Antigen neuron-specific enolase peptide for the diagnosis and treatment of autism

Abstract

This disclosure provides peptides that specifically bind to maternal autoantibodies produced in mothers or potential mothers against neuron-specific enolase (NSE) proteins targeting endogenous polypeptide antigens. The peptides described herein can be used to determine the risk of autism spectrum disorder (ASD) in offspring by detecting the presence of maternal autoantibodies in biological samples from mothers or potential mothers. The peptides or their mimic epitopes can also be administered to mothers or potential mothers to neutralize maternal autoantibodies by blocking the binding between maternal autoantibodies and their antigens.

Description

[0001] Cross-referencing of related applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 62 / 940175, filed November 25, 2019, which is incorporated herein by reference in its entirety for all purposes.

[0003] [Statement regarding rights to inventions developed through government-sponsored research or development]

[0004] This invention was completed with government funding from the National Institutes of Health (NIH), grant number 2P01ES011269-11. The government holds certain rights to this invention. [Background Technology]

[0005] Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders diagnosed early in childhood, classified according to the loss of social skills and communication abilities, as well as the presence of repetitive and restricted interests and behaviors. In 2018, the Centers for Disease Control and Prevention (CDC) estimated that 1 in 59 children in the United States is affected, making ASD a significant health problem and a substantial socioeconomic burden on affected families and healthcare systems. Currently available treatment interventions for ASD are behavior-oriented or symptom-based pharmacological therapies, applied only after diagnosis. Little is known about the causes of ASD, and while some treatments applied after early diagnosis have shown promise, there are currently no preventative alternatives.

[0006] It is known that activation of the maternal immune system in early fetal development can negatively impact brain development. For reasons unknown, some pregnant women's immune systems produce autoantibodies (proteins produced by the immune system in response to components of their own tissues), which may mistakenly identify parts of the fetal brain as foreign. Therefore, exposure to these maternal autoantibodies during pregnancy may lead to alterations in the neurodevelopmental characteristics of ASD. In fact, 23% of mothers who give birth to children with autism have circulating autoantibodies against seven proteins highly expressed in the developing brain, compared to only 1% of mothers who give birth to typically developing children. Each of these proteins is known to play a crucial role in neurodevelopment; interference with the level or function of more than one of them may synergistically alter the trajectory of brain development. See U.S. Patent No. 8,383,360.

[0007] Therefore, there is a need for early, non-genetic, epitope-specific biomarkers to determine maternal risk of ASD in children. Furthermore, there is an urgent need to address the etiology and treatment of ASD, not just its symptoms, by creating highly specific therapies and / or intervention tools. Early identification of these maternal autoantibodies in affected mothers will allow for early medical interventions to limit fetal exposure to these autoantibodies and the resulting risk of ASD in their children, thereby reducing the prevalence of ASD and improving the quality of life for children and their families who would otherwise be affected. This disclosure addresses these needs and provides relevant advantages. [Summary of the Invention]

[0008] This disclosure provides peptides (e.g., peptide epitopes) that specifically bind to maternal autoantibodies generated in mothers or potential mothers against neuron-specific enolase (NSE) peptides. The peptides described herein can be used to determine the risk of autism spectrum disorder (ASD) in offspring by detecting the presence of maternal autoantibodies in a biological sample from a mother or potential mother. The peptides can also be administered to the mother or potential mother to neutralize maternal autoantibodies by blocking the binding between maternal autoantibodies and their antigens. Furthermore, these peptides can be used for immunoadsorption to remove circulating autoantibodies from maternal plasma.

[0009] In a first aspect, this document provides an isolated peptide having at least about 80% sequence identity with any one of SEQ ID NOS: 1-6. In some embodiments, the peptide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous amino acids of any one of SEQ ID NOS: 1-6. In some embodiments, the peptide is bound to a parent antibody that binds to a neuron-specific enolase (NSE) protein.

[0010] In some embodiments, the peptide is about 15 to about 30 amino acids in length. In some embodiments, the peptide is more than about 25 amino acids in length. In a particular embodiment, the peptide comprises an amino acid sequence consisting of any one of SEQ ID NOS: 1-6.

[0011] In some embodiments, the peptide is a mimic epitope. In some embodiments, the mimic epitope comprises a D-amino acid. In other embodiments, the mimic epitope comprises one or more amino acid modifications (e.g., substitutions) relative to any one of SEQ ID NOS:1-6.

[0012] In some embodiments, the peptide further comprises a label, such as a biotin, fluorescent label, chemiluminescent label, and radioactive label. In other embodiments, the label is attached (e.g., covalently attached) to the peptide.

[0013] In another aspect, this disclosure provides a composition comprising the peptides or multiple peptides described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In particular embodiments, the peptides or multiple peptides in the composition are selected from the group consisting of SEQ ID NOS:1-6. In particular embodiments, the multiple peptides comprise at least 2, 3, 4, 5, or 6 different peptides. In some embodiments, the different peptides bind to the same parent antibody (e.g., an antibody against NSE).

[0014] In another aspect, this disclosure provides a kit comprising the peptide or multiple peptides described herein and a solid support. In some embodiments, the solid support is a multi-well plate, ELISA plate, microarray, chip, bead, porous tape, or nitrocellulose filter. In some embodiments, the peptide or multiple peptides are immobilized (e.g., covalently attached to) the solid support. In particular embodiments, the peptide or multiple peptides are selected from the group consisting of SEQ ID NOS:1-6. In particular embodiments, the multiple peptides comprise at least 2, 3, 4, 5, or 6 different peptides. In some embodiments, the different peptides bind to the same parent antibody (e.g., an antibody against NSE).

[0015] In some embodiments, the kit also includes instructions for use. In some cases, the instructions for use include instructions on contacting the solid carrier with a biological sample from a mother or potential mother. In other cases, the instructions for use include instructions on the association between the presence of maternal antibodies bound to one or more peptides and an increased risk of ASD in offspring, such as a fetus or child. In other embodiments, the kit also includes a labeled secondary antibody for detecting the presence of maternal antibodies bound to one or more peptides.

[0016] In other embodiments, the kit also includes negative and positive control samples. In some cases, the negative control sample is obtained from the mother of a child with normal development (TD). In other cases, the biological sample and / or control sample responds to full-length NSE. In still other cases, neither the biological sample nor the control sample responds to full-length NSE. In further embodiments, the kit also includes a secondary antibody directly or indirectly labeled with the detectable moiety.

[0017] In another aspect, this disclosure provides a method for determining the risk of autism spectrum disorder (ASD) in offspring, the method comprising: detecting in a biological sample from the mother or potential mother of the offspring the presence of maternal antibodies that bind to one or more peptides described herein, wherein the presence of maternal antibodies that bind to one or more peptides indicates an increased risk of ASD in the offspring.

[0018] In some embodiments of the method, the method further includes obtaining a sample from the mother or potential mother. In some embodiments, the sample is selected from the group consisting of blood, serum, plasma, amniotic fluid, breast milk, and saliva. In some embodiments, the peptide or multiple peptides are selected from the group consisting of SEQ ID NOS:1-6. In some embodiments of the method, the multiple peptides comprise at least 2, 3, 4, 5, or 6 different peptides. In particular embodiments, the different peptides bind to the same maternal antibody, for example, an antibody against NSE. In some embodiments of the method, the peptide or multiple peptides are attached to a solid carrier, such as a multiwell plate, ELISA plate, microarray, chip, beads, porous tape, or nitrocellulose filter.

[0019] In some embodiments of this method, the parent antibody is detected by techniques such as Western blot, dot blot, ELISA, radioimmunoassay, immunoprecipitation, electrochemiluminescence, immunofluorescence, FACS analysis, or multiplex bead analysis.

[0020] In some embodiments of this method, the presence of maternal antibodies in the test sample (i.e., a biological sample from the mother or potential mother) is not compared with a control sample. In other embodiments, the test sample is compared with a positive or negative control sample. In some cases, the test sample and / or control sample respond to full-length NSE. In other cases, neither the test sample nor the control sample responds to full-length NSE. In still other cases, the negative control is obtained from the mother of a child with TD. In some embodiments of this method, the mother or potential mother has a child with ASD. In some embodiments, the mother or potential mother has a family history of ASD or an autoimmune disease.

[0021] In another aspect, this disclosure provides a method for preventing or reducing the risk of autism spectrum disorder (ASD) in offspring, the method comprising: administering a therapeutically effective amount of the peptide or multiple peptides described herein to the mother or potential mother of the offspring, wherein the peptide or multiple peptides bind to antibodies circulating in the mother or potential mother to form a neutralizing complex, thereby preventing or reducing the risk of ASD in the offspring.

[0022] In some embodiments of the method, the method further includes removing the neutralizing complex from the mother or potential mother. In some embodiments of the method, the neutralizing complex is removed using affinity plasma purification technology.

[0023] In some embodiments of this method, the peptide or multiple peptides are administered intravenously. In some embodiments, the peptide or multiple peptides are selected from the group consisting of SEQ ID NOS:1-6. In particular embodiments, the multiple peptides comprise at least 2, 3, 4, 5, or 6 different peptides. In some embodiments, the different peptides bind to the same parent antibody, for example, an antibody against NSE.

[0024] Other objects, features, and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description. [Attached Image Description]

[0025] Figure 1A-1D Western blot (WB) was used to detect protein blots in the fetal monkey brain (FMB) using maternal plasma. Figure 1A Ponceau S-stained nitrocellulose membranes, comprising a sample of the first component collected from the prep cell isolate of FMB, and samples of every tenth component thereafter. Figure 1B ) Figure 1A The Western blot of the replicated membrane was detected using a pool of maternal plasma that responded to antigens of 37 kDa (LDH), 39 kDa (YBX1), 44 kDa (GDA), and 73 kDa (STIP1, CRMP1 / 2). Figure 1C Prep Cell fraction, containing proteins between 39-42 kDa. Fraction #12 is used for 2D gel electrophoresis. Figure 1D WB of FMB component #12 was detected using maternal plasma, which was unresponsive to LDHA-B, GDA, and YBX1. Channel 1: Secondary antibody control only; Channels 2-4: Maternal plasma showed reactivity to LDHA-B (green arrow), YBX1 (blue arrow), and GDA (black arrow). Channels 5-8: Maternal plasma pool #1, showing band reactivity to proteins near 39 kDa. Channels 10-14: Maternal plasma pool #2, showing band reactivity to two proteins near 37 and 39 kDa. Channel 9: Negative control of FMB antigen plasma sample. Abbreviations: FMB, fetal monkey brain; LDH A and B, lactate dehydrogenases A and B; YBX1, Y-box binding protein 1; GDA, guanine deaminase; CRMP1 and CRMP2, collapse reaction mediators 1 and 2; STIP1, stress-induced phosphorylated protein 1.

[0026] Figures 2A-2E Two-dimensional (2-D) gel electrophoresis and antigen selection for mass spectrometry analysis. Figure 2A Depicting the use of protein and FMB component #12 ( Figure 2B Anti-IgG staining gels compared with plasma pool 1 and plasma pool 2 ( Figure 2CImprinted membrane. Figure 2D Depicting Figure 2B and Figure 2C The merged image. Figure 2E Western blots (WB) of proteins bound by maternal IgG antibodies (mixed plasma 1 and 2) were performed, with each antibody labeled with a spot number. A total of 27 protein spots were selected and subsequently analyzed by mass spectrometry.

[0027] Figure 3 A heatmap of sequences with a mean reactivity (FI) greater than 50 for ELISA-positive samples. A sample is considered positive if FI > 200. Red letters indicate amino acid residues that are part of the major epitopes in ES 293-297, and ES 408-410 indicate amino acid sequences recognized only by the ASD group. The histogram on the right represents the reactivity of FI. Abbreviations: ES, epitope sequence; Autism spectrum disorder; TD, normal development; FI, fluorescence intensity.

[0028] Figure 4 The following describes the workflow of the methods used in the examples. The first three steps are used to identify proteins between 37-45 kDa that react with maternal plasma from mothers of children with ASD. The fourth step leads to the identification of target antigens (including NSE). The following steps demonstrate antigen characterization in the context of MAR ASD biomarkers.

[0029] Figure 5 A sequence heatmap of ELISA-negative samples with a mean reactivity (FI) greater than 50. A sample is considered positive if FI > 200. Amino acid residues that are part of the major epitope are highlighted in red. The histogram on the right represents the reactivity of FI. Abbreviations: ES, epitope sequence; ASD, autism spectrum disorder; TD, normal development; FI, fluorescence intensity.

Detailed Implementation Methods

[0030] I. Introduction

[0031] Autism spectrum disorder (ASD) is a significant health concern characterized by social and behavioral impairments, as well as restricted interests and repetitive behaviors. Previous research has identified maternal autoantibody-associated (MAR) autism as being associated with approximately 23% of ASD cases. Seven MAR-specific autoantigens have previously been identified, including CRMP1, CRMP2, GDA, LDHA, LDHB, STIP1, and YBX1, see, for example, International Patent Publication WO 2016 / 210137, the full text of which is incorporated herein by reference. The epitope peptide sequences recognized by maternal autoantibodies for each of the seven ASD-specific autoantigens have also been described.

[0032] This disclosure relates to other antigens recognized by ASD-specific maternal autoantibodies, and to the mapping of unique ASD-specific epitopes using microarray technology. Embryonic rhesus monkey brain tissue was isolated by molecular weight separation, and components containing bands between 37 and 45 kDa were analyzed by two-dimensional gel electrophoresis, followed by peptide mass spectrometry mapping using MALDI-TOF MS and TOF / TOF tandem MS / MS. Using this method, neuron-specific enolases (NSEs) were identified as target autoantigens and selected for epitope mapping. The complete NSE sequence was translated into a 15-mer peptide with 14 overlapping amino acids, placed on a microarray slide, and detected using maternal plasma from mothers of children with ASD and mothers of normally developing children (TD) (ASD = 27 and TD = 21). The obtained data were analyzed using a T-test. Using both T-test and SAM T-test, a total of 16 ASD-specific NSE peptide sequences were identified, of which four sequences were statistically significant (p<0.05): DVAASEFYRDGKYDL (SEQ ID NO:1) (SEQ ID NO:1) (p=0.047; SAM score 1.49), IEDPDQDDWAAWSK (SEQ ID NO:2) (SEQ ID NO:2) (p=0.049; SAM score 1.49), ERLAKYNQLMRIEEE (SEQ ID NO:3) (SEQ ID NO:3) (p=0.045; SAM score 1.57) and RLAKYNQLMRIEEEL (SEQ ID NO:4) (p=0.017; SAM score 1.82). All ASD-specific NSE peptide sequences had an odds ratio (OR) greater than 3, with SERLAKYNQLMRIEE (SEQ ID NO:6) (OR 10.1, C1 95% 0.5094 to 200.7) and ERLAKYNQLMRIEEE (SEQ ID NO:3) (OR 12.6, C1 95% 0.6408 to 247.7) being the two epitopes with the highest ORs. Five sequences were found to be recognized by both ASD and TD antibodies, indicating the presence of a large immunodominant epitope (DYPVVSIEDPFDQDDWAAW (SEQ ID NO:5)). Although both mothers of children with ASD and mothers of children with TD have maternal autoantibodies against NSE proteins, several ASD-specific epitopes may serve as biomarkers for MAR ASD.

[0033] II. Definition

[0034] As used herein, unless otherwise specified, the following terms have the meanings assigned to them.

[0035] The terms "autism spectrum disorder," "autism," or "ASD" refer to a range of neurodevelopmental disorders characterized by impairments in social interaction and communication, accompanied by repetitive and stereotyped behaviors. Autism encompasses a range of social interaction and communication impairments; however, based on the severity of these impairments, autism can be broadly categorized into "high-functioning autism" or "low-functioning autism." Individuals diagnosed with "high-functioning autism" have the mildest but identifiable social and communication impairments (i.e., Asperger's syndrome). More information about autism spectrum disorders can be found in, for example, Autism Spectrum Disorders: A Research Review for Practitioners, Ozonoff, et ah, eds., 2003, American Psychiatric Pub; Gupta, Autistic Spectrum Disorders in Children, 2004, Marcel Dekker Inc; Hollander, Autism Spectrum Disorders, 2003, Marcel Dekker Inc; Handbook of Autism and Developmental Disorders, Volkmar, ed., 2005, John Wiley; Sicile-Kira and Grandin, Autism Spectrum Disorders: The Complete Guide to Understanding Autism, Asperger's Syndrome, Pervasive Developmental Disorder, and Other ASDs, 2004, Perigee Trade; and Duncan, et al., Autism Spectrum Disorders [TwoVolumes]: A Handbook for Parents and Found in Professionals, 2007, and Praeger.

[0036] The terms “normal development” and “TD” refer to subjects who have not been diagnosed with autism spectrum disorder (ASD). Typically, normally developing children do not exhibit ASD-related impairments in communication, social interaction, or repetitive and / or stereotyped behaviors of a severity typically associated with an ASD diagnosis. While normally developing children may exhibit some behaviors displayed by children diagnosed with ASD, they do not exhibit behavioral clusters and / or severity that would support an ASD diagnosis.

[0037] When used for nucleic acids or proteins, the term "isolated" means that the nucleic acid or protein is substantially free of other cellular components associated with it in its natural state. It is preferably in a homogeneous state. It can be in a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. Proteins present as the main species in the formulation are essentially purified. In particular, the isolated gene is separated from the open reading frames flanking the gene and encoding proteins different from the gene of interest. The term "purified" means that the nucleic acid or protein essentially produces a single band in the electrophoresis gel. In particular, this means that the purity of the nucleic acid or protein is at least 85%, at least 95%, or at least 99%.

[0038] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term includes nucleic acids containing known natural nucleotide analogs that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, homologs, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or DNA residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0039] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues or the assembly of multiple polymers of amino acid residues. These terms apply to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as both naturally occurring and non-naturally occurring amino acid polymers.

[0040] The term "amino acid" includes naturally occurring α-amino acids and their stereoisomers, as well as non-natural (non-naturally occurring) amino acids and their stereoisomers. A "stereoisomer" of an amino acid refers to its mirror image, such as an L-amino acid or a D-amino acid. For example, a stereoisomer of a naturally occurring amino acid refers to its mirror image, namely a D-amino acid.

[0041] Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0042] Non-natural (not naturally occurring) amino acids include, but are not limited to, amino acid analogs, amino acid mimics, synthetic amino acids, N-substituted glycine, and N-methyl amino acids, which have an L- or D-configuration and function similarly to naturally occurring amino acids. For example, an "amino acid analog" is a non-natural amino acid with the same basic chemical structure as a naturally occurring amino acid—that is, an α-carbon bound to a hydrogen, carboxyl group, or amino group—but with a modified R (i.e., side chain) group or a modified peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonate. An "amino acid mimic" refers to a compound whose structure differs from the general chemical structure of an amino acid but whose function is similar to that of a naturally occurring amino acid.

[0043] Amino acids in this article may be represented by their known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. For example, in this article, L-amino acids may be represented by their known three-letter symbols (e.g., Arg for L-arginine) or by a capital one-letter amino acid symbol (e.g., R for L-arginine). In this article, D-amino acids may be represented by their known three-letter symbols (e.g., D-Arg for D-arginine) or by a lowercase one-letter amino acid symbol (e.g., r for D-arginine).

[0044] Regarding amino acid sequences, those skilled in the art will recognize that individual substitutions, additions, or deletions of a peptide, polypeptide, or protein sequence—that is, alterations, additions, or deletions of one or a small subset of amino acids in the coding sequence—are “conserved modification variants” in which such alterations result in the substitution of an amino acid by a chemically similar amino acid. Chemically similar amino acids include, but are not limited to, naturally occurring amino acids such as L-amino acids, stereoisomers of naturally occurring amino acids such as D-amino acids, and non-natural amino acids such as amino acid analogs, amino acid mimics, synthetic amino acids, N-substituted glycine, and N-methyl amino acids.

[0045] Providing a table of conservative substitutions for functionally similar amino acids is well known in the art. For example, substitutions can be made in which an aliphatic amino acid (e.g., G, A, I, L, or V) is substituted by another member of that group. Similarly, aliphatic polar uncharged groups, such as C, S, T, M, N, or Q, can be substituted by another member of that group; basic residues, such as K, R, or H, can be substituted for each other. In some embodiments, amino acids having acidic side chains, such as E or D, can be substituted by their uncharged counterparts, such as Q or N, respectively; and vice versa. Each of the following eight groups contains other exemplary amino acids that are conservatively substituted for each other:

[0046] 1) Alanine (A), glycine (G);

[0047] 2) Aspartic acid (D), glutamic acid (E);

[0048] 3) Asparagine (N), glutamine (Q);

[0049] 4) Arginine (R), Lysine (K);

[0050] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0051] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0052] 7) Serine (S), threonine (T); and

[0053] 8) Cysteine ​​(C), Methionine (M)

[0054] (For example, see Creighton, Proteins, 1993).

[0055] The term "amino acid modification" or "amino acid alteration" refers to the substitution, deletion, or insertion of one or more amino acids.

[0056] The "sequence identity percentage" is determined by comparing two best-aligned sequences in a comparison window, where the sequence portions in the comparison window (e.g., the peptides described herein) may include additions or deletions (i.e., gaps) to achieve optimal alignment of the two sequences compared to a reference sequence that does not include additions or deletions. The percentage is calculated by determining the number of matching positions in both sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100.

[0057] In the context of two or more polypeptide or peptide sequences, the term "identical" or percentage "identity" refers to two or more sequences or subsequences that are identical sequences. When comparing and aligning sequences through a comparison window or specified region, such as using one of the following sequence comparison algorithms or by manual alignment and visual inspection to obtain maximum correspondence, two sequences are considered "substantially identical" if they have the same percentage of specified amino acid residues (i.e., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity in a specified region, or, if not specified, throughout the entire length of a reference sequence). Regarding amino acid sequences, identity or substantial identity may be present in regions of at least 5, 10, 15, or 20 amino acids in length, optionally in regions of about 25, 30, 35, 40, 50, 75, or 100 amino acids in length, optionally in regions of about 150, 200, or 250 amino acids in length, or throughout the entire length of a reference sequence. For shorter amino acid sequences, such as those with 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.

[0058] For sequence comparisons, a reference sequence is typically used, and the test sequence is compared to it. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, with subsequence coordinates specified if necessary, and the sequence algorithm program parameters specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the program parameters. The BLAST and BLAST 2.0 algorithms are two examples of algorithms suitable for determining sequence identity and percentage sequence similarity, described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for BLAST analysis is publicly available from the National Center for Biotechnology Information.

[0059] When a first polypeptide or peptide exhibits an immunological cross-reactivity with an antibody generated against a second polypeptide or peptide, it indicates that the two polypeptide or peptide sequences are substantially identical. Therefore, the first polypeptide or peptide is generally substantially identical to the second polypeptide or peptide; for example, the two sequences may differ only due to conserved substitutions.

[0060] The term "antigen fragment" refers to a contiguous subsequence of a polypeptide that binds to an antibody. Antigen fragments may or may not be immunogenic; that is, they may induce an immune response or they may not.

[0061] The term "conformational antigen fragment" refers to a spatially contiguous region of a polypeptide or tetramer, which may or may not be composed of consecutive subsequences. Conformational antigen fragments may or may not be immunogenic.

[0062] The term "epitope" or "antigenic determinant" refers to a site on which B cells and / or T cells respond to a peptide or polypeptide. B cell epitopes can be formed from consecutive amino acids or from discontinuous amino acids arranged side-by-side in the tertiary or quaternary folds of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from tertiary or quaternary folds (i.e., conformationally determined) are generally lost upon treatment with denaturing solvents. In a distinctive spatial conformation, an epitope typically contains at least three, and more commonly, at least five or eight to ten amino acids. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn EMorris, Ed. (1996). Antibodies that recognize the same epitope can be identified by simple immunoassays, demonstrating the ability of one antibody to block the binding of another antibody to the target antigen (e.g., electrochemiluminescence assay, competitive ELISA, solid-phase radioimmunoassay (SPRIA), or blocking proteoblotting). T cells recognize a continuous epitope of approximately 9 amino acids on CD8 cells or approximately 13 to 15 amino acids on CD4 cells. T cells that recognize epitopes can be identified by in vitro assays measuring antigen-dependent proliferation, such as by the response of activated T cells to the epitope. 3 H-thymidine incorporation is identified (Burke et al, J.Inf.Dis.170, 1110-19 (1994)), recognition is achieved through antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al, J.Immunol. (1996) 156: 3901-3910), or recognition is achieved through cytokine secretion.

[0063] The terms "specific binding" or "specific targeting" refer to the preferential association between a T-cell receptor and / or antibody, wholly or partially, and a target peptide / peptide or its antigenic fragment, compared to other peptides / peptides. Of course, it is recognized that some degree of nonspecific interaction may occur between an antibody or T-cell receptor and non-target peptides / peptides. However, specific binding can be distinguished as being mediated by the specific recognition of a target peptide / peptide or its antigenic fragment. Typically, specific binding or specific targeting of an immune response results in a much stronger association between the target peptide / peptide and an antibody against the target peptide / peptide or T-cell receptor than between an antibody against the target peptide / peptide or T-cell receptor and a non-target peptide / peptide. Specific binding typically results in an approximately 10-fold (e.g., greater than 100-fold) increase in the amount of antibody binding to the target peptide / peptide (per unit time) compared to cells or tissues lacking the target peptide / peptide epitope. Specific binding between the target peptide / peptide and an antibody against the target peptide / peptide typically implies at least 10... 6 M -1 Affinity. Preferably greater than 10. 8 M -1 The affinity of the target peptide / peptide. Specific binding can be determined using any antibody binding assay known in the art, including but not limited to proteoblotting, dotblot, ELISA, flow cytometry, electrochemiluminescence, complex bead analysis (e.g., using Luminex or fluorescent microspheres), and immunohistochemistry. T cells specifically targeting the target peptide / peptide epitope typically exhibit antigen-induced proliferation in response to the target peptide / peptide, which is approximately twice (e.g., more than approximately 5 or 10 times) greater than antigen-induced proliferation in response to non-target peptides / peptides. T cell proliferation assays are known in the art and can be performed by... 3 The measurement was performed by incorporating H-thymidine.

[0064] The term "sample" refers to any biological sample obtained from a subject, such as a human subject. Samples include, but are not limited to, whole blood, plasma, serum, red blood cells, white blood cells, saliva, urine, feces, sputum, bronchoalveolar lavage fluid, tears, nipple aspiration, breast milk, any other bodily fluids, tissue samples such as placental biopsies, and their cellular extracts. In some embodiments, the sample is whole blood or a component thereof, such as plasma, serum, or cell clumps.

[0065] The terms “subject,” “individual,” or “patient” generally include humans, but may also include other animals, such as, for example, other primates, rodents, canines, felines, horses, sheep, porcupines, etc. In a particular embodiment, the subject is a human subject.

[0066] The term “increased risk of developing ASD” refers to an increased likelihood or probability of a fetus or child having symptoms of ASD when exposed to antibodies binding to one or more antigens (e.g., NSE) or when antibody levels against one or more antigens are above a predetermined threshold level, compared to the risk, likelihood, or probability of the fetus or child not being exposed to antibodies against one or more antigens or having antibody levels against one or more antigens below a predetermined threshold level.

[0067] The term “reduced risk of developing ASD” refers to a reduced likelihood or probability of developing ASD symptoms in a fetus or child exposed to antibodies binding to one or more antigens (e.g., NSE) or at antibody levels above a predetermined threshold for one or more antigens, whose mother has received treatment intervention—e.g., blocking, inactivating, or removing antibodies binding to antigens—compared to the likelihood or probability of developing ASD symptoms in a fetus or child exposed to antibodies against one or more antigens or at antibody levels above a predetermined threshold for one or more antigens, whose mother has not received treatment intervention.

[0068] The term “peptide epitope” or “antigen peptide” refers to a peptide or fragment of one or more antigens (e.g., NSE) described herein that mimics an epitope (e.g., bound by an antibody against the antigen), although there may not be explicit homology between the structure or sequence of such peptide epitopes and native antigenic epitopes. Rather, the mimicry of peptide epitopes relies on similarity in physicochemical properties and similar spatial organization. Screening and construction of peptide epitopes are known in the art. For example, peptide epitopes can be derived from known epitopes by sequence modification or developed de novo using a library of combined peptides, e.g., antibodies that bind to one or more antigens. See, for example, Yip and Ward, Comb Chem High Throughput Screen (1999) 2(3):125-128; Sharav, et al, Vaccine (2007) 25(16):3032-37; and Knittelfelder, et al., Expert Opin Biol Then (2009) 9(4):493-506.

[0069] The term "family history" refers to the presence of a medical condition (such as "ASD or autoimmune disease") in a family member. Family members can be immediate relatives, such as parents, children, grandparents, or close relatives, such as siblings, aunts, uncles, cousins, etc. Typically, family members are blood relatives who share a common genetic inheritance.

[0070] The term "therapeutic effective dose" refers to the amount of peptide described herein that achieves a therapeutic effect or desired outcome (i.e., a sufficient amount of peptide to block the binding of an antibody against the target antigen to the target antigen), preferably with minimal or no side effects. In some embodiments, a therapeutically acceptable amount does not cause or result in adverse side effects. Therapeutic effective doses can be determined by initially administering a low dose and then gradually increasing the dose until the desired effect is achieved. The "preventive effective dose" and "therapeutic effective dose" of the antibody blocker described herein can prevent the onset of ASD or reduce the severity of ASD. "Preventive effective doses" and "therapeutic effective doses" can also prevent or improve damage or disability resulting from disorders and diseases caused by maternal antibody activity, respectively.

[0071] The term "pharmaceutically acceptable carrier" refers to compounds, chemicals, or molecules that are useful in the preparation of pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, and that include substances that can be used for medicinal purposes in subjects. Suitable drug carriers are described in this article and in EW Martin's "Remington's Pharmaceutical Sciences".

[0072] As used herein, the term "administration" includes oral administration, local contact, administration as a suppository, intravenous injection, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained-release device, such as a microosmotic pump, into the subject. Administration can be performed via any route, including parenteral and transmucosal (e.g., oral, sublingual, palatal, gingival, nasal, vaginal, rectal, or percutaneous). Parenteral administration includes, for example, intravenous injection, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, ventricular, and intracranial administration. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusions, transdermal patches, etc. Those skilled in the art will recognize additional methods for administering a therapeutically effective amount of the peptide described herein, which is used to prevent or alleviate one or more symptoms associated with the presence or activity of a parent antibody. "Concomitant administration" means administering the peptide described herein concurrently with or before administration of a second drug.

[0073] As used herein, the term "treatment" refers to any indication of success in treating or improving a pathology or condition, including any objective or subjective parameters such as relief, remission, symptom reduction, or making the patient more tolerant of the pathology or condition, slowing the rate of degeneration or decline, reducing the degenerative endpoint to less debilitating, or improving the patient's physical or mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of physical examination, histopathological examination (e.g., biopsy tissue analysis), laboratory analysis or imaging of urine, saliva, tissue samples, serum, plasma, or blood.

[0074] The term "specific inhibition" refers to the ability of a reagent (such as the peptide described herein) to inhibit antibody binding against one or more antigens (e.g., NSE). Specific inhibition typically results in at least about 2-fold inhibition compared to background, for example, greater than about 10-fold, 20-fold, or 50-fold inhibition of antibody binding against the target antigen, for example, by comparing antibody binding in the absence of the reagent. In some embodiments, antibody binding to the target antigen is completely inhibited or blocked by the reagent (such as the peptide described herein). Typically, specific inhibition is a statistically significant reduction in antibody binding to the target antigen using appropriate statistical tests (e.g., p < 0.05).

[0075] The term "reagent" includes peptides (e.g., peptide epitopes), mimic epitopes, polypeptides (e.g., ligands, antibodies), nucleic acids, small organic compounds, etc.

[0076] The term "solid carrier" refers to any material suitable for performing the methods described herein, such as plastic or glass tubes, beads, glass slides, microtiter plates, porous filters or membranes, non-porous filters or membranes, non-magnetic beads, microspheres, glass slides, microarrays, etc.

[0077] The term "neutralizing complex" refers to a complex containing a maternal antibody that binds to a specific peptide described herein, thereby preventing / inhibiting / blocking the binding of the maternal antibody to its antigen (e.g., NSE). For example, a maternal autoantibody that specifically recognizes the NSE antigen may form a neutralizing complex with the NSE peptide described herein or its mimic epitope, thereby preventing the maternal autoantibody from binding to the NSE antigen.

[0078] The term "affinity plasma purification technology" refers to an extracorporeal blood purification procedure that removes harmful substances (such as pathogens) from a subject's plasma.

[0079] III. Detailed Description of the Embodiments

[0080] This disclosure provides peptides (e.g., peptide epitopes and their mimic epitopes) that specifically bind to maternal autoantibodies against endogenous autoantigen NSE proteins. This disclosure also provides compositions and kits comprising the peptides described herein. Additionally, this disclosure provides methods for determining the risk of autism spectrum disorder (ASD) in children or future offspring (e.g., pregnant or pre-pregnancy mothers or potential mothers) by detecting the presence of maternal autoantibodies in a biological sample of a mother or potential mother using the peptides described herein. This disclosure also provides methods for preventing or reducing the occurrence of ASD in offspring by administering a therapeutically effective amount of the peptides described herein to the mother or potential mother of the offspring to block the binding between maternal autoantibodies and their antigens.

[0081] A. Neuron-specific enolase (NSE)

[0082] NSE is one of the most abundant proteins in the brain, accounting for 0.4% to 2.2% of total soluble proteins depending on the brain region. It has different functions, including participating in glycolysis and gluconeogenesis, neuronal differentiation, activation and proliferation through the PI3K / Akt and MAPK / ERK signaling pathways. In addition, NSE plays a role in the activation of the RhoA kinase pathway, which can lead to neurodegeneration or neuroprotection depending on the strength of the signal. Furthermore, the upregulation of NSE expression in M1 microglia and reactive astrocytes indicates that NSE is involved in CNS inflammatory processes. Therefore, NSE plays several important roles in neurodevelopment and is also associated with neurodegeneration

[18] .

[0083] Measurements of plasma NSE levels have been used as biomarkers for a variety of applications

[17] . For example, it is a useful indicator of neural maturation, is currently the most widely used biomarker for small cell lung cancer (SCLC), and has been shown to have a direct effect on the in vitro growth and migration of different SCLC cell lines [28,29]. In addition, it is used for the diagnosis and prognosis of other types of cancer, such as non-small cell lung cancer (NSCLC), neuroendocrine tumors (NETs), neuroblastoma, brain cancer, and brain injury (TBI)

[30] . As illustrated in the examples in this paper, we address the value of NSE autoantibodies as potential biomarkers or risk factors for MAR ASD based on the concept that antibodies binding to NSE during neurogenesis may affect protein function and brain metabolism, with lasting effects on neural tissue function and development.

[0084] As described in the examples below, we found similar autoantibody response rates against NSE for both experimental groups (ASD and TD). This suggests that the complete NSE protein itself is not a biomarker, similar to previous studies that have demonstrated the necessity for autoantibodies to confer ASD specificity through reactivity to multiple rather than a single antigen [8, 12, 13, 31, 32]. When we first identified the initial seven autoantigens, we found that reactivity to specific combinations of antigens was highly significant as a biomarker of ASD risk, including LDH, STIP1, and CRMP1 (13% ASD vs 0% TD) and several other combinations of three or more autoantigens (with >98% specificity) [6, 8, 9]. Therefore, we tested NSE using a larger dataset and found that it increased the specificity and sensitivity of MAR ASD detection.

[0085] In a recent study, we performed microarray-based epitope mapping of CRMP1, CRMP2, GDA, LDHA / B, STIP1, and YBX1, and further described differential reactivity to several epitopes that are recognized only by autoantibodies from mothers of children with ASD

[15] . Additionally, we used our original set of autoantigen epitopes to create an endogenous antigen-driven mouse model of autism in which mice were immunized with peptide epitopes of LDHA, LDHB, CRMP1, and STIP1. This approach allows embryos to be continuously exposed to autoantibodies against MAR ASD-specific peptides throughout pregnancy. Thus, we created mouse models exhibiting ASD-related behaviors and demonstrated that exposure to this combination of autoantibodies leads to alterations in neurodevelopment

[11] .

[0086] As described in the examples below, we identified NSE as an additional MAR ASD autoantigen and discovered 16 epitope sequences recognized by maternal autoantibodies present only in the ASD group. Analysis using conventional t-tests and SAM score t-tests showed that four sequences were statistically significant compared to the control group. The epitope sequences (ES 408 and 409) SERLAKYNQLMRIEE (SEQ ID NO:6) and ERLAKYNQLMRIEEE (SEQ ID NO:3) had the largest OR values ​​(10.1 and 12.6, respectively), indicating a strong association between having autoantibodies against these sequences and the risk of having children with ASD. These ASD-specific epitope peptides can be used to create MAR ASD animal models to assess the effects of NSE ASD-specific peptides alone or in combination with pathogenic epitopes of other autoantigens, thereby better understanding the role of anti-NSE in autism pathology.

[0087] As a mechanism of action, we hypothesize that the presence of ASD-specific NSE epitope autoantibodies may inhibit appropriate protein function in two distinct ways: 1) by directly interfering with appropriate protein folding (tertiary and quaternary structures), or 2) by binding to key functional sites (catalytic or substrate sites) [33–36]. While anti-NSE antibodies in the developing brain may elicit responses in cells targeting these autoantibodies, we lack evidence of tissue destruction based on our previous rodent model. In contrast, the presence of MAR ASD autoantibodies against CRMP1, LDHA / B, and STIP1 appears to influence progenitor cell maturation and alterations in dendritic spines and structures in the adult brain [10, 13, 37]. However, the immunopathological mechanisms mediated by autoantibodies in the brain remain poorly understood.

[0088] The last area of ​​interest is exploring the relationship between ASD and non-ASD-specific peptide sequences and the epitope repertoire reported in the Immunological Epitope Database (IEDB)

[38] . This interest stems from the potential of peptide-mimicking recognition to provide some understanding of how autoantibodies against these self-proteins are generated. We found that the sequences DYPVVSIEDPFDQDD (SEQ ID NO:7), YPVVSIEDPFDQDDW (SEQ ID NO:8), PVVSIEDPFDQDDWA (SEQ ID NO:9), VVSIEDPFDQDDWAA (SEQ ID NO:10), and VSIEDDPFDQDDWAAW (SEQ ID NO:11) were recognized by antibodies in both experimental groups, indicating immunodominant epitopes recognized by the general population. As expected, these sequences share a high degree of homology with α and γ enolases (NNE and NSE), strictly 90%. Interestingly, they also share 80% homology with other proteins, including ORF73 from human gamma herpesvirus 8 (monocytosis pathogen), protein X from hepatitis B virus, and Serpin HI from humans, suggesting that direct contact with these substances may involve molecular mimicry.

[0089] B. Peptide epitopes

[0090] In some respects, this disclosure provides isolated peptides that specifically bind to maternal antibodies produced in mothers or potential mothers against neuron-specific enolase (NSE) proteins. NSEs are catalytic enzymes expressed in neurons and neuroendocrine tissues that mediate the conversion of glycerol 2-phosphate (2PG) to 2-phosphoenolpyruvate (2PEP) and the reverse reaction (2PEP to 2PG) in glycolysis and gluconeogenesis, respectively

[16] . For eukaryotic cells, there are three enolase isotypes encoded by different genes and expressed in tissue-specific ways: α-enolase (ENO 1) is widely expressed, γ-enolase (ENO 2) is found only in neurons, and β-enolase (ENO 3) is found only in muscle. Enolases exist in dimer form and their function depends on the natural cofactor Mg+ to regulate the conformation and catalytic activity of the enzyme

[17] . In the brain, NSEs are expressed as γγ in neurons and as αγ in microglia, astrocytes, and oligodendrocytes. In the early stages of development, non-neuronal enolases (NNE, αα-dimer) are observed in neural tissue, but during the differentiation and maturation of neurons and glial cells, they transform into γγ and αγ isoforms (NSE). They participate in cell metabolism, immune response regulation, neuroinflammation, neurodevelopment, and brain homeostasis by regulating cell survival / death signals

[18] . Therefore, given the well-established role of NSE in neurodevelopmental biology, there is ample evidence for the potential of NSE as a target for maternal autoantibodies in the context of ASD.

[0091] In a first aspect, the peptide has an amino acid sequence identity of at least about 50%, for example, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, with any one of SEQ ID NOs:1-6 (DVAASEFYRDGKYDL (SEQ ID NO:1); IEDPFDQDDWAAWSK (SEQ ID NO:2); ERLAKYNQLMRIEEE (SEQ ID NO:3); RLAKYNQLMRIEEEL (SEQ ID NO:4); DYPVVSIEDPFDQDDWAAW (SEQ ID NO:5); and SERLAKYNQLMRIEE (SEQ ID NO:6). In some embodiments, the peptide comprises SEQ ID NOs:1-6 (SEQ ID NO:1); IEDPFDQDDWAAW (SEQ ID NO:2); ERLAKYNQLMRIEEE (SEQ ID NO:3); RLAKYNQLMRIEEEL (SEQ ID NO:4); DYPVVSIEDPFDQDDWAAW (SEQ ID NO:5); and SERLAKYNQLMRIEE (SEQ ID NO:6). The amino acid sequence of any one of SEQ ID NOS:1-6 comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids. In other embodiments, the peptide (e.g., an antigen fragment thereof) has at least about 50% of the length of the amino acid sequence of any one of SEQ ID NOS:1-6, for example, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the peptide comprises an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID NOS:1-6. In other embodiments, the peptide includes one or more additional amino acid residues at the amino terminus and / or carboxyl terminus that correspond to amino acid residues at those positions in the NSE polypeptide sequence. In specific embodiments, the peptide binds to a parent antibody that binds to the NSE polypeptide.

[0092] In some embodiments, the peptide has a length of about 5 to about 45 amino acids, about 8 to about 45 amino acids, about 8 to about 25 amino acids, about 12 to about 45 amino acids, about 5 to about 40 amino acids, about 10 to about 40 amino acids, about 15 to about 30 amino acids, about 15 to about 25 amino acids, about 15 to about 22 amino acids, about 15 to about 20 amino acids, about 17 to about 25 amino acids, about 19 to about 25 amino acids, or about 45, 40, 35, 30, 25, 20, 15, 10, or 5 amino acids. For example, peptides can be approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more amino acids long. Generally, the length of a peptide should not exceed the length allowed to form tertiary structures, such as, for example, greater than 45 amino acids (if present in the form of a separate molecule). However, if fused to a larger molecule, such as an antibody or another protein or macromolecule, the peptide may exceed 45 amino acids, which may prevent the formation of tertiary structures within the peptide. If the peptide is a divalent peptide with first and second peptide fragments that bind to different parent antibodies, the peptide may also exceed 45 amino acids. In certain embodiments, the peptide length is up to about 15, 20, 25, 30, 35, 40, or 45 amino acids.

[0093] In some embodiments, the peptide further includes a label, such as a detectable label. In some cases, the label is selected from the group consisting of biotin, fluorescent labels, chemiluminescent labels, and radioactive labels. In some other cases, the label is covalently bound to the peptide.

[0094] In other embodiments, the peptide includes variants with further modifications to enhance its resistance to proteolytic degradation, optimize its solubility, or make it more suitable as a therapeutic agent. For example, the peptide may also include analogs containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. The D-amino acids may be partially or completely substituted with amino acid residues.

[0095] In some embodiments, the peptide comprises natural and / or non-natural amino acids. Examples of non-natural amino acids include, but are not limited to, D-amino acids, ornithine, diaminobutyric acid ornithine, norleucine ornithine, pyridylalanine, thienylalanine, naphthylalanine, phenylglycine, α and α-disubstituted amino acids, N-alkyl amino acids, lactic acid, halogenated derivatives of naturally occurring amino acids (e.g., trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, p-I-phenylalanine, etc.), L-allylglycine, β-alanine, La-aminobutyric acid, Lg-aminobutyric acid, and La-amino... Isobutyric acid, Le-aminohexanoic acid, 7-aminoheptanoic acid, L-methionine sulfone, L-norleucine, L-norvaline, p-nitro-L-phenylalanine, L-hydroxyproline, L-thioproline, and methyl derivatives of phenylalanine (e.g., 1-methyl-Phe, pentamethyl-Phe, L-Phe(4-amino), L-Tyr(methyl), L-Phe(4-isopropyl), L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), L-diaminopropionic acid, L-Phe(4-benzyl), etc.). Peptides can be further modified. For example, one or more amide bonds may be substituted with an ester or alkyl backbone. N- or C-alkyl substituents, side chain modifications, or restrictions, such as disulfide bonds or side chain amide or ester bonds, may be present.

[0096] In some embodiments, peptides include both modified peptides and synthetic peptide analogs. Peptides can be modified to improve formulation and storage performance, or to protect unstable peptide bonds by binding to non-peptide structures.

[0097] In other embodiments, the peptide may be cyclized. A method well known in the art is to introduce a cyclic structure into the peptide to select for and provide conformational constraints to the structure, thereby improving stability. For example, a C- or N-terminal cysteine ​​residue may be added to the peptide so that upon oxidation the peptide will contain a disulfide bond, thus generating a cyclic peptide. Other peptide cyclization methods include the formation of thioethers, carboxyl-terminal and amino-terminal amides and esters. Numerous synthetic techniques have been developed to generate synthetic cyclic peptides (e.g., see Tam et al, Protein Sci., 7:1583-1592 (1998); Romanovskis et al, J. Pept. Res., 52:356-374 (1998); Camarero et al, J. Amer. Chem. Soc., 121:5597-5598 (1999); Valero et al, J. Pept. Res., 53(1):56-67 (1999)). Generally speaking, cyclic peptides have two functions: (1) reducing hydrolysis in the body; and (2) thermodynamically destabilizing the unfolded state and promoting the formation of secondary structures.

[0098] In some embodiments, the present invention provides a plurality of peptides comprising at least two identical or different peptides covalently or non-covalently linked. For example, in some embodiments, at least two, three, four, five, or six identical or different peptides are covalently linked, for example, to give them appropriate size and / or binding properties, but to avoid unwanted aggregation.

[0099] The peptides described herein can be produced by any suitable method known in the art or subsequently discovered, such as in vitro synthesis, purification or substantially purification from natural sources, recombinant production from eukaryotic or prokaryotic cells, etc.

[0100] Peptides can be synthesized in vitro using conventional methods known in the art. For example, peptides can be produced chemically, such as using solid-phase techniques and / or automated peptide synthesizers. In some cases, peptides can be synthesized using a solid-phase strategy on an automated peptide synthesizer (Abimed AMS422) employing 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. The peptide can then be purified by reversed-phase high-performance liquid chromatography and lyophilized. By using the synthesizer, naturally occurring amino acids can be replaced by non-natural amino acids. The specific preparation sequence and method will be determined by convenience, economy, desired purity, etc. Peptides can also be prepared additionally by cleaving longer peptides or full-length protein sequences.

[0101] Peptides can also be isolated and purified using conventional recombinant synthesis methods. Lysis products can be prepared using an expression host, and these products can be purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. Methods well-known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcription / translation control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / gene recombination. Alternatively, RNA capable of encoding the peptide of interest can be chemically synthesized. Those skilled in the art can readily provide suitable coding sequences for any peptide described herein using well-known codon usage tables and synthetic methods. See, for example, Sambrook and Russell, *Molecular Cloning: A Laboratory Manual*, 3rd Ed., 2001, Cold Spring Harbor Laboratory Press; and Ausubel, et al., *Current Protocols in Molecular Biology*, 1987–2009, John Wiley Interscience.

[0102] In other respects, this disclosure provides compositions comprising any one or more of those described herein. As a non-limiting example, the composition comprises a plurality of peptides that bind to a parent antibody against NSE. As another non-limiting example, the composition comprises one or more peptides selected from the group consisting of SEQ ID NOS:1-6. As a further non-limiting example, the composition comprises peptides corresponding to SEQ ID NOS:1-6.

[0103] C. Simulated epitope

[0104] In some aspects, this disclosure provides mimicry epitopes that mimic the peptide epitopes described herein (e.g., peptides that bind to a parent antibody that binds to an NSE protein). In some embodiments, the mimicry epitope is an immunomimicry peptide epitope and a peptide sequence that has sequence homology with an antigen site. In other embodiments, the mimicry epitope is an immunomimicry peptide epitope having a three-dimensional conformation but not having sequence homology with an antigen site.

[0105] In some embodiments, the antibody response induced by a mimic epitope is similar to that induced by a peptide epitope. In some cases, the antibody response to a mimic epitope corresponds to binding to the same antigenic site on the parent antibody that binds to the peptide epitope. The ability of a mimic epitope to bind to a parent antibody as a molecular mimic can be used to prevent the antibody from binding to its original target antigen (e.g., the NSE protein).

[0106] In some embodiments, mimic epitopes are obtained from phage display libraries through biological screening. Phage display libraries suitable for screening and identifying candidate mimic epitopes are typically multiple phages that express random amino acid sequences of less than 100 amino acids, less than 75 amino acids, less than 50 amino acids, or less than 25 amino acids in length at locations where antibodies may bind, particularly in the range of about 3 to about 25 amino acids.

[0107] In other embodiments, mimic epitopes are obtained by screening peptide libraries. In some cases, the peptide library is an overlapping peptide library. In other cases, the peptide library is a truncated peptide library that can be used to identify the shortest amino acid sequence required for activity. In other cases, mimic epitopes are obtained by alanine scanning, where alanine is used to sequentially replace each residue to identify the specific amino acid residue responsible for peptide activity. In a further case, mimic epitopes are obtained by position scanning, which identifies the amino acid of interest at a single position and replaces it one by one with all other natural amino acids at a time to identify the preferred amino acid residue for increasing peptide activity at that position. In relevant cases, position scanning may include a two-position combination scan or a three-position combination scan. Other methods for designing, screening, and identifying mimic epitopes are described in U.S. Patent No. 4,833,092, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0108] In a further embodiment, the simulated epitope may be a peptide sequence that is structurally more restricted than the linear form of the sequence. Unsubstituted linear peptides, such as free peptides in solution, are typically capable of exhibiting a wide range of different conformations. In contrast, structurally restricted peptides, which may have one or usually two or more substituents that reduce the number of possible conformations they can exhibit, are also within the scope of this invention.

[0109] Substituents, such as covalent bonds or intramolecular bonds with further peptide chains, will structurally restrict the peptide. For example, a peptide can form part of the primary structure of a larger polypeptide that contains its amino acid sequence. In some cases, peptides include cyclic peptides.

[0110] Other substituents include covalent bonds with other parts, such as macromolecular structures (both biological and abiotic). Examples of biological structures include, but are not limited to, carrier proteins. Examples of abiotic structures include lipid vesicles, such as liposomes, micelles, lipid nanoparticles, etc.

[0111] In some embodiments, the carrier protein binds to a mimic epitope. Many carriers are known for this purpose, including various protein-based carriers such as albumins (e.g., bovine serum albumin (BSA)), keyhole cyanin (KLH), ovalbumin (OVA), tetanus toxoid (TT), high molecular weight protein (HMP) from untyped Haemophilus influenzae, diphtheria toxoid, or bacterial outer membrane proteins, all of which are available from biochemical or pharmaceutical supply companies or prepared using standard methods.

[0112] In other embodiments, mimic epitopes are components of the vaccine. A vaccine may contain one or more mimic epitopes, each capable of binding to the same or different parental antibodies to prevent the antibodies from binding to their original target antigen (e.g., the NSE protein). Multiple mimic epitopes may be combined, for example, using polylysine bound to each mimic epitope.

[0113] In certain embodiments, peptide mimic epitopes are designed using single amino acid substitutions, followed by affinity testing on each peptide structure to determine which peptide mimics are capable of blocking autism-specific maternal autoantibodies. In some cases, D-amino acids are used when synthesizing peptide mimic epitopes because peptides synthesized from D-amino acids are more resistant to proteolytic digestion and have a longer half-life in vivo. In other cases, peptide mimic epitopes for each autoantigen are fused to a polyethylene glycol (PEG) scaffold, resulting in heteropolymers capable of neutralizing autism-specific maternal autoantibodies. See, for example, Kessel et al, Chem Med Chem. 4(8):1364-70, 2009.

[0114] Because peptides on PEG scaffolds are less immunogenic than single peptides, mimic epitope peptides attached to PEG scaffolds can be used as antibody blockers. Peptide mimic epitopes can be chemically synthesized on suitable polyethylene glycol (PEG)-PS resin (GenScript Corporation; Piscataway, NJ) using an automated peptide synthesizer (Pioneer; Applied Biosystems; Foster City, CA) with 9-fluorenyl-methoxy-carbonyl-protected amino acids. The peptides can be cleaved from the resin and the protecting groups removed from the side chains using trifluoroacetic acid and a scavenging agent. The crude peptides can be processed by reversed-phase high-performance liquid chromatography using a pre-concentrated C44-dimethylformamide (PCC) solution. 18 The column was purified using a gradient of solvent A [95% / 5%, H₂O (0.1% trifluoroacetic acid) / acetonitrile] and solvent B (100% acetonitrile). Then, high-performance liquid chromatography (HPLC) was used to analyze C... 18 The purity of the peptides was analyzed by column chromatography. The properties of the synthetic peptides were also confirmed by matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry. In some cases, peptide mimic epitopes can be PEGylated using a strategy involving the reversible protection of specific residues on the peptide. This procedure is only applicable to peptides, as they typically contain only a few nucleophilic groups and are more stable than full-length proteins under the harsh chemical treatments involved in the process. The method involves three steps: (1) protecting residues known to be important for activity with appropriate reagents and ultimately purifying the desired isomer; (2) PEGylation at the level of a single unprotected reactive target residue; and (3) removal of all protecting groups.

[0115] To determine whether a polypeptide mimic epitope binds to anti-brain autoantibodies in a patient's serum, an ELISA assay can be used. ELISA can also be used to determine whether a polypeptide mimic epitope inhibits antigen-antibody interactions against native antigen proteins. This can be achieved by pre-incubating maternally antibody-positive plasma with the heteropolymer prior to performing the ELISA.

[0116] Animal models can be used to examine the efficacy, safety, and / or pharmacokinetic performance of peptide mimics in vivo. As a non-limiting example, a mouse model of maternal autoantibody-associated (MAR) autism can be used. See, for example, Example 5 in International Patent Publication WO 2016 / 210137. MAR autistic mothers and control mothers (i.e., pregnant female mice) can be randomly assigned to one of two treatment conditions: gestational mimic epitope treatment or saline control. Once tolerance in the MAR autistic mothers is broken, the mothers assigned to the treatment group can be administered the mimic epitope peptide via intravenous injection. The efficacy of the mimic epitope in vivo can be determined by injecting 200 μg of the mimic epitope into the mothers four times every 24 hours. The reduction in mouse autoantibody titers following treatment can be determined using an ELISA analysis targeting the entire target antigen protein. A series of treatment trials can be performed to determine the number of treatments required to reduce / block maternal antibodies in mice during gestation. After determining the ideal treatment regimen, the mothers can be bred to produce offspring for subsequent behavioral analysis.

[0117] In a particular embodiment, the peptide is a mimic epitope comprising D-amino acids at some (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or all positions of an amino acid sequence and / or comprising amino acid modifications (e.g., substitutions) at one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) positions of an amino acid sequence relative to any one of SEQ ID NOS: 1-6.

[0118] D. Reagent kit

[0119] This disclosure also provides kits for diagnosis or prognosis to determine whether there is an increased risk of autism spectrum disorder (ASD) in offspring, such as fetuses or children. Relatedly, these kits can also be used to diagnose or predict whether a mother or potential mother has an increased risk of giving birth to a child who will develop ASD.

[0120] Materials and reagents for performing these different methods can be provided in the kit to facilitate their execution. As used herein, the term "kit" includes a combination of items that facilitate a process, assay, analysis, or manipulation. In particular, kits comprising the peptides or compositions described herein can be used for a wide range of applications, including diagnostics, prognosis, immunotherapy, and more.

[0121] In certain embodiments, the kit comprises any one or more peptides described herein (e.g., peptide epitopes and their mimic epitopes) that specifically bind to a parent antibody against the NSE antigen and a solid carrier. In some cases, the kit comprises a peptide (e.g., a peptide epitope and / or its mimic epitope) corresponding to any one of SEQ ID NOS:1-6, or a combination thereof.

[0122] In some embodiments, the solid support comprises at least one peptide, such as at least 1, 2, 3, 4, 5, or 6 peptides. In some cases, the solid support comprises an NSE peptide epitope. In some embodiments, the solid support comprises one or more peptides having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the peptides described herein (e.g., with the amino acid sequence of any one of SEQ ID NOS: 1-6). In other embodiments, the solid support comprises one or more peptides having the amino acid sequence of any one of SEQ ID NOS: 1-6 or a fragment thereof.

[0123] In some embodiments, the peptide or multiple peptides may be immobilized on a solid support. In other embodiments, the solid support is a multiwell plate, ELISA plate, microarray, chip, bead, porous tape, or nitrocellulose filter. Immobilization can be performed by covalent or nonvalent binding. In some embodiments, immobilization is performed by a capture antibody that specifically binds to one or more peptides. In some cases, the solid support provided in the kit is prepared with one or more immobilized peptides.

[0124] In some embodiments, the multiple peptides in the kit comprise at least 2, 3, 4, 5, or 6 different peptides, for example, selected from SEQ ID NOS:1-6 and their mimic epitopes. In some cases, each different peptide in the kit binds to the same parent antibody; for example, all different peptides bind to a parent antibody against the NSE antigen.

[0125] In certain embodiments, the multiple peptides in the kit constitute one or more groups, wherein each group contains a combination of peptides that bind to a parent antibody against the NSE antigen. As a non-limiting example, each group contains a combination of peptides that bind to a parent antibody against the NSE antigen. As another non-limiting example, the kit includes one or more combinations of peptides selected from SEQ ID NOS:1-6, wherein each combination contains 2, 3, 4, 5, or 6 peptides selected from SEQ ID NOS:1-6.

[0126] The kit may include chemical reagents and other components. Additionally, the kit described herein may include, but is not limited to, user instructions for the kit, equipment and reagents for sample collection and / or purification, equipment and reagents for product collection and / or purification, reagents for bacterial cell transformation, reagents for eukaryotic cell transfection, previously transformed or transfected host cells, sample tubes, scaffolds, trays, racks, plates, solutions, buffers, or other chemical reagents, and suitable samples for standardization, normalization, and / or control samples. The kit described herein may also be packaged for easy storage and safe transport, for example, in a covered box.

[0127] In some embodiments, the kit further includes a labeled secondary antibody for detecting the presence of maternal autoantibodies that bind to one or more peptides. The secondary antibody binds to the constant region or “C” region of different classes or isotypes of immunoglobulins IgM, IgD, IgG, IgA, and IgE. Typically, the kit includes a secondary antibody targeting the constant region of IgG, such as a secondary antibody targeting one of the IgG subclasses, such as IgG1, IgG2, IgG3, and IgG4. The secondary antibody can be labeled with any directly or indirectly detectable portion, including fluorophores (e.g., fluorescein, phycoerythrin, quantum dots, luminescent beads, fluorescent beads), enzymes (e.g., peroxidase, alkaline phosphatase), and radioisotopes (e.g., [missing information]). 3 H, 32 P, 125 I) or chemiluminescent components. The labeling signal can be amplified using a complex of biotin and its binding moiety (e.g., avidin, streptavidin, neutral avidin). Fluorescently labeled anti-human IgG antibodies are available from Molecular Probes, Eugene, OR. Enzyme-labeled anti-human IgG antibodies are available from Sigma-Aldrich, St. Louis, MO and Chemicon, Temecula, CA.

[0128] The kit may also include instructions for contacting the solid carrier with a biological sample from the mother or potential mother, and instructions for associating the presence of maternal antibodies or maternal antibody levels above a threshold with an increased probability of ASD in the fetus or child of the mother or potential mother.

[0129] In some embodiments, the kit further includes negative and positive control samples for detecting maternal antibodies. In some cases, the negative control sample is obtained from the mother of a child with TD. In other cases, the negative and / or positive control samples react to the NSE antigen. In still other cases, the negative and / or positive control samples do not react to the NSE antigen. In some embodiments, the kit includes a sample for preparing a titration profile of maternal antibodies in the sample to assist in assessing the quantitative level of antibodies in the test biological sample. In specific embodiments, the kit package includes one or more peptides described in SEQ ID NOS:1-6, such as peptides 1, 2, 3, 4, 5, or 6 described in SEQ ID NOS:1-6.

[0130] This kit can be used to provide diagnosis or prognosis for any woman of childbearing age. Diagnosis or prognosis can be determined before, during, or after pregnancy. Maternal antibodies can be detected during one or more of the early, middle, and / or late stages of pregnancy. In some embodiments, detection of maternal antibodies is performed on biological samples from women carrying fetuses whose brains have begun to develop, for example, after approximately 12 weeks of pregnancy. In some embodiments, the presence or quantification of maternal antibodies is assessed once or more postpartum, for example, in the first four weeks after birth and / or while the mother is breastfeeding the child. In some embodiments, the presence or quantification of maternal antibodies is assessed once or more in women before pregnancy or in any woman who has not been pregnant.

[0131] E. Patients undergoing diagnosis or treatment

[0132] The methods described herein can be applied to any mammal, such as humans, non-human primates, laboratory mammals (e.g., mice, rats, rabbits, hamsters), domesticated mammals (e.g., cats, dogs), or agricultural mammals (e.g., cattle, sheep, pigs, horses). In some embodiments, the patient is a woman or a human.

[0133] Any woman capable of having children can benefit from the methods described herein. A child may or may not be conceived; that is, a woman can become pregnant, but does not have to. In some embodiments, the woman has a newborn. In some embodiments, the woman is of reproductive age, meaning she has begun menstruating and has not reached menopause.

[0134] In some embodiments, the diagnostic, preventive, and / or treatment methods described herein are performed on women carrying a fetus (i.e., pregnant women). These methods can be performed at any time during pregnancy. In some embodiments, these methods are performed on women carrying a fetus whose brain has begun to develop. For example, the fetus may be around 12 weeks of gestation or later. In some embodiments, the female subject receiving treatment or diagnosis is in the second or third trimester. In some embodiments, the female subject receiving treatment or diagnosis is in the first trimester. In some embodiments, the woman is postpartum, for example, within 6 months after delivery. In some embodiments, the woman is in the postpartum lactation period.

[0135] Women who benefit from the current approach may, but do not need to, have a family history of ASD or an autoimmune disease. For example, a woman may have ASD, or a family member (e.g., a parent, child, grandparent, maternal grandparent) may have ASD. In some embodiments, a woman may have an autoimmune disease, or a family member (e.g., a parent, child, grandparent, maternal grandparent) may have an autoimmune disease.

[0136] In some embodiments, the methods described herein include steps to determine the suitability of a diagnosis or treatment for a patient, for example, based on prior medical history or family history or pregnancy status or any other relevant criteria.

[0137] F. Methods for determining the risk of developing autism spectrum disorder

[0138] In some aspects, this disclosure provides methods for determining the likelihood or risk of autism spectrum disorder (ASD) in a fetus or child, including identifying the presence of maternal autoantibodies binding to NSE antigens in a biological sample from the mother or potential mother of the fetus or child. The method includes detecting the presence in the biological sample of maternal autoantibodies binding to any one or more peptides described herein, wherein the presence of maternal autoantibodies binding to said peptides or peptides indicates an increased likelihood or risk of ASD in the fetus or child.

[0139] For biological samples collected from the mother or potential mother, any fluid sample containing antibodies can be used. For example, biological samples can be blood, serum, plasma, amniotic fluid, urine, breast milk, or saliva. Of course, antibodies that specifically bind one or more peptides in one or more different bodily fluids can be evaluated.

[0140] In certain embodiments, the presence of a biological sample in which a parent antibody specifically binds to at least one or more of the peptides described herein (e.g., SEQ ID NOS: 1-6), such as at least 1, 2, 3, 4, 5, or 6 of the peptides described in SEQ ID NOS: 1-6, is evaluated. In some embodiments, the presence of a parent antibody in a sample that specifically binds to the NSE antigen is detected using one or more of the peptides described herein (e.g., SEQ ID NOS: 1-6). As a non-limiting example, one or more peptides, such as 1, 2, 3, 4, 5, or 6 different peptides described in SEQ ID NOS: 1-6, can be used to detect the presence of a parent antibody in a sample.

[0141] In some cases, the presence of maternal antibodies against NSE can be detected using peptides 1, 2, 3, 4, 5 or 6 of SEQ ID NOS:1-6 or their antigen fragments.

[0142] In some embodiments, the detection of maternal antibodies (compared to the absence of maternal antibodies) indicates an increased likelihood that the fetus or child has or will develop ASD.

[0143] In some embodiments, the level or titer of maternal antibodies in a biological sample is compared to a threshold level or titer. A level or titer of antibodies in a biological sample greater than the threshold level or titer indicates an increased likelihood that the fetus or child has or will develop ASD. Similarly, a level or titer of antibodies in a biological sample less than the threshold level or titer does not indicate an increased probability that the fetus or child has or will develop ASD (i.e., it does not indicate an increased probability). The threshold level or titer of maternal antibodies in a particular biological fluid can be determined by assessing maternal antibody levels in a pregnant population and comparing the antibody levels or titers in the mother's biological fluid when the child has ASD to the antibody levels or titers in the mother's biological fluid when the child does not have ASD. The threshold level or titer can also be determined at different points in time during pregnancy, such as every four weeks, every two weeks, or weekly during fetal development. The threshold antibody level or titer can also be measured after the child's birth, for example, in the first four weeks after birth and / or while the mother is breastfeeding the child.

[0144] The presence or quantitative level of maternal antibodies against the NSE antigen can be determined before, during, or after pregnancy. When determined during pregnancy, maternal antibody testing can be performed once, twice, three times, four times, or more at any time during pregnancy, as appropriate. For example, maternal antibodies can be detected during one or more of the early, middle, and / or late stages of pregnancy. In some embodiments, maternal antibody testing is performed on biological samples from women carrying fetuses whose brains have begun to develop, for example, after approximately 12 weeks of pregnancy. In some embodiments, the presence or quantitative level of maternal antibodies is assessed once or more postpartum, for example, in the first four weeks after birth and / or while the mother is breastfeeding the child. In some embodiments, the presence or quantitative level of maternal antibodies is assessed once or more in women who are not pregnant or before pregnancy.

[0145] The presence of maternal antibodies may be determined once or multiple times as needed or desired. In some embodiments, the presence or quantification of maternal antibodies may be assessed every four weeks, every two weeks, or weekly during pregnancy, or more or fewer times as appropriate.

[0146] In some embodiments, determining the presence of maternal antibodies does not require comparing the test sample (i.e., a biological sample from the mother or potential mother) with a control sample. In other embodiments, the test sample is compared with a control. The control may be from the same individual at different time points. For example, the test sample may be collected during pregnancy, and the control sample may be collected from the same individual before pregnancy. In some cases, the test sample will be collected relatively late in pregnancy, and the control sample will be collected from the same individual earlier in pregnancy. In such cases, if the level of maternal antibodies in the test sample is higher than that in the control sample, the risk of ASD in the fetus or child is increased. If multiple samples are evaluated during pregnancy, an increase in maternal antibody levels or titers during pregnancy indicates an increased risk of ASD in the fetus or child. Similarly, a absence or decrease in maternal antibody levels or titers during pregnancy indicates a lower or reduced risk of ASD in the fetus or child.

[0147] Controls can also be derived from different individuals known to have a maternal antibody status. Controls can be calculated values ​​from a set of individuals known to have a maternal antibody status. Controls can be positive or negative controls. In some cases, negative controls are obtained from mothers of children with TD. In other cases, negative and / or positive control samples react to the NSE antigen. In still other cases, negative and / or positive control samples do not react to the NSE antigen.

[0148] In some embodiments, the control is a negative control from another individual or set of individuals. If the known status of the control sample is antibody negative, a higher level of maternal antibodies in the test sample indicates an increased risk of ASD in the fetus or child. A similar level of maternal antibodies in the test sample to the negative control sample indicates no increased risk of ASD in the fetus or child, i.e., a lower or reduced risk.

[0149] In some embodiments, a control is a positive control from another individual or set of individuals, or a control reflects a predetermined antibody threshold level. If the known status of a control sample is antibody positive, a maternal antibody level in the test sample that is similar to or higher than that in the positive control sample indicates an increased risk of ASD in the fetus or child. A lower maternal antibody level in the test sample relative to the control sample indicates no increased risk of ASD in the fetus or child, or a lower or reduced risk of ASD.

[0150] The difference between the control sample or the test sample only needs to be sufficient to be detectable. In some embodiments, an increase in maternal antibody levels in the test sample is determined to be an increased risk of ASD when antibody levels increase by at least 10%, 25%, 50%, 1-fold, 2-fold, 3-fold, 4-fold, or more compared to a negative control or a previously measured control.

[0151] To diagnose an increased likelihood of ASD in a fetus or child, it is possible to determine the presence of maternal antibodies against any subtype, isotype, or isoenzyme of the NSE antigen.

[0152] The maternal antibody can be detected using any method known in the art. Exemplary methods include, but are not limited to, Western blotting, dot blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence, and complex bead analysis (e.g., using Luminex or fluorescent microbeads).

[0153] Peptides can be antigenic fragments of NSE antigens. Peptides can be derived from known antigenic epitopes of NSE antigens, wherein one or more amino acids are substituted, deleted, added, or otherwise modified. Peptides can be purified or substantially purified from natural sources, or produced through recombinant or synthetic methods.

[0154] In some embodiments, the peptide for detecting the maternal antibody can be immobilized on a solid support. The solid support can be, for example, a multi-well plate, microarray, chip, bead, porous tape, or nitrocellulose filter. Immobilization can be achieved through covalent or non-covalent linkage. In some embodiments, immobilization is achieved by capturing antibodies that specifically bind to the target peptide.

[0155] To detect maternal antibodies, one or more peptides described herein can be incubated with the sample under conditions sufficient to allow specific binding of any antibody to one or more target antigens present in the sample (e.g., time, temperature, sample concentration). One or more peptides can be bound to a solid carrier. For example, one or more peptides can be exposed to the sample for about 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 hours, or overnight, or about 8, 10, 12, 14, or 16 hours. However, the incubation time can be more or less dependent on, for example, the composition of one or more peptides, the composition of one or more target antigens, the sample dilution, and the incubation temperature. Incubation times are shorter when using samples with lower dilutions and higher temperatures. Incubation is typically performed at room temperature (about 25°C) or biological temperature (about 37°C), but can also be performed in a refrigerator (about 4°C). Washing is performed before adding the secondary antibody to remove unbound sample, according to known immunoassay methods.

[0156] Labeled secondary antibodies are typically used to detect antibodies in samples that bind to one or more peptides described herein. Secondary antibodies bind to the constant region or “C” region of different classes or isotypes of immunoglobulins IgM, IgD, IgG, IgA, and IgE. Typically, secondary antibodies targeting the IgG constant region are used in this method. Secondary antibodies targeting IgG subclasses, such as IgG1, IgG2, IgG3, and IgG4, may also be used in this method. Secondary antibodies can be labeled with any directly or indirectly detectable portion, including fluorophores (e.g., fluorescein, phycoerythrin, quantum dots, luminescent beads, fluorescent beads), enzymes (e.g., peroxidase, alkaline phosphatase), and radioisotopes (e.g., [missing information]). 3 H, 32 P, 125 I) or chemiluminescent components. The labeling signal can be amplified using a complex of biotin and its binding moiety (e.g., avidin, streptavidin, neutral avidin). Fluorescently labeled anti-human IgG antibodies are available from Molecular Probes, Eugene, OR. Enzyme-labeled anti-human IgG antibodies are available from Sigma-Aldrich, St. Louis, MO and Chemicon, Temecula, CA.

[0157] The method for detecting the presence or difference of autoantibodies in a sample will be consistent with the choice of secondary antibody labeling. For example, if one or more peptides described herein are transferred to a membrane matrix suitable for immunoblotting, the detectable signal (i.e., the blot) can be quantified using a digital imager if enzyme labeling is used, or using an X-ray film developer if radioisotope labeling is used. In another example, if one or more peptides described herein are transferred to a multi-well plate, the detectable signal can be quantified using an automated plate reader capable of detecting and quantifying fluorescence, chemiluminescence, and / or chromaticity signals. Such detection methods are well known in the art.

[0158] General immunoassay techniques are well known in the field. Guidelines for parameter optimization can be found, for example, in Wu, Quantitative Immunoassay: A Practical Guide for Assay Establishment, Troubleshooting, and Clinical Application, 2000, AACC Press; Principles and Practice of Immunoassay, Price and Newman, eds., 1997, Groves Dictionaries, Inc.; The Immunoassay Handbook, Wild, ed+, 2005, Elsevier Science Ltd.; Ghindilis, Pavlov and Atanassov, Immunoassay Methods and Protocols, 2003, Humana Press; Harlow and Lane, Using Antibodies: A Laboratory Manual, 1998, Cold Spring Harbor Laboratory Press; and Immunoassay Automation: An Updated Guide to Systems, Chan, ed., 1996, Academic Press.

[0159] In some embodiments, the presence or increased presence of maternal antibodies is indicated by a detectable signal in an immunoassay (e.g., blotting, fluorescence, chemiluminescence, color, radioactivity), wherein a biological sample from a mother or potential mother is contacted with one or more peptides described herein. This detectable signal may be compared to a signal or threshold from a control sample. In some embodiments, an increased presence of ASD is detected when the detectable signal of maternal antibodies in the test sample is at least 10%, 20%, 30%, 50%, or 75% higher than the maternal antibody signal or a predetermined threshold in the control sample, indicating an increased risk of ASD. In some embodiments, an increased presence of ASD is detected when the detectable signal of maternal antibodies in the test sample is at least 1, 2, 3, 4, or more times higher than the maternal antibody signal or a predetermined threshold in the control sample, indicating an increased risk of ASD.

[0160] In some embodiments, the results of maternal antibody determination are recorded in a physical medium. For example, the results of current diagnostic analysis (e.g., observing the presence or increased presence of maternal antibodies) and the diagnosis determining whether there is an increased risk of ASD can be recorded on, for example, paper or electronic media (e.g., audio tape, computer disk, CD, flash drive, etc.).

[0161] In other embodiments, these methods further include the step of providing a diagnosis to a patient (i.e., a mother or potential mother) based on maternal antibody determination results to determine whether the patient’s fetus or child has an increased risk of developing ASD.

[0162] G. Risk reduction through the application of peptide epitopes

[0163] In some aspects, this disclosure provides a method for preventing and / or reducing the risk of autism spectrum disorder (ASD) in a fetus or child by administering an inhibitor (such as the NSE peptide or its mimicry epitope described herein, which specifically binds to maternal autoantibodies associated with ASD) to the mother or potential mother. The inhibitor prevents maternal antibodies from specifically binding to endogenous NSE autoantigens present in the fetus or child.

[0164] In some embodiments, the method includes administering at least one blocking agent to the mother or potential mother, the blocking agent comprising at least one or more peptides described herein (e.g., SEQ ID NOS:1-6) or their mimicry epitopes, such as at least 1, 2, 3, 4, 5, or 6 of the peptides described in SEQ ID NOS:1-6 or their mimicry epitopes. In some cases, the blocking agent comprises a peptide corresponding to SEQ ID NOS:1-6 or its mimicry epitopes and combinations thereof. In some cases, the blocking agent specifically binds to maternal antibodies that recognize NSE antigens.

[0165] The preventive and / or therapeutic methods of this disclosure, using one or more blocking agents, may be provided to women before, during, or after pregnancy. In some embodiments, one or more blocking agents may be administered once, twice, three times, four times, or more, as appropriate, at any time during pregnancy. For example, one or more blocking agents may be administered during one or more periods of early, mid, and / or late pregnancy. In some embodiments, one or more blocking agents are administered to women carrying a fetus whose brain has begun to develop, for example, after approximately 12 weeks of pregnancy. In some embodiments, one or more blocking agents are evaluated after delivery, for example, during the first four weeks after birth and / or while the mother is breastfeeding the child. In some embodiments, one or more blocking agents are administered before pregnancy, for example, to women who test positive for maternal antibodies and women attempting to conceive.

[0166] In some embodiments, a plurality of reagents comprising two or more peptides or their mimic epitopes are applied. The reagents may be applied individually or together. The reagents may be a pool of single peptides or mimic epitopes. In some embodiments, two or more peptides or mimic epitopes with different epitopes are chemically linked. Multiple antigenic epitopes may originate from the same or different antigenic polypeptides. In this case, the chemical linking may be a direct linking of peptides or a linking process using a chemical scaffold or linker. In some embodiments, two or more peptides or mimic epitopes with different peptide epitopes are fused together. Peptide epitope fusion may be recombinant expression or chemically synthesized.

[0167] In some embodiments, the method further includes the step of administering a treatment or prevention regimen of one or more blocking agents (e.g., one or more peptides of SEQ ID NOS: 1-6 or their mimic epitopes) to the mother or potential mother to reduce, inhibit, or prevent the binding of maternal autoantibodies to NSE antigens.

[0168] In some cases, the applied blocking agent or multiple blocking agents that reduce, inhibit, or prevent the binding of maternal antibodies to NSE peptides comprise one, two, three, four, five, or six peptides as described in SEQ ID NOS:1-6, or antigenic fragments or mimic epitopes thereof. In other cases, the applied blocking agent or multiple blocking agents that reduce, inhibit, or prevent the binding of maternal antibodies to NSE peptides comprise one, two, three, four, five, or six peptides as described in SEQ ID NOS:1-6, or antigenic fragments or mimic epitopes thereof.

[0169] Administered inhibitors may contain modifications that reduce or minimize their immunogenicity. Modifications to amino acids in peptides or mimic epitopes include, but are not limited to, amide moieties or pyroglutyl residues, or the addition of polyethylene glycol chains (PEGylation). These modifications may help reduce the tendency to form R-sheet conformations or contribute to reduced peptide stability, solubility, and immunogenicity. In some cases, peptides that are more stable, more soluble, and less immunogenic are desired. Many peptides modified with a CONH2 (amide) group at the C-terminus appear to be resistant to carboxypeptidase attack, while many peptides with a pyroglutyl residue at the N-terminus are more resistant to broad-specific aminopeptidase attack. PEGylated peptides have been shown to increase plasma half-life and reduce immunogenicity compared to unmodified peptides. Furthermore, sequence analysis of inhibitors will allow for minimization of known T-cell epitopes through conserved modifications. The peptides described herein also include cyclic peptides that are resistant to both carboxypeptidase and aminopeptidase attack. Additionally, oral administration of inhibitors may help minimize immunogenicity.

[0170] In some embodiments, prevention and / or treatment methods include the step of first determining the presence or increased presence of maternal antibodies binding to the NSE antigen in the mother or potential mother using the detection methods described herein. Women who test positive or at a level above the threshold for maternal antibody presence are candidates for receiving one or more blocking agents that specifically bind to maternal antibodies. Women who test negative or at a level below the threshold for maternal antibody presence do not need to receive one or more blocking agents that specifically bind to maternal antibodies.

[0171] Suitable pharmaceutical compositions for use in this disclosure include compositions containing a therapeutically effective amount of the active ingredient. Of course, the amount of the composition administered depends on the subject being treated, the subject's weight, the severity of suffering, the method of administration, and the prescribing physician's judgment. Determining the effective amount is entirely within the capabilities of those skilled in the art, especially based on the detailed disclosure provided herein. Typically, the effective amount or amount of one or more parent antibody blockers is determined as follows: a low dose or small amount of the blocker is initially administered, followed by a gradual increase in the dose, and / or the addition of one or more second blockers as needed, until the desired effect is observed in the treated subject, such as elimination or reduction of the presence of unbound or free parent antibodies to below a predetermined threshold level, with minimal or no toxicity or adverse side effects. Applicable methods for determining the appropriate dosage and timing of administration of the pharmaceutical compositions disclosed herein are described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., Bmnton, et al, Eds., McGraw-Hill (2006), and Remington: The Science and Practice of Pharmacy, 21st Ed., University of the Sciences in Philadelphia (USIP), 2005, Lippincott, Williams and Wilkins.

[0172] Dosage and intervals can be individually adjusted to provide sufficient plasma or tissue levels of one or more blockers to maintain therapeutic efficacy. Single or multiple administrations of a composition containing an effective amount of one or more blockers can be performed according to a dosage level and pattern selected by the treating physician. Dosage and administration timing can be determined and adjusted, for example, based on maternal antibody levels in the mother or potential mother, and can be monitored throughout treatment according to methods commonly used by clinicians or methods described herein. In some embodiments, therapeutic levels will be achieved by administering a single daily dose. In other embodiments, administration timing may include multiple daily dosing schedules. In other embodiments, this disclosure includes administration every other day, every half week, or weekly.

[0173] For example, one or more blocking agents may be administered monthly, every two weeks, weekly, or daily as needed. In some embodiments, the level of maternal antibodies in the mother or potential mother is monitored, and one or more blocking agents are administered if maternal antibodies are present or if the level of maternal antibodies exceeds a predetermined threshold level. One or more blocking agents may be administered for approximately 1, 2, 3, 4, 5, 10, 12, 15, 20, 24, 30, 32, or 36 weeks, or for a longer or shorter period as appropriate. For example, if the level of maternal antibodies falls below a predetermined threshold level, administration of one or more blocking agents may be discontinued. One or more blocking agents may be administered throughout the pregnancy, or during one or more periods of the early, middle, or late stages of pregnancy. Administration may begin before pregnancy or continue after birth, while the mother is breastfeeding the child.

[0174] In some embodiments where one or more blockers are peptides or their mimic epitopes, typical doses can be from about 0.1 μg / kg body weight to about 1 g / kg body weight, and include a range from about 1 g / kg body weight, for example, from 1 μg / kg body weight to about 500 mg / kg body weight. In some embodiments, the dose of the peptide or mimic epitope is about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg body weight.

[0175] The exact dosage depends on various factors described herein, including the specific inhibitor, disease severity, and route of administration. Determining the exact therapeutically effective dosage can be done by a clinician without excessive experimentation and may include any dosage within the range disclosed above.

[0176] One or more blocking agents are administered via a route of administration that allows the blocking agent to bind to maternal antibodies and prevents the antibodies from binding to endogenous autoantigens associated with the risk of developing ASD, and minimizes the immune response to the agent. Typically, these drugs are administered systemically. In some embodiments, one or more agents are administered via a parenteral route, such as intravenous injection or intraamniotic administration (i.e., direct entry into the amniotic sac). Alternatively, one or more agents may be administered orally.

[0177] One or more blockers for parenteral administration can be formulated by injection (e.g., by bolus injection or continuous infusion). For injection, one or more blockers can be formulated as follows: one or more blockers can be dissolved, suspended, or emulsified in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oil, synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol; and, if desired, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives can be used. In some embodiments, the combination of blockers can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer. Injectable formulations can be presented in a single dosage form, such as in ampoules or multi-dose containers, with added preservatives. The composition can be in the form of a suspension, solution, or emulsion in an oily or aqueous carrier, and may contain formulations such as suspending agents, stabilizers, and / or dispersants.

[0178] Pharmaceutical formulations intended for parenteral administration include aqueous solutions of one or more inhibitors in water-soluble form. Alternatively, suspensions of one or more inhibitors may be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to prepare a high-concentration solution. Alternatively, one or more inhibitors may be in powder form to be mixed with a suitable carrier, such as sterile, pyrogen-free water, prior to use.

[0179] Treatment with one or more blocking agents is considered effective if the level or titer of maternal antibodies that actively bind to NSE antigens is reduced or eliminated in a biological sample from an individual after one or more administrations of one or more blocking agents compared to before administration. For example, a reduction of approximately 10%, 25%, 50%, 75%, or 100% in maternal antibodies that actively bind to NSE antigens in a sample after one or more administrations of one or more blocking agents indicates that administration of one or more blocking agents is effective. Where a threshold level has been determined, treatment with one or more blocking agents is considered effective if the level or titer of maternal antibodies that actively bind to NSE antigens decreases below the threshold level. Maternal antibodies that actively bind to NSE antigens can be measured using any method known in the art, including the method described herein.

[0180] H. Methods to reduce risk by removing maternal antibodies

[0181] In some aspects, this disclosure provides a method for preventing or reducing the risk of autism spectrum disorder (ASD) in offspring, such as fetuses or children, by removing maternal antibodies from a biological fluid of the mother or potential mother in vitro, and then returning the biological fluid with reduced or eliminated maternal antibody levels to the mother or potential mother.

[0182] In some embodiments, the biofluid containing maternal antibodies may be removed from the mother or potential mother and contacted with one or more peptides described herein. In other embodiments, one or more peptides described herein may be administered to the mother or potential mother to block the binding between maternal autoantibodies and their own antigens in the biofluid, thereby neutralizing the maternal autoantibodies, and the neutralizing complex present in the biofluid may be removed using in vitro therapies, such as affinity plasma purification techniques.

[0183] In some embodiments, a biofluid from the mother or potential mother is contacted with one or more peptides immobilized on a solid support. The solid support can be, for example, a multiwell plate, ELISA plate, microarray, chip, bead, column, porous tape, membrane, or nitrocellulose filter. Immobilization can be covalent or non-covalent. In some embodiments, immobilization is performed by a capture antibody that specifically binds to a target peptide epitope. The one or more peptides linked to the solid support form a stationary phase that captures maternal antibodies in the biofluid, thereby separating the biofluid with reduced or eliminated maternal antibody levels from the solid support, i.e., as a mobile phase, and returning it to the mother or potential mother.

[0184] In some embodiments, the biofluid processed in vitro is plasma, and maternal antibodies are removed by plasma removal methods, a process well known in the art. The plasma is contacted with a solid carrier having one or more immobilized peptides. Maternal antibodies in the plasma bind to the immobilized peptides. Plasma with reduced or eliminated maternal antibody levels is then returned to the mother or potential mother.

[0185] In vitro removal of maternal antibodies can be performed before, during, or after a woman's pregnancy. In some embodiments, maternal antibodies may be removed from the biological fluid once, twice, three times, four times, or more at any time during pregnancy, as appropriate. For example, maternal antibodies may be removed at one or more stages of early, mid, and / or late pregnancy. In some embodiments, maternal antibodies are removed from women carrying fetuses whose brains have begun to develop, for example, after approximately 12 weeks of pregnancy. In some embodiments, maternal antibodies are removed once or more postpartum, for example, in the first four weeks after birth and / or while the mother is breastfeeding the child. In some embodiments, maternal antibodies are removed once or more before pregnancy, for example, by women who test positive for maternal antibodies and women attempting to conceive.

[0186] As needed, one, two, three, four, or more in vitro maternal antibody removal procedures may be performed to eliminate or reduce maternal antibodies from the mother or potential mother. In vitro removal of maternal antibodies may be performed daily, weekly, bi-weekly, monthly, or bi-monthly, as appropriate. In some embodiments, maternal antibody levels in the mother or potential mother are monitored, and in vitro maternal antibody removal is performed if the presence of maternal antibodies exceeds a predetermined threshold level. In vitro maternal antibody removal may be performed at 1, 2, 3, 4, 5, 10, 12, 15, 20, 25, 35, 36 weeks or longer or shorter timeframes, as appropriate. For example, if maternal antibody levels fall below a predetermined threshold level, in vitro maternal antibody removal may be discontinued. In vitro maternal antibody removal may be performed throughout pregnancy or during one or more periods in the early, middle, or late stages of pregnancy. Maternal antibody removal may begin before pregnancy and may continue after birth, for example, while the mother is breastfeeding the child.

[0187] Biofluids containing maternal antibodies are typically blood, serum, plasma, or breast milk. In some embodiments, the biofluid is amniotic fluid.

[0188] Example

[0189] This disclosure will be described in more detail through specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit this disclosure in any way.

[0190] Example 1. Materials and Methods

[0191] 1.1 Study Subjects

[0192] This study included mothers who participated in the CHARGE study (Genetic and Environmental Risks for Childhood Autism) at the MIND Institute at UC Davis.

[19] Participants in the CHARGE study included mothers diagnosed with ASD (n=246) and mothers of children (normal development, TD; n=149) from the general population. We used the recruitment, eligibility, and psychometric assessment procedures described above.[7, 19] The diagnosis of ASD was validated by the MIND Institute in accordance with the Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5).

[20] All procedures were approved by the California Human Subject Protection Committee and Institutional Review Committee of UC Davis and UCLA. Twenty participants provided written informed consent in English or Spanish prior to participation. Demographic information associated with these samples is shown in Table 1.

[0193] Table 1 shows the demographics of the study population. It indicates the average age of the mothers at birth and the average age of the children at the time of sample collection.

[0194]

[0195] Abbreviations: ASD, Autism Spectrum Disorder; TD, Normal Development; SD, Standard Deviation; Max, Maximum Age; Min, Minimum Age. a Participants in the CHARGE study on the risk of childhood autism from genetics and environment.

[0196] 1.2 Sample Collection and Preparation

[0197] Blood was collected in a citrate-glucose (BD-diagnostic) solution, and the plasma was separated, coded, aliquoted, and stored at -80°C. Before use, the samples were thawed and centrifuged at 13,000 RPM for 10 minutes.

[0198] 1.3 Preparation of fetal brain antigen

[0199] Tissue processing was performed as described above [8]. Briefly, we used 152-day-old embryonic rhesus monkey brains (FMBs) from the California National Primate Research Center. The FMBs were mechanically homogenized with buffer using a Polytron 3000 homogenizer (Brinkman), sonicated for 3 minutes, and centrifuged at 3000×g for 10 minutes. The supernatant was then collected, concentrated by ultrafiltration, and its protein content was measured by the quinoline carboxylic acid assay (BCA).

[0200] 1.4 Cell Preparation (Prep Cell)

[0201] Protein separation was performed as described above [8]. Briefly, 40 mg FMB was electrophoresed and separated by molecular weight as follows: 17 hours on a 10% polyacrylamide gel at 12 watts using a Prep Cell device (Bio-Rad, Hercules, CA). Protein fractions were collected every 5 minutes at a flow rate of 0.75 ml / min. A total of 110 fractions were obtained, concentrated to 5 mg / ml by ultrafiltration, and their molecular weight and antigenic reaction were determined by Western blotting (WB). Figure 1A-1D Ponceau S staining confirmed protein enrichment and fractionation, ranging from approximately 5 kDa per fraction. Fraction #12 contained proteins between 37 and 45 kDa and was therefore selected for antigen recognition. Figures 2A-2E ).

[0202] 1.5 Protein Blotting

[0203] To test the reactivity of autoantibodies to FMB component #12, which contains proteins between 37 and 45 kDa, the component was detected using maternal plasma samples, as previously described. Figure 1D[8]. In summary, 200 μg of protein was denatured by heating in SDS buffer at 100 °C for 10 min and separated on a 12% SDS-PAGE gel at 200 V for 1 h. The protein was transferred to a 0.2 μm nitrocellulose membrane overnight at 4 °C (10 V for 16 h). To confirm the transfer, the membrane was stained with Ponceau S dye and cut into 3 mm strips, which were labeled with 1% casein buffer and blocked. Plasma samples were then diluted (1:400) and added to the strips, incubated at RT for 1.5 h, followed by five washes and incubation with 1:20,000 goat anti-human IgG HRP for 30 min. After five washes, detection was performed by adding 800 μl of Super Signal substrate and the strips were placed on a glass plate and imaged using a FluoroChem 8900 imager. If negative, the image score was 0, and if positive, the image score was 1.

[0204] 1.6 Two-dimensional (2-D) gel electrophoresis

[0205] As previously described, maternal autoantibody-targeted protein fractions were separated by two-dimensional electrophoresis [8]. Briefly, 300 μg of protein fractions in the 30–40 kDa range were labeled with Cy2 (GE Life Sciences, Pittsburgh, PA, USA) to prepare for two-dimensional electrophoresis (all gels were repeated). First, 15 μg of each sample was separated by its isoelectric point using a 3–10 isoelectric focusing band (GE Healthcare, Piscataway, NJ, USA). The bands were then loaded onto 2 x 10.5% polyacrylamide gels (GE Healthcare) for two-dimensional electrophoresis. Images were captured using Quant software (version 6.0, GE Healthcare). One of the gels was transferred to a nitrocellulose membrane to determine maternal plasma that reacted to the bands near 37–39 kDa but did not react to GDA, LDHA / B, and YBX1 by Western blotting. The resulting positive spots were mapped back to repeated two-dimensional gels stained with Cy2, extracted from the gels, and digested with trypsin (Promega, Madison, WI, USA) to prepare for mass spectrometry analysis.

[0206] 1.7 Mass Spectrometry

[0207] Mass spectrometry analysis was performed as described in our previous report [8]. Digested peptides were desalted (Zip-tip Cl8, Millipore, Billerica, MA, USA) and formed spots on MALDI plates (ABI 01-192-6-AB type). MALDI-TOF MS and TOF / TOF tandem MS / MS data were obtained using an ABI 4700 mass spectrometer (Applied Biosystems, Framingham, MA). The obtained peptide mass and associated fragment spectra were analyzed using a GPS Explorer workstation equipped with the MASCOT search engine (Matrix Science, Boston, MA, USA) and used for BLAST search on NCBI. Candidates with a protein score confidence interval (CI%) or ion CI% greater than 95 were considered positive (Table 1).

[0208] The top four commercially available antigens identified by mass spectrometry were selected at 100 CI for further evaluation. To assess antibody reactivity against our top hits, including NSE, NNE, ALDOC, and CKB, as previously described, 2 μg of recombinant protein was detected by Western blotting in diluted maternal plasma (1:800) (Novus Biological, Littleton, CO).

[0209] 1.8 Enzyme-linked immunosorbent assay (ELISA)

[0210] Once NSE was identified as a viable antigen candidate by Western blotting (WB), we evaluated a larger set of NSE-reactive samples using an ELISA method. We tested plasma from 418 mothers of children with at least one ASD (n=232) participating in the CHARGE study, or control samples from mothers of normally developing children (TD; n=186). Microtiter plates were incubated overnight at 4°C with 100 μl NSE (Novus Biological, Littleton, CO) (2 μg / ml in carbonate-coated buffer at pH 9.6), washed four times with 0.05% PBST, and blocked for 1 hour at room temperature (RT) with 2% Super Block (Thermo Scientific, Rockford, 1L). Plasma samples were diluted 1:500 and run in duplicate. After dilution, 100 μl of diluted sample was added to each well, incubated for 1.5 hours, washed four times, and then incubated for 1 hour with 1:10,000 goat anti-human IgG HRPIgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MA). The plate was then washed and detected by adding 100 μl of BD-optEIA fluid substrate for ELISA (BD Biosciences, San Jose, CA). After 4 minutes, the reaction was stopped with 50 μl of 2N HCl. The absorbance was measured at 490–450 nm using an iMark microplate absorbance reader (Biorad, Hercules, CA, USA).

[0211] 1.9 Receiver Operating Characteristic (ROC) Curve

[0212] For ELISA analysis, ROC curves were used to determine the positive cutoff value for NSE reactivity. ROC curves were created by plotting the true positive rate and false positive rate under various threshold settings. Therefore, we created our curve using seven positive samples (labeled +) from mothers with children suffering from ASD who were also WB-positive (true positive samples) and the test sample. By using positive samples as reference events, the cutoff value had higher specificity (fewer false positives), although some sensitivity (limit of detection) was sacrificed. ROC plots were used to graph the sensitivity versus 1-specificity for each value, creating the area under the curve (AUC) representing test accuracy. The Udon index was used to calculate the cutoff value [21, 22].

[0213] 1.10 Microarray Screening

[0214] The complete NSE sequence (NP_001966.1) was obtained from NCBI and translated into a library of consecutive 15-mer peptides on a microarray slide, with each peptide overlapping by 14 amino acids (aa). As previously mentioned, the peptide microarray was found to be synthesized by PEPperPRINT

[23] , thereby allowing the targeted 15-mer peptide sequences to be directly printed onto the slide in duplicate using solid-phase Fmoc chemistry (PEPperPRINT, Heidelberg, Germany). Peptides derived from human influenza hemagglutinin (HA) (YPYDVPDYAG) and polio vaccine (KEVPALTAVETGAT) were also included as positive controls.

[0215] To test antibody reactivity against the printed peptide, we used plasma probe arrays from mothers participating in the CHARGE study (ASD=27 and TD=22) according to the manufacturer's instructions. Microarray slides were first incubated for 10 minutes with standard buffer (PBS containing 0.05% Tween 20, pH 7.4) and then blocked at RT for 45 minutes (Rockland Blocking Buffer MB-070; Rockland Immunochemicals Inc.). The slides were then incubated overnight with a single maternal plasma sample diluted 1:250 in staining buffer at 4°C with shaking, followed by washing three times in standard buffer. For signal detection, the slides were incubated at RT for 30 minutes with goat anti-human IG (H+L)-DyLight649 diluted 1:5000 in staining buffer (standard buffer containing 10% blocking buffer). After incubation with the secondary antibody, the microarray was imaged using a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, California).

[0216] Following the manufacturer's recommendations, fluorescence signal quantification of spot intensity (FI) and peptide annotation was performed using PEPperPRINT Analyser software (PEPperPRINT). Data preprocessing methods were as reported in previous peptide microarray studies. Briefly, net fluorescence intensity (FI) was calculated using the correction method reported by Zue et al. [24,25]. A 3x2 window was set for each spot, and the median of the six spots was used as the “neighborhood background” for the center spot. To normalize the net fluorescence intensity (FI), a 3x1 “sliding window” was set for each spot, and the median of the three was used as the normalized signal for the center spot [24,25]. The corrected net intensity was calculated by subtracting the corrected background from the normalized signal. If the background signal in the background was higher than that of the spot (negative FI), the signal was set to 1, following similar studies [26,27].

[0217] Finally, after background correction and signal normalization, the corrected net signal was obtained by calculating the median of the repeating signals, and the coefficient of variation was calculated. Samples with a CV higher than 50% were labeled and corrected. Due to non-specific binding, FI values ​​below 200 were considered negative, and only sequences with values ​​above 200 were considered positive for statistical analysis [26, 27].

[0218] Statistical analysis

[0219] To thoroughly examine the significantly different sequence data between the diagnostic groups and to identify specific epitopes for a particular group (TD or ASD), we used two different analytical methods: 1) the T-test—a parametric test that allows us to compare two independent samples by means of difference, assuming a normal distribution of the data; and 2) microarray significance analysis (SAM)—a permutation-based method used to measure the strength of the relationship between epitope expression and response variables, in this case, ASD and TD diagnoses. SAM scores are directly proportional to the significance of the data relationship (maximum score = 2). T-tests were performed using XLSTAT 2015.1 software (Addinsoft, Paris, France), and SAM analyses were run using the R statistical computing environment. Additionally, we compared the generality of epitope reactivity between the ASD and TD groups using Fisher's exact test. A p-value < 0.05 was considered significant. The odds ratio (OR 95% CI) of important sequences was calculated using GraphPad Prism software (GraphPad Software, San Diego, CA).

[0220] Example 2. Antigen recognition

[0221] Fetal monkey brain (FMB) was divided into 110 fractions according to molecular weight, and fraction #12 (Figures 1b and 1C), which contained proteins with molecular weights of 37-45 kDa, was analyzed by 2-D gel / protein blot pair analysis. Figures 2A-2E A gel was transferred onto a nitrocellulose membrane and used to verify the autoantibody reactivity of mothers of children with ASD to proteins in the 37-45 kDa range (Figures IB and 1C), which were negative by Western blotting for autoantigens (GDA, LDHA, LDHB, and YBX1) within the aforementioned molecular weight range. Figure 1D Multiple spots were observed, and all identified spots were collected from a second matched 2D gel for mass spectrometry analysis. Figures 2A-2E Proteins in the 37-45 kDa range were selected for validation using 100% CI. Table 2 lists the detailed mass spectrometry results for the validation antigens.

[0222] Table 2 Figure 2E Overview of mass spectrometry results for each selected spot

[0223]

[0224] The proteins in spots 1, 6, 8 and 21-25 are involved in the glycolysis-gluconeogenesis pathway.

[0225] The top four commercially available proteins recognized by the maternal autoantibody were selected with 100% CI for further evaluation, including neuron-specific enolase (NSE), non-specific enolase (NNE), fructose-2-bisphosphate aldolase C (ALDOC), and creatinine kinase B (CKB). Each protein was tested using recombinant proteins to assess the reactivity of the maternal autoantibody against a single antigen. Subsequently, NSE was recognized by the maternal sample as corresponding to the 37–45 kDa band, exhibiting the highest specificity among the identified tested samples, and was therefore selected as the most likely candidate for additional MAR ASD target autoantigens.

[0226] Example 3. Antigen Validation

[0227] NSE was identified by mass spectrometry as a potential target for maternal autoantibodies, and based on its crucial role in neurodevelopment, we chose to further evaluate NSE as a potential biomarker for MAR ASD. Recombinant NSE reactivity was first verified by Western blotting followed by ELISA. Reactivity was observed in 26 of 232 mothers of children with ASD (6.2%) and in 21 of 186 mothers of normally developing children (TD, 5%), indicating that NSE alone is not a biomarker for MARASD. Therefore, we used a method similar to that described previously for the seven MAR autoantigens to probe samples to identify differential epitopes between the ASD and TD groups.

[0228] Example 4. Tabletop plotting

[0229] The complete NSE sequence (NP_001966.1) was translated into 434 distinct 15-mer peptides with 14 overlapping amino acids (AAs), and two copies were printed on a glass microarray. These were then tested using diluted plasma from mothers in the ASD group and control group. After data preprocessing, samples were categorized into two groups based on ELISA reactivity (positive: samples with antibodies against NSE; negative: samples negative for the native form of NSE but potentially reactive to occult epitopes) for statistical analysis. For ELISA(+) samples, we found 16 sequences with ASD specificity (0% TD), and 5 sequences were recognized by both antibody groups (FI > 200). Among the 16 ASD-specific sequences, 4 sequences were statistically significant using t-tests and SAM t-tests (Table 3). DVAASEFYRDGKYDL (SEQ ID NO:1) (p = 0.047; SAM score 1.49), IEDPDQDDWAAWSK (SEQ ID NO:2) (p = 0.049; SAM score 1.49), ERLAKYNQLMRIEEE (SEQ ID NO:3) (p = 0.045; SAM score 1.57) and RLAKYNQLMRIEEEL (SEQ ID NO:4) (p = 0.017; SAM score 1.82).

[0230] Table 3. Overview of Important NSE Epitopes Recognized by Maternal Autoantibodies (ELISA Positive)

[0231]

[0232] Abbreviations: ES, Epitope Sequence; ASD, Autism Spectrum Disorder; TD, Normal Development.

[0233] Additionally, to assess the relevance of epitope sequences to a given group, we used Fisher's exact test, which found no significant differences, possibly due to our small sample size. Instead, we calculated the odds ratio (OR) for each individual peptide's 95% confidence interval (95% CIs). We found that all ASD-specific sequences had ORs greater than 3, with SERLAKYNQLMRIEE (SEQ ID NO:6) (OR 10.1, CI 95% 0.5094 to 200.7) and ERLAKYNQLMRIEEE (SEQ ID NO:3) (OR 12.6, CI 95% 0.6408 to 247.7) being the two epitopes with the highest ORs. Figure 3As described above, we found five consecutive epitope sequences recognizable in the plasma of both sample groups, indicating that large immunodominant epitopes include the printed sequences DYPVVSIEDPFDQDD (SEQ ID NO:7), YPVVSIEDPFDQDDW (SEQ ID NO:8), PVVSIEDPFDQDDWA (SEQ ID NO:9), VVSIEDPFDQDDWAA (SEQ ID NO:10), and VSIEDDPFDQDDWAAW (SEQ ID NO:11) (Table 3). Figure 3 As shown, the sequences highlighted in red represent the conserved amino acids recognized by the antibodies in each of the five different peptide epitopes. Responses to the large, dominant immunogenic epitope were also observed in ELISA(-) samples, indicating that it is a mimic epitope primarily recognized by the general population (Table 4). Interestingly, we also found an ASD-specific epitope sequence, QDFVRDYPVVSIEDP (p = 0.054, SAM score 1.97, OR 12.6, CI 95% 0.6408 to 247.7; SEQ ID NO: 23), which was confirmed by ELISA(-) samples, suggesting that it may not respond to the native structure of NSE and is more likely to bind to a recessive determinant (Table 4).

[0234]

[0235] Example 5. Bioinformatics

[0236] To better understand the potential reactive sources of the recently identified epitopes, we used the Immunoepitaxy Database (IEDB) tool to analyze the homology of the epitopes with all epitopes reported in the IEBDB database. We performed BLAST searches at 90% and 80% sequence homology settings and found that each identified sequence was homologous to other enolases, primarily α-enolases, with 90% homology (Table 5). The epitopes DYPVVSIEDPFDQDD (SEQ ID NO:7) and DFVRDYPVVSIEDPF (SEQ ID NO:16) each showed 90% homology to the protein ORF73 of human gamma herpesvirus 8 (monocytosis pathogen), and DVAASEFYRDGKYDL (SEQ ID NO:1) showed 90% homology to the outer surface protein A of Borrelia burgdorferi (Lyme disease pathogen). Other sequences share 80% homology with peptides from different organisms, including the hepatitis C virus genomic polyprotein, the human β-herpesvirus 6B viral particle packaging protein UL25, the Streptococcus aureus Alt a6, the Vibrio cholerae ATP-dependent RNA helicase RhlB, and the hepatitis B virus protein X (Table 5).

[0237]

[0238]

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[0278] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or alterations will be suggested to those skilled in the art based thereon and will be included within the spirit and scope of this application and the appended claims. All publications, patents, patent applications and serial numbers cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. An isolated peptide, wherein the complete amino acid sequence of the peptide is as shown in SEQ ID NO:3 or 4.

2. The peptide of claim 1, wherein the peptide is bound to a parent antibody that binds to neuron-specific enolase (NSE) protein.

3. The peptide according to claim 1 or 2, wherein the peptide is a mimic epitope.

4. The peptide of claim 3, wherein the mimic epitope comprises a D-amino acid.

5. The peptide according to claim 1 or 2, wherein the peptide further comprises a label.

6. The peptide according to claim 5, wherein the label is selected from the group consisting of biotin, fluorescent label, chemiluminescent label, and radioactive label.

7. A composition comprising a peptide or multiple peptides according to any one of claims 1 to 6.

8. The composition according to claim 7, wherein the composition further comprises a pharmaceutically acceptable carrier.

9. The composition according to claim 7 or 8, wherein the plurality of peptides comprises at least two different peptides.

10. The composition of claim 9, wherein the different peptides bind to the same parent antibody.

11. A kit comprising a peptide or multiple peptides according to any one of claims 1 to 6 and a solid carrier.

12. The kit according to claim 11, wherein the solid carrier is a multi-well plate, ELISA plate, microarray, chip, bead, porous tape or nitrocellulose filter.

13. The kit according to claim 11 or 12, wherein the peptide or multiple peptides are immobilized on the solid support.

14. The kit according to claim 11 or 12, wherein the plurality of peptides comprises at least two different peptides.

15. The kit of claim 14, wherein the different peptides bind to the same parent antibody.

16. The kit according to claim 11 or 12, further comprising instructions for use.

17. The use of the peptide or multiple peptides according to any one of claims 1 to 6 in the preparation of a diagnostic reagent for detecting maternal antibodies bound to said peptide or multiple peptides to predict the risk of autism spectrum disorder (ASD) in offspring, wherein said detection is performed on a biological sample from the mother or potential mother of said offspring.

18. The application according to claim 17, wherein the sample is blood, serum, plasma, amniotic fluid, breast milk, or saliva.

19. The application according to claim 17 or 18, wherein the plurality of peptides comprises at least two different peptides.

20. The application according to claim 19, wherein the different peptides bind to the same parent antibody.

21. The application according to claim 17 or 18, wherein the peptide or peptides are attached to a solid carrier.

22. The application according to claim 21, wherein the solid carrier is a porous plate, ELISA plate, microarray, chip, bead, porous tape or nitrocellulose filter.

23. The application according to claim 17 or 18, wherein the parent antibody is detected by Western blotting, dot blot, ELISA, radioimmunoassay, immunoprecipitation, electrochemiluminescence, immunofluorescence, FACS analysis or multiplex bead analysis.

24. The application according to claim 17 or 18, wherein the mother or potential mother has a child with ASD.

25. The application according to claim 17 or 18, wherein the mother or potential mother has a family history of ASD or an autoimmune disease.

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