to autoantibodies in the subject’s blood.

[0098] Preferably, the blocking peptides are capable of binding to (and blocking) autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen, Sm Antigen, ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CPNE6, CRAT, PLCD4, DHX58, DMD, HSPA13, SRP19, and ELOB / TCEB2, and optionally also PRDX2.

[0099] The blocking peptides are designed to block the interaction between the autoantibody and the autoantigen, i.e. the subject’s self antigen or protein. Such blocking peptides are also known as mimotopes. The blocking peptide may, for example, comprise a fragment of the subject’s self protein, e.g. a peptide fragment of the subject’s self protein which corresponds to a continuous epitope to which the autoantibody binds, or a peptide fragment which mimics a discontinuous epitope on the subject’s self protein to which the autoantibody binds. The specific blocking peptide, or set of blocking peptides, for the subject may be selected to target the set of autoantibodies detected by a method of the invention.

[0100] One or more or a plurality of blocking peptides may be used to block a particular autoantibody. For example, a plurality of blocking peptides may be used to block a particular autoantibody, wherein each of the blocking peptides individually mimics a different part of the epitope on the subject’s self protein to which the autoantibody binds, and preferably wherein the plurality of blocking peptides collectively mimic all parts of the epitope on the subject’s self protein to which the autoantibody binds. The blocking peptides may be administered intravenously into the subject. Preferably, the blocking peptides are stable, water soluble, do not form aggregates and have extended serum half-life.

[0101] In another embodiment, the invention provides a composition comprising a plurality of blocking peptides, as defined herein, for use in preventing cancer in a subject. In yet another embodiment, the invention provides the use of a plurality of blocking peptides as defined herein in the manufacture of a medicament for the prevention of a cancer in a subject.

[0102] The plurality of blocking peptides may, for example, be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18 19, 20, 21 , 22, 23,24, 25 or 26; preferably at least 5, 10, 15, 20 or 25 blocking peptides.

[0103] Methods of preventing cancer may also be implemented by removing memory B cells which express one or more of the autoantibodies as defined herein from the subject’s blood.

[0104] In another embodiment, therefore, the invention provides a method of preventing a cancer in a subject at risk of developing a cancer, the subject being one who has autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen, Sm Antigen, ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CPNE6, CRAT, PLCD4, DHX58, DMD, HSPA13, SRP19, and ELOB / TCEB2, and optionally also PRDX2, the method comprising the step of:

[0105] (a) removing memory B cells which express one or more of the autoantibodies from the subject’s blood. In some preferred embodiments, the memory B cells are removed by: (a) administering to the subject an effective amount of one or more cytotoxic conjugates, wherein each cytotoxic conjugate comprises a targeting moiety and a cytotoxic moiety, wherein each targeting moiety comprises a peptide (e.g. a blocking peptide) capable of forming an epitope which is capable of being bound by a B-cell receptor which expresses one of the autoantibodies.

[0106] The cytotoxic moiety is one which is capable of being internalised into a B cell and is capable of causing or inducing cell death of the B cell. The cytotoxic moiety may, for example, be a membrane-permeable cytotoxin or apoptosis-inducing agent (e.g. Volkman et a / ., J Exp Med. 1982 Aug 1 ; 156(2):634-9).

[0107] An appropriate treatment in the context of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. Preferred routes of administration of treatments in accordance with the invention may include oral, intravenous, intrathecal, intracerebroventricular, intraperitoneal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0108] Alternatively, treatments may be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration. The blocking peptides are preferably administered orally or by injection, depending on specific formulations. The cytotoxic conjugates are preferably administered by injection. Preferably, the blocking peptides are encapsulated in a gastro-resistant capsule which is removed in the higher pH in the intestines. The identification by the Applicant of a set of autoantibodies which are associated with cancer or the future development of cancer in a subject enables the production of diagnostic devices which can be used to specifically detect those autoantibodies. Such devices may be used in the methods of the invention. In this context, the autoantibodies may be treated as antibodies.

[0109] In yet another aspect, therefore, the invention provides a device for determining the presence of two or more antibodies in a biological sample, the device comprising means for detecting, in the biological sample, the presence of antibodies against a plurality of antigens or proteins selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen, Sm Antigen, ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CPNE6, CRAT, PLCD4, DHX58, DMD, HSPA13, SRP19, and ELOB / TCEB2, and optionally also PRDX2.

[0110] In some embodiments, the means for detecting the presence of the antibodies against the plurality of the selected antigens or proteins comprises specific binding partners for the plurality of antibodies. In some embodiments, the means for detecting the presence of the plurality of antibodies (e.g. 2 or more) comprises a plurality (e.g. 2 or more) of the antigens or proteins or antigenic fragments thereof.

[0111] As used herein, the term “plurality of antibodies” includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27 antibodies, preferably at least 5, 10, 15, 20, 25 or 26 antibodies.

[0112] Multiplex Flow-based ELISA is the gold standard for the detection of multiple autoantibodies used in routine clinical practice at the present time. Such multiplex assays are derivatives of ELISA assays which use beads for binding to the autoantibodies. The beads may be used to simultaneously measure multiple autoantibodies in a single experiment. The beads may be magnetic or coloured (e.g. comprising a fluorescent tag). The results can be read by flow cytometry because the beads are distinguishable, e.g. by their fluorescent tags. In some embodiments, therefore, the device is a multiplex-ELISA device, wherein the means for detecting the presence of antibodies against a plurality of the antigens or proteins comprises at least two sets of beads, wherein each set of beads is coated with a specific binding partner for one of the antibodies (e.g. the antigen or protein or an antigenic fragment thereof to which the antibody binds).

[0113] Lateral flow devices may also be used to detect the autoantibodies. In other embodiments, therefore, the device is a lateral flow device, wherein the means for detecting the presence of the antibodies against a plurality of the antigens or proteins comprises specific binding partners for the plurality of antibodies (e.g. the antigens or proteins or antigenic fragments thereof to which the antibodies are capable of binding).

[0114] In some embodiments, the lateral flow device comprises a conjugate zone wherein antigens or proteins or antigenic fragments thereof to which the plurality of antibodies are capable of binding are present in the conjugate zone, and wherein the antigens or proteins or antigenic fragments thereof are labelled with detectable labels.

[0115] In some embodiments, the lateral flow device comprises a detection zone wherein antigens or proteins or antigenic fragments thereof to which the plurality of antibodies are capable of binding are present in the detection zone.

[0116] In some embodiments the lateral flow device comprises: (a) a sample receiving zone, to which the biological sample is applied or is capable of being applied; (b) a conjugate zone, wherein first specific binding partners are linked to detectable labels, and wherein the first specific binding partners are not immobilised; (c) a detection zone, wherein second specific binding partners are immobilised in the detection zone; and optionally one or both of: (d) a control zone, and (e) an absorbent zone, wherein the above zones are joined in (fluid) communication, in the above-mentioned order, wherein the first or second specific binding partners are antigens or proteins or antigenic fragments thereof to which the plurality antibodies are capable of binding. (The other specific binding partners may, for example, be secondary (e.g. Fc-binding) antibodies). In some embodiments, the detectable labels are coloured moieties, gold nanoparticles or fluorescent labels.

[0117] The disclosure of each reference set forth herein is specifically incorporated herein by reference in its entirety.

[0118] BRIEF DESCRIPTION OF THE FIGURES

[0119] Figure 1. Age distribution of pediatric cancers and autoantibodies. (A) Age incidence in Norway 1980-2019 of the cancers included in this study. Data are plotted as 5-year moving averages for better visibility. The arrows indicate the peaks of incidence in children. The bar below indicates the pediatric (0-19), intermediate (20-29), and adult (30+) age groups. ALL - acute lymphoblastic leukemia; GLM - glioma; NBL - neuroblastoma; RMS - rhabdomyosarcoma; OS - osteosarcoma; WT - Wilm’s tumor; UPS - undifferentiated pleomorphic sarcoma; LPS - liposarcoma. Data courtesy of the Norwegian Cancer Registry. (B) Results from gene set enrichment analysis GSEA. (C) Number and distribution of autoantigens according to age. Each bar shows the total number of autoantigens per patient from the four categories enriched in children with cancer. The cases are sorted by age at diagnosis in ascending order, controls are sorted by sampling age. The small squares below the x axis indicate each case, coloured according to cancer type as shown in the legend, asterisks indicate samples taken after initial chemotherapy.

[0120] (D) Number and distribution of autoantigens according to the age in healthy controls. Each bar shows the total number of autoantigens from the same four categories as in b per control. The controls are sorted by age in ascending order. (E) Autoantibodies detected in cord plasma from children diagnosed with cancer in the first 2 years of life (left) and from controls who did not have cancer in their first 18 years (right). Samples from cases are sorted by age at diagnosis in months in ascending order, and the type of cancer indicated by the colour of the squares below as in b. Each bar shows the total number of autoantigens per case from the same four categories as in b and c. B=Cord blood from child who got benign tumour at 10 months. Figure 2: Heat map data showing the frequencies (%) and number of autoantigens of the core list of autoantigens within the groups enriched in pediatric cancer patients versus healthy controls. For the 17 less shared DNA repair autoantigens only aggregate data are shown. Blank fields indicate 0. The autoimmunity markers are not cloned peptides but affinity-purified antigens in clinical use.

[0121] Figures 3-4: ELISA assessment of autoantibodies against antigens of interest in cases and controls sera. The dot plots show individual differences between cases and controls. Samples were analyzed in triplicate and results are expressed as fluorescence intensity (a.u.) using a green filter. Significant differences between cases and controls were evaluated by unpaired t-test (****<0.0001). Results are expressed as mean±SD.

[0122] EXAMPLES

[0123] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0124] The following Materials and Methods were used in one or more of the Examples.

[0125] Materials and Methods

[0126] Patient samples

[0127] The main cohort, 78 adult sarcoma patients and 37 paediatric were part of the NoSarC Norwegian Sarcoma Study, a collection of tumour and blood samples from >700 sarcoma patients from all Norwegian health regions, collected from 2014-2017. All patients have provided informed consents according to the ethical approval 17866 from the Regional Committee for Medical Ethics, who also approved amendments for inclusion of anonymous samples from healthy controls and additional samples from paediatric patients from the General Cancer Biobank and from the National Pediatric Cancer Biobank, both at Oslo University Hospital as part of the same approval number.

[0128] The umbilical cord plasma samples were from the Norwegian Mother, Father, and Child Cohort Study (MoBa) Study54, which were collected in 2000-2002, and the cancer diagnoses in the children have been identified by the Cancer Registry of Norway. The use of these anonymous samples and connected cancer diagnoses has been approved by the Regional Committee for Medical Ethics approvals 17866 (to Ola Myklebost, University of Bergen) and 2009 / 784 (to Per Minor Magnus at the Norwegian Institute of Public Health).

[0129] Anonymous control samples were obtained from blood donors, the Bergen Growth Study 255, and surplus samples from small children visiting the Helse Bergen Trauma Clinic. Their use in this study was approved by an extension to approval 17866.

[0130] Proteome microarray analysis.

[0131] HuProt v4.0 Human Proteome Microarrays were purchased from Cambridge Protein Arrays Ltd. The probing of the arrays and the statistical data analysis were performed according to the Manufacturer’s protocol. Specifically, the arrays were blocked with 2% BSA at 4°C overnight. Sera samples were diluted 1 :1000 in blocking buffer and incubated with the arrays with gentle shaking for 2 hours at RT. The arrays were washed 3 times in rapid succession and 2 times for 5 minutes. Next, the arrays were incubated with anti-human IgG secondary antibody for 2 hours at RT, following by the washing like described before. The detection was performed on the GenePix scanner, with the same setting kept throughout entire study. Statistical analysis was performed according to the manufacturer’s protocol. For the z-score positive proteins, the cut off was set at 2.5 corresponding to p-value 0.0214; for the sample-to-control-ratio (Prot s / c) the cut off was set at 3.5, as recommended. Cell lines

[0132] The panel of osteosarcoma cell lines were either derived by the NoSarC project from patient derived xenografts (PDXes) from respective patient tumours (NSC0474, NSC0387), or older cell lines (U2OS, SJSA-1 , IOR / OS18, IOR / OS15) available from the EuroBoNeT collection 56. Fibroblasts (BJ, MRC5) were from ATCC, immortalised mesenchymal cells were made in-house (iMSC3)57and primary NHOst osteoblasts were from Lonza.

[0133] Confocal microscopy

[0134] Cells were seeded on coverslips in 12-well plate sin a regular culture medium. Next day, the medium was replaced with a fresh culture medium containing donor serum at 1 :40 dilution. Cells were left in incubation at 37 degrees for 2 hours. After the incubation, cells were washed twice with PBS and fixed with 4% formaldehyde solution for 15 minutes; washed twice with PBS and permeabilized with 0.02% Tween-100 solution for 10 minutes; washed twice with PBS and blocked for 30 minutes with 2% BSA solution. Cells were incubated with anti-human IgG antibody overnight at 4 degrees. The day after, cells were either incubated for 2 hours at room temperature with primary antibodies against protein of interest, followed by secondary antibodies, or incubated for 30 minutes with Phalloidin. Finally, coverslips were mounted on the slides using Vectashield mounting medium with DAPI.

[0135] IgG isolation

[0136] IgG were purified from blood serum using Nab Protein G Spin Kit according to the Manufacturer protocol (Thermo scientific).

[0137] Example 1 : Sera from paediatric cancer patients are enriched in IgG against autoimmune disease biomarkers and proteins maintaining genome integrity

[0138] Osteosarcoma is more frequent in children and young people, with an incidence that peaks around 19 years of life and then flattens towards older ages (Fig. 1a). Our discovery cohorts consisted of 62 osteosarcoma patients between 2 and 76 years at diagnosis, and 30 healthy controls between 22 and 69 years. Serum samples were screened on high density protein arrays to determine the repertoire of circulating IgG autoantibodies. Large numbers of autoantibodies were detected in both cancer patients and healthy controls. After filtering out autoantibodies present in healthy controls, the osteosarcoma patients were divided into three age groups: 0-19 (paediatric),20-29(young adults),30-76(adult), and statistically significant clusters of autoantigens by gene set enrichment analysis (GSEA) were determined using the Canonical pathways collection17. Surprisingly, the DNA repair pathway was among the top enriched clusters in the 0-19 age group (P = 6.39E-06), while it was absent from the other groups. In the >30 group, the most significant categories were NOS3 and AURKA activation as well as mitotic division-related processes, and in the intermediate age group, the only enriched pathway was RNA polymerase II transcription.

[0139] To determine if these autoantibody patterns were specific for osteosarcoma or had more general significance, additional paediatric cohorts were analysed: glioma, neuroblastoma, Wilms’ tumour, and some rare cancers in the first year of life, as well as two additional age-spanning cohorts: rhabdomyosarcoma (RMS) and acute lymphoblastic leukaemia (ALL), plus two adult types of sarcomas: undifferentiated pleomorphic sarcoma (UPS) and dedifferentiated liposarcoma (LPS). Moreover, the healthy control cohort was extended by adding 18 new samples between 0.6-14 years of age. The analysis of the autoantigens that were most shared between paediatric cancer patients again showed an enrichment of the DNA repair pathway according to the GSEA Hallmark collection (P = 1.98E-04), while there was no such enrichment in the adults. For a more exhaustive analysis, a manual functional classification of all detected autoantigens was performed, based on information in the Human Protein Atlas18, GeneCards19, the MD Andersen list of DNA repair Genes*, and PubMed. Unlike previous reports on autoantigens in cancer, a large set of autoantigens were found that were shared not only between patients with the same cancer type, but across different paediatric and young patients (Figure 2). Among the autoantibodies that were most enriched and shared between paediatric patients were those against RAD52, a pivotal component in transcription-associated homologous recombination repair20, and HSPA13, a master regulator of plasma cell production and antibody secretion21, which was detected also in the cord blood of children who later developed cancer, but not in control samples. Two other clusters over-represented and highly shared in paediatric patients were a set of autoimmune disease-associated autoantibodies which are used as biomarkers in clinical diagnostics, and lipid metabolism, which was not detected in healthy controls (except for one in cord blood). Finally, a group of autoantigens were found with unrelated biological functions that also were widely shared by paediatric patients, but almost absent in the adult patients and controls (referred to as Autoantigen Group 4, Figure 2). Notably, antibodies to SRP19, in this latter group, and NUMA1 , in our core genome maintenance group, are involved in autoimmune disease2223Furthermore, a missense variant of the repair DNA polymerase p (POLB) in the core group, which is involved in the somatic processing of immunoglobulin genes, is associated with antinuclear antibodies and lupus-like disease in mice24.

[0140] The frequencies of the core list of 13 genome maintenance autoantigens detected in at least 10% of patients in at least 3 types of paediatric cancer are shown in Figure 2. Altogether, on average, paediatric and young patients had 5-fold more of these autoantigens per person than adult cancer patients and 7- fold more than healthy controls. In addition to the core list of highly shared autoantigens, we found 19 genome maintenance proteins that were shared at least by 5% of cases in two cancer types (summary data in Figure 2). Some of these autoantigens were occasionally observed in adult patients, mostly in those below 30 years of age.

[0141] For most cases, serum was collected before any treatment, thus avoiding therapy- induced autoantibodies. Some sarcoma patients received chemotherapy before serum sampling, as indicated in Figure 1 c, but this did not appear to have any systematic effect on the profiles. The lists of enriched autoantigens were compared with public tumour mutation data and with exome data available and it was found that only occasional proteins on our antigen lists were mutated in some cases of the corresponding tumour type.

[0142] This systematic and deep investigation of autoantibodies and their effect on cells in a large cohort of patients and healthy controls has revealed that paediatric patients sharply diverged from the adult patients and healthy controls in number and repertoire of autoantibodies targeting critical genome maintenance proteins. Thus, it has surprisingly been discovered that the enriched autoantibodies in the serum of patients with paediatric cancers can be used as biomarkers in order to aid prediction of and diagnosis of paediatric cancers.

[0143] Example 2: Autoantibodies from paediatric patients penetrate live cells with specific patterns of intracellular localisation

[0144] It is well documented that some autoantibodies can penetrate live cells and affect intracellular processes25’30. The enriched autoantibodies in our paediatric patients may serve a pathogenic role and this was tested here. The ability of IgG in the serum samples to penetrate live cells was investigated. In total serum from 65 patients and 30 controls were screened using normal and cancer cell lines. The sera were heat-treated to inactivate the complement system prior to the incubation and diluted 1 :40 for the initial tests and 1 :60 subsequently. The autoantibody uptake was concentrationdependent, but the 1 :10 dilutions revealed the same patterns as with 1 :40.

[0145] The first group of samples was analysed using a scanner microscope which allows visualisation of large areas with many cells. To be able to determine the subcellular localisation of penetrating autoantibodies, confocal microscopy was employed. Penetration into live cells was observed as a general phenomenon present both in patients and healthy controls regardless of age. However, profound differences were detected between serum from paediatric and adult patients or healthy controls. Only 33% of tested controls were positive, while the positivity reached 92% with pediatric patients and 62% in the adult ones.

[0146] The penetration capacity of autoantibodies was highly cell-type dependent, with BJ fibroblasts the most resistant. The most salient difference was nuclear and “Golgi-like” patterns of IgG staining, as well as a cytotoxic effect, with a cellular morphology compatible with pyroptosis or necroptosis, which were strictly paediatric patient-specific. All three patterns were detected also with purified patient IgG. In mesenchymal cells, fibroblasts, and osteoblasts, the cytoplasmic dots and “Golgi-like” patterns were observed in a larger proportion of cells, while nuclear and cytotoxic patterns were less frequent. The “Golgi-like” autoantibodies did not colocalize with Golgi protein 58K or Golgin-97 (GOLGA1). Similarly, the cytoplasmic dots did not colocalize with endosomal marker EEA1 . Only homogeneous and to a lesser extent dotted cytoplasmic staining was observed also in some controls. All positive cases gave penetration of only a small fraction of these cells. In addition to intracellular staining patterns that we consider relevant for the main hypothesis of interference with genome maintenance, we observed numerous patterns of penetrating autoantibodies that may be relevant to other processes or pathologies.

[0147] Example 3: Penetrating autoantibodies co-localize with their antigens or displace genome maintenance proteins

[0148] To assess whether the penetrating IgG can target and possibly inhibit their antigens, the colocalization of some of the genome maintenance proteins with internalized patient IgG were investigated. Of 5 proteins tested, the co-localization of antibodies specific to INO80E and EEPD1 were detected in three positive patient serum samples. The colocalization with INO80E and EEPD1 was detected in fibroblasts. The cells positive for EEPD1 appeared normal, but some of the fibroblasts positive for INO80E showed clear signs of cellular damage.

[0149] While investigating the co-localization, other phenotypes induced by the penetrating autoantibodies were observed. The nucleolar protein RSL1 D1 was completely dispersed from nucleoli in BJ fibroblasts penetrated by IgG from patient serum positive for that autoantibody. On the other hand, nuclear protein CGGBP1 , which was weakly expressed and dispersed in primary osteoblasts, formed bright foci in the nuclei of cells penetrated by IgG from a case positive for this autoantigen. Neither co-localization nor altered localisation of specific antigens were detected in cells treated with serum from healthy controls or adult cancer patients.

[0150] Thus, it is shown in Examples 2 and 3 that autoantibodies from paediatric patients can penetrate cells and co-localize with or alter the subcellular localization of genome maintenance proteins, as well as target other intracellular structures that were not observed in adult patients or healthy controls. Example 4: Cord blood from children who contracted cancer in their first two years of life had autoantibody profiles similar to paediatric cancer patients

[0151] Since maternal IgG are efficiently transported across the placenta during gestation, the autoantibody profiles of the smallest children in the cohort, although similar to those of older children, might be maternal of origin. To assess if such autoantibodies are present already at birth, the autoantibody profiles of umbilical cord blood were investigated from 15 children diagnosed with cancer in their first two years, and from 6 who did not contract cancer in their first 18 years. In most of the cases, autoantibodies typical of paediatric patients were found, although only two had many (Figure 1 e). We also found some novel autoantibodies that were only detected in cord blood from children later diagnosed with leukaemia.

[0152] Surprisingly, we detected a high percentage of positivity and one case with an elevated number of autoantibodies against DNA-repair proteins also in the control group.

[0153] Example 5: ELISA assessment of autoantibodies against antigens

[0154] High-binding ELISA plates (384 well, Greiner, Austria) were coated with target proteins (2 ng / pl, 10 pl, purchased from CDI labs, MD, US) followed by overnight incubation at 4°C. The plate was then washed once and blocked with 2 % BSA (in 1 x PBS). After blocking, the plate was washed once with PBST (0,05 % Tween-20) and incubated with sera (cases / control) at 1 :2 dilution for 1 .5 hr. at 37° C. After three washes, anti-human IgG (AF 488, 1 :1000, #A-11013, see confocal microscopy) was added, and incubated for 1 h at room temperature. After three washes, fluorescence was read using a green filter set (Excitation: 485 / 20, Emission: 528 / 20) in a BioTek Synergy H1 reader (Agilent Technologies, CA, US). Anti-CGGBP1 (1 :200, confocal microcopy) and 2% BSA was used as positive control and blank, respectively. The results are shown in Figures 3-4.

[0155] REFERENCES

[0156] 1. Shome, M., et al. Serum autoantibodyome reveals that healthy individuals share common autoantibodies. Cell Rep 39, 110873 (2022). 2. Kapsogeorgou, E.K. & Tzioufas, A.G. Autoantibodies in Autoimmune Diseases: Clinical and Critical Evaluation. Isr Med Assoc J 18, 519-524 (2016).

[0157] 3. Satoh, M., et al. Prevalence and sociodemographic correlates of antinuclear antibodies in the United States. Arthritis Rheum 64, 2319-2327 (2012).

[0158] 4. Nilsson, B.O., et al. Antinuclear antibodies in the oldest-old women and men. J Autoimmun 27, 281-288 (2006).

[0159] 5. Guo, Y.P., et al. The prevalence of antinuclear antibodies in the general population of china: a cross-sectional study. Curr Ther Res Clin Exp 76, 116-119 (2014).

[0160] 6. Hilario, M.O., et al. Frequency of antinuclear antibodies in healthy children and adolescents. Clin Pediatr (Phila) 43, 637-642 (2004).

[0161] 7. Marin, G.G., Cardiel, M.H., Cornejo, H. & Viveros, M.E. Prevalence of antinuclear antibodies in 3 groups of healthy individuals: blood donors, hospital personnel, and relatives of patients with autoimmune diseases. J Clin Rheumatol 15, 325-329 (2009).

[0162] 8. Wananukul, S., Voramethkul, W., Kaewopas, Y. & Hanvivatvong, O. Prevalence of positive antinuclear antibodies in healthy children. Asian Pac J Allergy Immunol 23, 153- 157 (2005).

[0163] 9. Lastwika, K.J., et al. Posttranslational modifications induce autoantibodies with risk prediction capability in patients with small cell lung cancer. Sci Transl Med 15, eadd8469 (2023).

[0164] 10. Didona, D., G, D.I.Z. & Joly, P. Paraneoplastic autoimmune multiorgan syndrome. Ital J Dermatol Venerol 156, 174-183 (2021).

[0165] 11. Gastaldi, M., et al. Paraneoplastic Neurological Syndromes: Transitioning Between the Old and the New. Curr Oncol Rep 24, 1237-1249 (2022).

[0166] 12. Fiorentino, D.F. & Casciola-Rosen, L. Autoantibodies and Cancer Association: the Case of Systemic Sclerosis and Dermatomyositis. Clin Rev Allergy Immunol (2022).

[0167] 13. Chmielecki, J., et al. Genomic Profiling of a Large Set of Diverse Pediatric Cancers Identifies Known and Novel Mutations across Tumor Spectra. Cancer Research 77(2017).

[0168] 14. Grdbner, S.N., et al. The landscape of genomic alterations across childhood cancers. Nature 555, 321-327 (2018).

[0169] 15. Ma, X., et al. Pan-cancer genome and transcriptome analyses of 1 ,699 paediatric leukaemias and solid tumours. Nature 555, 371-376 (2018). 16. Zhang, J., et al. Germline Mutations in Predisposition Genes in Pediatric Cancer. N Engl J Med 373, 2336-2346 (2015).

[0170] 17. Subramanian, A., et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102, 15545- 15550 (2005).

[0171] 18. Thul, P.J., et al. A subcellular map of the human proteome. Science 356(2017).

[0172] 19. Safran, M., et al. The GeneCards Suite, in Practical Guide to Life Science Databases (eds. Abugessaisa, I. & Kasukawa, T.) 27-56 (Springer Nature Singapore, Singapore, 2021).

[0173] 20. Yasuhara, T., et al. Human Rad52 Promotes XPG-Mediated R-loop Processing to Initiate Transcription-Associated Homologous Recombination Repair. Cell 175, 558-570 e511 (2018).

[0174] 21. He, Y., et al. Hspa13 Promotes Plasma Cell Production and Antibody Secretion. Front Immunol 11 , 913 (2020).

[0175] 22. Arcani, R., et al. Anti-NuMA antibodies: clinical associations and significance in patients with primary Sjogren's syndrome or systemic lupus erythematosus. Rheumatology (Oxford) 60, 4074-4084 (2021).

[0176] 23. Wang, Q., Duan, F., Liu, P., Wang, P.F. & Wang, M.X. Expression of anti-SRP19 antibody in muscle tissues from patients with autoimmune necrotizing myopathy. Genet Mol Res 15 (2016).

[0177] 24. Sawyer, D.L. & Sweasy, J.B. DNA Polymerase beta in the Context of Cancer. Crit Rev Oncog 27, 17-33 (2022).

[0178] 25. Alarcon-Segovia, D., Ruiz-Arguelles, A. & Fishbein, E. Antibody to nuclear ribonucleoprotein penetrates live human mononuclear cells through Fc receptors. Nature 271 , 67-69 (1978).

[0179] 26. Turchick, A., Liu, Y., Zhao, W., Cohen, I. & Glazer, P.M. Synthetic lethality of a cell penetrating anti-RAD51 antibody in PTEN-deficient melanoma and glioma cells. Oncotarget 10, 1272-1283 (2019).

[0180] 27. Hansen, J.E., et al. Targeting cancer with a lupus autoantibody. Sci Transl Med 4, 157ra142 (2012). 28. Noble, P.W., Young, M.R., Bernatsky, S., Weisbart, R.H. & Hansen, J.E. A nucleolytic lupus autoantibody is toxic to BRCA2 -deficient cancer cells. Sci Rep 4, 5958 (2014).

[0181] 29. Liu, H., Zhang, Y., Liu, H., Huang, Q. & Ying, Y. Anticentromere antibody induced by immunization with centromere protein and Freund's complete adjuvant may interfere with mouse early-stage embryo. Reprod Biol Endocrinol 19, 127 (2021).

[0182] 30. Golan, T.D., Gharavi, A.E. & Elkon, K.B. Penetration of autoantibodies into living epithelial cells. J Invest Dermatol 100, 316-322 (1993).

[0183] 31. Noble, P.W., et al. DNA-damaging autoantibodies and cancer: the lupus butterfly theory. Nat Rev Rheumatol 12, 429-434 (2016).

[0184] 32. Turchick, A., Hegan, D.C., Jensen, R.B. & Glazer, P.M. A cell-penetrating antibody inhibits human RAD51 via direct binding. Nucleic Acids Res 45, 11782-11799 (2017).

[0185] 33. Vlahakos, D., et al. Murine monoclonal anti-DNA antibodies penetrate cells, bind to nuclei, and induce glomerular proliferation and proteinuria in vivo. J Am Soc Nephrol 2, 1345-1354 (1992).

[0186] 34. Weisbart, R.H., et al. DNA-dependent targeting of cell nuclei by a lupus autoantibody. Sci Rep 5, 12022 (2015).

[0187] 35. Huang, K.L., et al. Pathogenic Germline Variants in 10,389 Adult Cancers. Cell 173, 355-370 e314 (2018).

[0188] 36. Ge, Q., et al. Antinuclear antibodies in healthy population: Positive association with abnormal tissue metabolism, inflammation and immune dysfunction. Int Immunopharmacol 113, 109292 (2022).

[0189] 37. Thatikonda, V., et al. Comprehensive analysis of mutational signatures reveals distinct patterns and molecular processes across 27 pediatric cancers. Nat Cancer (2023).

[0190] 38. Sali, A.D., Karakasiliotis, I., Evangelidou, M., Avrameas, S. & Lymberi, P. Immunological evidence and regulatory potential for cell-penetrating antibodies in intravenous immunoglobulin. Clin Transl Immunology 4, e42 (2015).

[0191] 39. Biermann, M.H., et al. The role of dead cell clearance in the etiology and pathogenesis of systemic lupus erythematosus: dendritic cells as potential targets. Expert Rev Clin Immunol 10, 1151-1164 (2014).

[0192] 40. Waidhauser, J., Schuh, A., Trepel, M., Schmalter, A.K. & Rank, A. Chemotherapy markedly reduces B cells but not T cells and NK cells in patients with cancer. Cancer Immunol Immunother 69, 147-157 (2020).

[0193] 41. Lee, J.Y., et al. Neurotoxic autoantibodies mediate congenital cortical impairment of offspring in maternal lupus. Nat Med 15, 91-96 (2009).

[0194] 42. Fox, E., Amaral, D. & Van de Water, J. Maternal and fetal antibrain antibodies in development and disease. Dev Neurobiol 72, 1327-1334 (2012).

[0195] 43. Wahren-Herlenius, M. & Sonesson, S.E. Specificity and effector mechanisms of autoantibodies in congenital heart block. Curr Opin Immunol 18, 690-696 (2006).

[0196] 44. Greeley, S.A., et al. Elimination of maternally transmitted autoantibodies prevents diabetes in nonobese diabetic mice. Nat Med 8, 399-402 (2002).

[0197] 45. Farnam, J., Lavastida, M.T., Grant, J.A., Reddi, R.C. & Daniels, J.C. Antinuclear antibodies in the serum of normal pregnant women: a prospective study. J Allergy Clin Immunol 73, 596-599 (1984).

[0198] 46. Ballinger, M.L., Mitchell, G. & Thomas, D.M. Surveillance recommendations for patients with germline TP53 mutations. Current opinion in oncology 27, 332-337 (2015).

[0199] 47. Stepanov, A.V., et al. Design of targeted B cell killing agents. PLoS One 6, e20991 (2011).

[0200] 48. Havasi, A., et al. Blocking peptides and molecular mimicry as treatment for kidney disease. Am J Physiol Renal Physiol 312, F1016-F1025 (2017).

[0201] 49. Karnaukhov, V., et al. HLA variants have different preferences to present proteins with specific molecular functions which are complemented in frequent haplotypes. Front Immunol 13, 1067463 (2022).

[0202] 50. Miller, M.B., et al. Somatic genomic changes in single Alzheimer's disease neurons. Nature 604, 714-722 (2022).

[0203] 51. Erickson, R.P. Somatic gene mutation and human disease other than cancer: an update. Mutat Res 705, 96-106 (2010).

[0204] 52. Poduri, A., Evrony, G.D., Cai, X. & Walsh, C.A. Somatic mutation, genomic variation, and neurological disease. Science 341 , 1237758 (2013).

[0205] 53. Gollob, M.H., et al. Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation. N Engl J Med 354, 2677-2688 (2006).

[0206] 54. Magnus, P., et al. Cohort Profile Update: The Norwegian Mother and Child Cohort Study (MoBa). Int J Epidemiol 45, 382-388 (2016). 55. Madsen, A., et al. Reference Curves for Pediatric Endocrinology: Leveraging Biomarker Z-Scores for Clinical Classifications. J Clin Endocrinol Metab 107, 2004-2015 (2022).

[0207] 56. Ottaviano, L., et al. Molecular characterization of commonly used cell lines for bone tumor research: A trans-European EuroBoNet effort. Genes, Chromosomes and Cancer 49, 40-51 (2010).

[0208] 57. Skarn, M., et al. Generation and characterization of an immortalized human mesenchymal stromal cell line. Stem Cells Dev 23, 2377-2389 (2014).

Claims

CLAIMS1 . A method of detecting or predicting cancer in a subject, the method comprising the step:(a) determining, from a biological sample obtained from:(i) the subject;(ii) the subject’s mother during the peripartum period; or(iii) the subject’s mother whilst pregnant with the subject; the presence of autoantibodies against all of the following subject’s (self) antigens or proteins: APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, ACOT12, CPNE6, CRAT, PLCD4, DHX58, ELOB / TCEB2 and PRDX2; wherein the presence of autoantibodies against all of the selected antigens or proteins within the biological sample is indicative of the presence of a cancer in the subject or is predictive of the future development of a cancer in the subject.

2. A method of detecting or predicting cancer in a subject, the method comprising the step:(a) determining, from a biological sample obtained from:(i) the subject;(ii) the subject’s mother during the peripartum period; or(iii) the subject’s mother whilst pregnant with the subject; the presence of autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen, Sm Antigen, ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CPNE6, CRAT, PLCD4, DHX58, DMD, HSPA13, SRP19, ELOB / TCEB2, CGGBP1 and CCT4, and optionally also PRDX2, wherein the presence of autoantibodies against the selected antigens or proteins within the biological sample is indicative of the presence of a cancer in the subject or is predictive of the future development of a cancer in the subject.

3. A method as claimed in claim 2, wherein the subject’s (self) antigens or proteins are or are selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen, Sm Antigen, ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CPNE6, CRAT, PLCD4, DHX58, DMD, HSPA13, SRP19, and ELOB / TCEB2, and optionally also PRDX2.

4. A method as claimed in claim 3, wherein the plurality of the subject’s (self) antigens or proteins is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27 of the subject’s (self) antigens or proteins, preferably at least 5, 10, 15, 20 or 27, more preferably at least 26 or 27 of the subject’s (self) antigens or proteins.

5. A method as claimed in claim 2, wherein the subject’s (self) antigens or proteins are or are selected from the group consisting of APBBI , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, ACOT12, CPNE6, CRAT, PLCD4, DHX58, ELOB / TCEB2, PRDX2, CGGBP1 and CCT4.

6. A method as claimed in claim 2, wherein the subject’s (self) antigens or proteins are or are selected from the group consisting of APBBI , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, ACOT12, CPNE6, CRAT, PLCD4, DHX58, ELOB / TCEB2 and PRDX2.

7. A method as claimed in claim 6, wherein the plurality of the subject’s (self) antigens or proteins is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 or 17 of the subject’s (self) antigens or proteins, preferably at least 5, 10, 15, more preferably 16 or all 17 of the subject’s (self) antigens or proteins.

8. A method as claimed in any one of claims 2-7, wherein the selected antigens or proteins are all tumour suppressor proteins.

9. A method as claimed in claim 2 or claim 3, wherein the selected antigens or proteins are all tumour suppressor proteins and wherein the tumour suppressor proteinsare or are selected from the group consisting of APBB1 , TP53I3, ACOT12, CRAT, PLCD4 and DMD.

10. A method as claimed in claim 2, wherein:(i) the plurality of the subject’s (self) antigens or proteins are at least 5 antigens or proteins, preferably wherein the selected antigens or proteins are or comprise RAD52, UBE2N, CRAT, PLCD4 and HSPA13;(ii) the plurality of the subject’s (self) antigens or proteins are at least 10 antigens or proteins, preferably wherein the selected antigens or proteins are or comprise APBB1 , NUMA1 , RAD52, UBE2N, ACOT12, CRAT, PLCD4, DHX58, HSPA13 and SRP19;(iii) the plurality of the subject’s (self) antigens or proteins are at least 15 antigens or proteins, preferably wherein the selected antigens or proteins are or comprise ALKBH7, APBB1 , DMC1 , MSH5, NUMA1 , RAD52, TP53I3, UBE2N, ACOT12, CRAT, PLCD4, DMD, DHX58, HSPA13 and SRP19;(iv) the plurality of the subject’s (self) antigens or proteins are at least 20 antigens or proteins, preferably wherein the selected antigens or proteins are or comprise ALKBH7, APBB1 , DMC1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, UBE2N, ACOT12, CRAT, PLCD4, DMD, DHX58, HSPA13, SRP19, TCEB2 and ADRM1 ; or(v) the plurality of the subject’s (self) antigens or proteins are all of the antigens or proteins listed in claim 1 (a).

11. A method as claimed in claim 2 or claim 3, wherein the antigens or proteins are or are selected from the group consisting of ADRM1 , ALKBH7, APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, and UBE2N, and optionally also PRDX2.

12. A method as claimed in claim 2 or claim 3, wherein the plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of ACOT12, CPNE6, CRAT and PLCD4.

13. A method as claimed in claim 2 or claim 3, wherein the plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of Jo-1 antigen, Sm / RNP Complex, Scl-70 Antigen and Sm Antigen.

14. A method as claimed in claim 2 or claim 3, wherein the plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of DHX58, DMD, HSPA13, SRP19 and ELOB / TCEB2.

15. A method as claimed in any one of the preceding claims, wherein the biological sample is a blood sample, preferably blood serum or blood plasma.

16. A method as claimed in any one of the preceding claims, wherein the subject is a human subject, preferably a paediatric subject.

17. A method as claimed in claim 16, wherein the subject is a foetus, neonate, infant or adolescent.

18. A method as claimed in any one of the preceding claims, wherein the cancer is a paediatric cancer, preferably selected from the group consisting of blood, brain, spinal cord, lymphatic system, muscle, bone, nerve cells, kidney and eye cancers.

19. A method as claimed in claim 18, wherein the cancer is neuroblastoma, glioma, Wilms’ tumour, leukaemia, osteosarcoma, rhabdomyosarcoma or Ewing’s sarcoma.

20. A method as claimed in claim 18, wherein:(i) the plurality of antigens or proteins are or are selected from the group consisting of Scl-70 Antigen, RAD52, PLCD4, HSPA13 and SPR19, and the cancer is neuroblastoma;(ii) the plurality of antigens or proteins are or are selected from the group consisting of Scl-70 Antigen, RAD52, CRAT, PLCD4 and HSPA13, and the cancer is glioma;(iii) the plurality of antigens or proteins are or are selected from the group consisting ofScl-70 Antigen, MSH5, UBE2N, CRAT, DMD and HSPA13, and the cancer is Wilms’ tumour;(iv) the plurality of antigens or proteins are or are selected from the group consisting of CRAT, DHX58 and DMD, and the cancer is leukaemia, preferably ALL; or(v) the plurality of antigens or proteins are or are selected from the group consisting of Scl-70 Antigen, TP53I3, ACOT12 and CRAT, and the cancer is osteosarcoma, rhabdomyosarcoma or Ewing’s sarcoma, preferably osteosarcoma or rhabdomyosarcoma.21 . A method of detecting or predicting leukaemia in an adult subject, the method comprising the step:(a) determining, from a biological sample obtained from the adult subject, the presence of autoantibodies against a plurality of the subject’s (self) proteins selected from the group consisting of ALKHB7, APBB1 , MSH5, NUMA1 , POLR2H, RAD52, RCHY1 , CRAT, DMD and HSPA13, wherein the presence of autoantibodies against the selected proteins within the biological sample is indicative of the presence of leukaemia in the subject or is predictive of the future development of leukaemia in the subject.

22. A method of obtaining information relevant to the diagnosis of cancer in a subject, the method comprising the step:(a) determining, from a biological sample obtained from:(i) the subject;(ii) the subject’s mother during the peripartum period; or(iii) the subject’s mother whilst pregnant with the subject; the presence of autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from one of the groups as defined in any one of claims 1 -6.

23. A method of preventing cancer in a subject, the method comprising the steps:(a) determining, from a biological sample obtained from:(i) the subject;(ii) the subject’s mother during the peripartum period; or(iii) the subject’s mother whilst pregnant with the subject; the presence of autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from one of the groups as defined in any one of claims 1 -6, wherein the presence of autoantibodies against the selected antigens or proteins within the biological sample is predictive of the future development of a cancer in the subject; and(b) if a prediction of the future development of a cancer in the subject has been obtained, administering to the subject a prophylactic treatment appropriate for preventing the cancer.

24. A method of preventing the future development of a cancer in a subject, the method comprising the steps of:(a) administering a prophylactic treatment appropriate for preventing the future development of a cancer to the subject, wherein, prior to administration, an indication of the future development of a cancer in the subject, has been obtained by a method comprising the steps of(b) determining, from a biological sample obtained from:(i) the subject;(ii) the subject’s mother during the peripartum period; or(iii) the subject’s mother whilst pregnant with the subject; the presence of autoantibodies against a plurality of the subject’s (self) antigens or proteins selected from one of the groups as defined in any one of claims 1 -6, wherein the presence of autoantibodies against the selected antigens or proteins within the biological sample is predictive of the future development of a cancer in the subject.

25. A method as claimed in any one of claims 20-22, wherein the plurality of the subject’s (self) antigens or proteins are or are selected from the group consisting of APBB1 , DMC1 , HMGN1 , MSH5, NUMA1 , POLB, POLR2H, RAD52, RCHY1 , TP53I3, ACOT12, CPNE6, CRAT, PLCD4, DHX58, ELOB / TCEB2 and PRDX2, and optionally also CGGBP1 and CCT4.

Images