Molecular dissection of an immunodominant epitope in Kv1.2-exclusive autoimmunity

Introduction Subgroups of autoantibodies directed against voltage-gated potassium channel (Kv) complex components have been associated with immunotherapy-responsive clinical syndromes. The high prevalence and the role of autoantibodies directly binding Kv remain, however, controversial. Our objective was to determine Kv autoantibody binding requirements and to clarify their contribution to the observed immune response. Methods Binding epitopes were studied in sera (n = 36) and cerebrospinal fluid (CSF) (n = 12) from a patient cohort positive for Kv1.2 but negative for 32 common neurological autoantigens and controls (sera n = 18 and CSF n = 5) by phospho and deep mutational scans. Autoantibody specificity and contribution to the observed immune response were resolved on recombinant cells, cerebellum slices, and nerve fibers. Results 83% of the patients (30/36) within the studied cohort shared one out of the two major binding epitopes with Kv1.2-3 reactivity. Eleven percent (4/36) of the serum samples showed no binding. Fingerprinting resolved close to identical sequence requirements for both shared epitopes. Kv autoantibody response is directed against juxtaparanodal regions in peripheral nerves and the axon initial segment in central nervous system neurons and exclusively mediated by the shared epitopes. Discussion Systematic mapping revealed two shared autoimmune responses, with one dominant Kv1.2-3 autoantibody epitope being unexpectedly prevalent. The conservation of the molecular binding requirements among these patients indicates a uniform autoantibody repertoire with monospecific reactivity. The enhanced sensitivity of the epitope-based (10/12) compared with that of the cell-based detection (7/12) highlights its use for detection. The determined immunodominant epitope is also the primary immune response visible in tissue, suggesting a diagnostic significance and a specific value for routine screening.

Introduction: Subgroups of autoantibodies directed against voltage-gated potassium channel (K v ) complex components have been associated with immunotherapy-responsive clinical syndromes.The high prevalence and the role of autoantibodies directly binding K v remain, however, controversial.Our objective was to determine K v autoantibody binding requirements and to clarify their contribution to the observed immune response.
Methods: Binding epitopes were studied in sera (n = 36) and cerebrospinal fluid (CSF) (n = 12) from a patient cohort positive for K v 1.2 but negative for 32 common neurological autoantigens and controls (sera n = 18 and CSF n = 5) by phospho and deep mutational scans.Autoantibody specificity and contribution to the observed immune response were resolved on recombinant cells, cerebellum slices, and nerve fibers.
Results: 83% of the patients (30/36) within the studied cohort shared one out of the two major binding epitopes with K v 1.2-3 reactivity.Eleven percent (4/36) of the serum samples showed no binding.Fingerprinting resolved close to identical sequence requirements for both shared epitopes.K v autoantibody response is directed against juxtaparanodal regions in peripheral nerves and the axon initial segment in central nervous system neurons and exclusively mediated by the shared epitopes.
Discussion: Systematic mapping revealed two shared autoimmune responses, with one dominant K v 1.2-3 autoantibody epitope being unexpectedly prevalent.
The conservation of the molecular binding requirements among these patients indicates a uniform autoantibody repertoire with monospecific reactivity.The enhanced sensitivity of the epitope-based (10/12) compared with that of the cellbased detection (7/12) highlights its use for detection.The determined immunodominant epitope is also the primary immune response visible in tissue, suggesting a diagnostic significance and a specific value for routine screening.KEYWORDS autoantibodies, K v channel, KCNA2, autoimmune encephalitis, epitope mapping, immunodominant antigen, dementia, critical

Introduction
Neurological diseases associated with autoantibodies are increasingly recognized as new medical entities (1) but frequently remain to be fully resolved at the molecular level.The identification of exact underlying disease-defining autoantibody epitopes is critical for understanding and addressing the root cause of these clinical entities.Advanced peptide microarray technologies demonstrated a remarkable success in resolving comprehensive linear epitope landscapes from raw patient samples (2,3).Here, we used a peptide microarray-based readout (4) for analyzing the largest so far studied cohort with K v 1.2-exclusive immune response in molecular detail.
Autoantibodies directed against K v 1 channel complexes have been identified in several neurological diseases, including autoimmune encephalitis, limbic encephalitis, and Morvan's syndrome (5,6).Within this group, anti-leucine-rich glioma inactivated 1 (LGI1) and anti-Contactin-associated protein-like 2 (Caspr2) autoantibodies are among the most prevalent, and these have been associated with clinical syndromes that are immunotherapy-responsive (7,8).Previous studies also identified highly prevalent intracellular binding of K v 1 antibodies, many of which are targeting intracellular epitopes.Only a fraction of the intracellular positive K v 1 patients (27%) showed a sustained immunotherapy benefit (7).The subgroup of K v 1 channels play a critical role in regulating neurotransmission in both the central and peripheral nervous system by controlling the flux of potassium ions from the neuron during the action potential.K v 1.3 was related not only to astrocyte activation in experimental autoimmune encephalitis but also to CD4 + T-cell differentiation during inflammatory immunemediated disease (9).Pharmacological and knockout blockade of this channel has been shown to suppress these functions (10,11).K v 1.2 knockout mice show seizures in early developmental stages (12).Furthermore, K v 1.2 dysfunction has been associated not only with epilepsy (13-15) and developmental disorders (16,17) but also with multiple sclerosis (18) and neuroinflammatory disorders due to its crucial role in T-lymphocytes (19).The role of K v 1.2 in autoimmune disorders remains to be fully explored (20).K v 1.2 autoantibodies were shown to exacerbate an epileptic phenotype in rodent models (21), and cases of K v 1 autoantibody-associated limbic encephalitis were reported to be immunotherapy-sensitive (22).In vitro evidence suggests that autoantibodies can potentially reach their intracellular epitopes through Fc receptor-mediated internalization (23,24), consequently leading to a smoldering autoimmunity.The high prevalence and the functional role of anti-K v 1.2 autoantibodies in vivo and their association with specific clinical phenotypes are currently undefined, thereby potentially limiting diagnostic and therapeutic options.This is in part due to the lack of molecular knowledge on the involved epitopes and their contribution to the observed immune response.
Here, we report the screening, mapping, and validation of two K v 1.2 epitopes in 32 patients, thereby providing detailed molecular information on K v 1.2 autoimmunity and a basis for the development of diagnostic approaches.
Liquid chromatography-mass spectrometry (LC-MS) ( 27) was carried out using peptide quality controls that were cleaved from the solid support.To ensure cleavage, a Rink amide linker (Iris) suitable for Solid Phase Peptide synthesis (SPPS) on cellulose support was introduced during the first coupling cycle.In an acidic environment, the quality controls were cleaved off the solid support.To isolate the quality controls, 150 µL of the supernatant was transferred to 1.5-mL reaction tubes, followed by the addition of 700 µL of diethyl ether.The samples were then vortexed, and the peptides were allowed to precipitate by incubation at −20°C overnight.After centrifugation at 13,300×g and 4°C for 10 min, the supernatant was discarded, and 500 µL of diethyl ether was added.The mixture was vortexed and centrifuged for 10 min, and the supernatant was decanted.This process was repeated twice, and the peptides were left to dry for 60 min.Finally, the Rink amides were dissolved in 50 µL of 50% acetonitrile and 0.1% formic acid (v/v) and vortexed briefly before centrifugation at 13,300×g and RT.For analysis, the quality controls were diluted 1:3 and analyzed via LC-MS (Agilent technologies).

Microarray printing and binding assay
Peptide-cellulose conjugate (PCC) solutions were mixed 2:1 with saline-sodium citrate buffer [150 mM NaCl and 15 mM trisodium citrate (pH 7.0)] and transferred to a 384-well plate.For transfer of the PCC solutions to white-coated CelluSpot blank slides (76 mm × 26 mm, Intavis AG Peptide Services GmbH and Co. KG), a SlideSpotter (CEM GmbH) was used.After completion of the printing procedure, slides were left to dry overnight.
Recombinant expression of K v 1 in human embryonic kidney 293 cells 2, and K v 1.6 were expressed in HEK293 cells, following a previously described protocol (Miske et al., 2023).To summarize, genomic DNA was extracted from HEK293 cells and utilized as a template for K v 1.2 coding sequence amplification via polymerase chain reaction (PCR).Respective DNA oligonucleotides were employed to introduce the required enzyme restriction sites (Table 1).Indicated enzymes were used to digest the resulting PCR fragments and subsequently ligated with NcoI/XhoI-linearized pTriEx-1 (Merck).Prior to transfection, HEK293 cells were plated on sterile poly-L-lysine-treated coverslips.Transient expression of K v 1-encoded proteins was accomplished through Polyethylenimine (PEI)-mediated transfection (PEI 25K ™ ) following the manufacturer's instructions (Polysciences, Europe).After 48 h of transfection, cells were fixed, permeabilized with acetone, and rinsed with PBS before conducting the immunofluorescence described below.

Mouse brain and sciatic nerve processing
All animal procedures were approved by the Landesamt für Gesundheit und Soziales (LaGeSo) Berlin, Germany (approval numbers T-CH 0009/22), and conducted in compliance with the German and international guidelines for care and humane use of animals.Unfixed brain processing and sciatic nerve teased fiber preparations were performed as previously described (29).In brief, male C57BL/6 mice were used at an age of 10-12 weeks.Unfixed brains were dissected and frozen in 2-methylbutan.Cryostat-cut 20µm sections were mounted on glass slides and used for tissue-based immunofluorescence.Murine sciatic nerves were dissected from hind limbs, fixed in 4% paraformaldehyde, and washed in PBS.The epineurium was removed prior to teasing.Teased fibers were airdried and stored at −20°C until further usage.

Cell and tissue neutralization of anti-K v sera
Neutralization assays were adapted from established protocols (Miske et al., 2023).In brief, peptides were solubilized in PBS at a final concentration of 1 mg/mL.Neutralization assays were performed on transfected HEK293 cells and on mouse tissues.Sera were diluted in PBS-T to the abovementioned concentrations.The peptide antigen was added in a dilution of 1:10.Sera and peptides were thereby preincubated for 1 h at RT and subsequently added to slides and cells incubating for 1 h at RT.After washing with PBS-T, cells and slides were stained with secondary Alexa488-labeled antibodies for 30 min at RT.After washing with PBS-T, cell nuclei were stained with DAPI.Costaining with the commercial K v 1.2 antibody (K14/16 NeuroMab) and secondary Alexa594-labeled antibody was accomplished through sequential staining rounds, thereby avoiding potential cross-reactivity of secondary antibodies to primary mouse or human antibodies.

Data availability
Data are available upon reasonable request to qualified investigators for the purposes of replicating procedures and results.

Patient samples
Screening, mapping, and validation of K v 1.2 epitopes was based on 36 samples from patients (74.3% men, median disease onset age of 65, median high sera titer of 1:1,000, with four out of the 96 positive healthy controls positive in this readout) positive for K v 1.2 but negative for 33 common neurological autoantigens in cell-based assays.Patients showed heterogeneous neuropsychiatric phenotypes ranging from dementia, to epilepsy, autoimmune encephalitis, ischemic strokes, and peripheral neuropathies.Patient ID 19 had anti-AP3B2 IgG (titer 1:32) and patient ID 30 anti-NMDAR IgM (1:1,000) autoantibodies in serum; in the rest of the cohort, no other co-existing antibodies were detected.

Single-amino acid resolution mapping of two immunodominant K v 1.2 epitopes
To identify anti-K v 1.2 epitopes, the qualified patient samples were screened in peptide microarray format.Here, the entire primary sequence of K v 1.2 was displayed in the form of 20-mer peptides with 17-residue overlap (Figure 1A).First, the array approach was validated using the commercial anti-mouse K v 1.2 (NeuroMab clone K14/16) antibody.The microarray defined the sequence NEDFRE as the core motif (Figure 1B), thereby recapitulating the expected ( 463 EGVNNSNEDFREENLKTA 480 ) epitope.Next, 22 seropositive (Figure 1C) and 13 seronegative (control) (Figure 1D) sera were probed on the K v 1.2 array.Here, autoantibody binding was detected using anti-human IgG coupled to HRP for chemiluminescence detection.The screening identified two prominent intracellular (Figure 1E) epitopes: Epitope 1 (E1) ( 469 NEDFREENLKTANCTLA 485 ) and Epitope 2 (E2) ( 481 NCTLANTNYVNITK 495 ).Notably, none of the tested sera shared both epitopes.An additional array library with a single-AA shift of 15-mer peptides (Figure 1F) defined 478 KTANCTLA 485 as the E1 core motif, which was shared among 30 patients (Serum IDs 1-6, 8-10, 14-21, 23-34, and 36) and 485 ANTNYVNITK 495 as the minimal required E2 core motif, which was shared by two patients (Serum IDs 11 and 24) (Table 2).
Remarkably, the identified epitopes both overlap and include a validated phosphorylation site ( 389 Tyr; PhosphoSitePlus: P16389).In order to delineate the differences in the mode of binding between these two antibodies and specifically their dependency on phosphorylation, we next probed the corresponding phosphotyrosine K v 1.2 library.Whereas autoantibody binding of E1 was not affected, E2 binding was completely abolished upon phosphorylation (Figure 1G).

Autoantibody binding profiles hint toward a common molecular motif
To further resolve the binding requirements for autoantibodies, we conducted a deep mutational scan of the newly defined minimal binding epitopes.These libraries comprised all possible single-point variants of 18-mer peptides that harbored the minimal core motifs.The subsequent fingerprint analysis of the resulting 2 × 342 peptide variants (Figure 2A) confirmed both the previously defined minimal core motifs (Figure 1C) and the impact of the Tyr 489 phosphorylation (Figure 1G).
Notably, for E1, all patient samples displayed a seemingly identical binding requirement for their K v 1.2 autoantibodies (Figures 2B, C).More precisely, within the mapped core motif 78 KTANCTLA 485 (Figure 1C) residues, 478 Lys, 481 Asn, and 483 Thr were characterized by strict conservation with no tolerance toward any AA exchange (Figure 2B) for all tested patients (Figure 2C).For E2, both patients were fingerprinted (Figure 2D).Here, the finemapped core motif 485 ANTNYVNITK 495 (Figure 1C) was also recapitulated by both patients in the same way.In addition to the mapping, the fingerprint further highlights a strong conservation of the C-terminal part of the core motif 489 Tyr to 495 Lys and strict conservation of 490 Val, 491 Asn, and 494 Lys.In line with the analysis of the phosphorylated peptides (Figure 1G), the phophomimetic exchange of Tyr 489 with Glu resulted in complete loss of autoantibody recognition, thereby confirming our previous finding of orthogonal recognition of this Tyrosine depending on its phosphorylation status.In summary, the profiling highlights and substantiates a shared autoimmune response toward two distinct K v 1.2 epitopes.The unexpectedly high similarities in the relative binding responses within the patient cohort and the strict conservation of identical residues suggest a homogenous autoantibody repertoire within the tested patient group and further hint toward a shared molecular origin of the observed epitopes.
The resolved binding profiles prompted us to next explore K visoform specificity of the immunodominant antibody.Comparing the alignment of K v subfamily members 1.1 to 1.4 with the previous binding requirements highlights the conservation of critical residues within these four subfamily members (Figure 2E), specifically between K v 1.1, K v 1.2, and K v 1.3.We therefore displayed and probed overlapping C-terminal peptides of all four members of this subfamily in microarray format and found that autoantibody binding to E1 is maintained within K v 1.2 and K v 1.3 but not K v 1.1 and K v 1.4 (Figure 2F).The additional binding capacity for K v 1.3 is in line with the previously resolved binding requirements of E1 (Figure 2C).Vice versa, the lack of binding of K v 1.1 that shares high homology in this region highlights the need for experimental validation of putative binders.

Array detection complements cellbased assay
To resolve the predictive value of the specific epitope signals, we correlated seropositivity with the observed disease phenotype.To this end, the Euroimmun pre-screening determined 4% of the samples from healthy individuals as anti-K v 1.2-positive (four out of the 96 samples, data not shown).Our microarray readout recapitulated the reactivity and deep mutational scan analysis and attributed it toward E1, thus suggesting a similar broad occurrence (4%) of these monoreactive autoantibodies within healthy individuals (Supplementary Figure 1).In this light, we focused on the CSF samples obtained from 12 patients exploring both the diagnostic value and the sensitivity of the array screening in comparison to cell-based screening and its dependence of key parameters of the peptide display (Figure 3).The CSF of first five patients was analyzed with longer peptides and larger offset (20-mers, offset of 3) (Figure 3A), and CSF from seven patients was screened using shorter peptides with shorter offset (15-mers, offset of 1) (Figure 3B) (Table 2).Complementation of the arraybased screening with cell-based screens showed equal in several cases even superior sensitivity for both display variants (7/12 CBA and 10/ 12 array detected patients) (Figure 3C).Specifically, CSFs from patients 2, 25, and 36 were tested positive in microarray and

Patient ID Serum -array CSF -array
Ctrl ID Serum/ CSF-array  Deep mutational scans reveal common binding profiles for both shared epitopes.(A) Scheme of the K v 1.2 microarray profiling of E1 and E2 core motifs.From both epitopes, each possible positional substitution was generated (342 variants for each epitope) and printed in microarray format.
(B) Fingerprint analysis for sera containing autoantibody in the E1 group.Heat map overview of the major epitope fingerprint from serum 1; the wt sequence was sequentially scanned from N to C terminal by exchanging each position into each proteinogenic amino acid.Subsequently, the residue binding contribution was depicted in blue-white-red shades, where white corresponds to no variation over the wt [1], blue shades depict a loss, and red shades a gain of binding intensity.The most conserved residues for E1 were K 478 , N 481 , and T 483 .(C) Fingerprint analysis revealed a shared profile in the E2 group.General overview of the binding profile for E2 (patient 11) shows matching conserved residues.Here, Y 489 , V 490 , N 491 , and K 494 were the most conserved residues.(D) Recapitulated minimal motifs for E1 and E2.(E) Alignment of K v 1 channels shows high homology for the 1.1, 1.2, and 1.3 subunits.Residues "PQTP" on K v 1.3 are the known Cortactin interface, where the "TDV" sequence is a known PDZ-binding interface (F) C-terminal K v 1 peptides display reveals novel co-occurring autoantigen "sub-scenario" for E1.Combined K v 1.1, K v 1.2, K v 1.3, and K v 1.4 peptides were probed with patient 1 sera from the E1 group.In addition to the already mapped K v 1.2 epitope, the K v 1.3 peptides showed strong binding as depicted in the heat map.In line with the previous fingerprinting analysis, the R to K exchange in-between K v 1.1 and K v 1.2/1.3abolishes binding.
negative in CBA under the tested conditions.In addition, patient 12, negative from the arrays, resulted positive for K v 1.2 and K v 1.1 in cellbased assay (data not shown), potentially bearing a conformational epitope.Importantly, the CBA-determined positivity showed an association with cognitive impairment (30).Here, the array showed improved sensitivity over the CBA-based assay.We therefore conclude that, for K v autoimmunity patients, clinicians should consider combining array-and CBA-based testing for autoantibody confirmation, when an autoimmune pathogenesis is suspected.
The identified K v 1.2 epitopes are the exclusive mediators of the observed immune response A commonly assumed limitation of array-based antibody screenings is the risk of missing autoantigen contributions from certain conformational and discontinuous epitopes not represented by the linear peptide display.In addition, anti-K v 1.2 autoimmune response was previously observed to co-occur with unrelated Higher sensitivity of the microarray over cell-based detection for CSF anti-K v 1.2.neurological diseases (30,31) as well as other K v 1 complex-directed (7, 8) autoantibodies.To clarify the isoform specificity, K v 1.1, K v 1.2, and K v 1.6 were expressed in HEK293 cells and probed against the serum IDs 1, 3, and 27 and then compared with the respective control antibodies (Supplementary Figure 2).In line with the deep mutational scans (Figure 2), K v 1.1 and K v 1.6 showed no binding, thus confirming K v 1.2 specificity of the serum antibodies.To resolve contributions from possible additional antibodies binding via discontinuous epitopes that could not be resolved using peptide microarrays, we next conducted neutralization experiments of the identified epitope/autoantigen on chips and on cells.Soluble peptides were synthesized and purified on a preparative scale.
On-chip neutralization resulted in a strongly reduced signal, thereby supporting a specific and exclusive binding through the identified epitopes toward K v 1.2 in this format (Figures 4A, B).To search for additional, e.g., conformational, epitopes, we expressed K v 1.2 in HEK293 cells (Figures 4C, D) and tested the neutralization of the observed immune response for the two identified epitopes and the commercial control antibody (Figure 4C).Transfected HEK293 cells were stained with serum pre-treated with neutralizing peptides, non-neutralizing peptides, and buffer only (Figure 4C, D).Patient's sera were selected on the basis of their different epitopes for neutralization.Consequently, no residual binding was detected for either neutralized sample bearing E1 (Figure 4C) or E2 (Figure 4D).The non-neutralizing peptide had no impact on autoantibody binding.Notably, because no residual binding was detected, we conclude that K v 1.2 autoimmunity is primarily mediated by autoantibodies that recognize the previously highlighted linear motif, without any contributions from additional linear or conformational epitopes.

Peptide-based autoantibody neutralization on nerves and brain sections
Prompted by the confirmation of the identified epitopes as sole driver of the observed immune response in transfected recombinant cells, we next explored autoantibody binding toward the native  autoantigens in their cellular context.Here, we applied the neutralized sera on teased fibers (Figure 5A).Commercial antibody (K14/16) signals recapitulate the expected juxtaparanodal binding on teased fibers (Figure 5B).Sera applied together without peptide (Figure 5C) and with non-neutralizing peptide (Figure 5D) recapitulated the same labeling.In stark contrast, sera pre-treated with neutralizing peptide display a complete loss of binding signal (Figure 5E).Thus, complete anti-K v 1.2 epitope-specific neutralization was achieved using only the minimal K v 1.2 epitope.Remarkably, no residual autoantibody binding was detected in the nerve tissues, and the tested sera did not cross-react with additional autoantigens co-expressed peripherally.This corroborates the hypothesis of selective K v 1.2 autoimmunity without co-existing peripheral autoantibodies, thus contrasting previous observations (7,8,30,31).The neutralization in peripheral nerves was complemented by neutralization in tissue slices from the central nervous system, specifically mouse cerebellum, where K v 1.2 expression is high at the axon initial segment (AIS) of Purkinje cells.MAb K14/16 served as a K v 1.2 antibody control for specific binding of autoantibodies.Here, the typical axonal initial segment stainings were observed on  the Purkinje cell layer.E1 (Figure 5F) and E2 (Figure 5G) positive serum was applied in presence of several neutralizing and nonneutralizing peptide variants.
Taken together, the neutralization data in cell and tissues confirmed the high selectivity for the identified peptide epitopes, showing no detectable residual binding.Thus, leading to the exciting conclusion that a single, broadly shared epitope may contribute significantly to the often-reported K v 1 autoimmunity (7,8,30,31), in some cases, even without coexisting autoantigens or other conformational epitopes.In addition, the disease association of the detection of the here defined immunodominant epitope in CSF suggests implications for diagnosis, possibly even the pathology of a subgroup of autoimmune neuropsychiatric phenotypes.

Conclusion
Among K v 1 complex-directed autoantibodies, anti-K v 1 are among the most prevalent (7, 8); compared with LGI1 and CASPAR2 autoantibodies subgroups, their association with clinical syndromes and their immunotherapy responsiveness, however, appears less clear.Despite the association of K v 1 subfamily autoantibodies to neurological autoimmune diseases (5,6,9) and their pathology (7,8,10,11,(20)(21)(22) and their resulting diagnostic and therapeutic potential the involved K v 1 epitopes remained largely undefined.Here, we provide a first K v 1.2 autoantibody epitope landscape within a cohort of 36 K v 1.2exclusive neuropsychiatric patients and 18 healthy controls.In contrast to structural (24) and recombinant protein-based approaches (32)(33)(34), the array approach (2-4) combined with cell-based and tissue-based studies enabled the high-throughput molecular characterization of the autoantibodies directly from patient samples.Our data depict an unexpectedly monospecific and uniform autoantibody repertoire with two shared responses including one immunodominant K v 1.2 and K v 1.3 autoantibody epitope common to most of the patients tested here.Binding to additional subfamily members K v 1.1, K v 1.4, and K v 1.6 has been excluded by array or cellular assays.Moreover, the notable similarity in binding responses and the preservation of the required residues between patients implies a shared molecular genesis, which may include viral or bacterial antigens.In line with a possibly elevated immunogenic potential, K v positivity was reported in swine abattoir workers negative for both anti-LGI1 and anti-CASPR2 (35) as well as co-occurring with nonimmunogenic neurological disease (30,31).Sequences with high similarity to "EENLKTANCTLANTNYVNITK," namely, "EESLKTGNAG" and "ANTIYVNITKMLT," were previously reported (36, 37) as highly immunogenic.Autoantibody response is exclusively directed against K v 1.2 as substantiated by juxtaparanodal reactivity on teased nerves and AIS labeling in Purkinje cells.Importantly, the mapped epitopes enabled the complete neutralization of the observed reactivity, thereby establishing the outlined auto-antigen region as the primary mediator and even sole driver, of the observed autoimmune response.It remains to be seen whether these antibodies can bind in vivo (23, 24) to interfere with Kv channel function or protein-protein interactions such as the scaffolds PSD-95 and Cortactin, which are reported to bind near the N-terminal "PQTP" (38) or C-terminal "TDV" (39).On the other hand, a direct functional effect on the K v 1.2 channel has been shown to exacerbate a pro-epileptic state (21) and could potentially modulate the excitability of entire complex (40)(41)(42).The high prevalence of the here identified K v 1 epitope argues for future detailed studies on a possible intracellular action or indirect mechanisms such as T-cell cytotoxicity (43).Investigation of the relevant T-cell subpopulation involved and the HLA association, together with a possible tolerance mechanism, could shed light on the identified disease-specific antigen and why it is shared by several patients, similar to multiple sclerosis (44).CSF was previously reported to harbor enhanced diagnostic value in autoimmune neurological diseases (45, 46), but detection of low autoantibody titers remains challenging for conventional ELISA (enzyme-linked immunosorbent assay) and cell-based assay approaches that are limited in the density of the antigen display and further require the successful expression and immobilization of the antigens.Notably, the here reported prevalent and immunodominant K v 1 epitope achieves sensitivity and specificity for autoantibody detection in CSF.Significantly, the detected presence of autoantibodies in CSF associates with the clinical symptoms of cognitive impairment, thus highlighting its value for K v 1.2 autoantibody confirmation.This study provides a prevalent and immunodominant epitope together with the underlying autoantibody binding requirements in K v 1 autoimmunity in neuropsychiatric patients.Thus, setting the stage for future investigation of the molecular origin and a potential intracellular action of the here identified mono-specific K v 1 antibodies.These studies may focus on the analysis of clinical presentations, longitudinal samples, and immunotherapy responsiveness.Finally, the reported epitope also provides a means for isolating or even depleting the potentially diseasedefining autoantibodies or their respective B cells.
In summary, our study defines an immunodominant epitope as single determinant in K v 1 autoimmunity and thus outstanding diagnostic potential.
written informed consent to participate in this study.Ethical approval was not required for the studies on animals in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.

1
FIGURE 1Peptide microarray screening of anti-K v 1.2-positive patient samples reveals two shared binding epitopes.(A) Scheme of the K v 1.2 microarray display.K v 1.2 complete primary sequence was displayed in microarray format as 20-mer peptides with 17-residue overlap, and autoantibody binding was detected using goat anti-human IgG coupled to HRP for chemiluminescence readout.(B) Array validation using a commercial antibody.The microarray reports the residues NEDFRE as the core motif for Anti-mouse K v 1.2 (NeuroMab clone K14/16) that was raised against immunogen (Fusion K v 1.2 residues: 428-499) and previously mapped for the following sequence EGVNNSNEDFREENLKTA.(C) Identification of two distinct epitopes from sera.Epitope mapping of 22 patient sera.The sera reactivity of 22 positive samples and (D) 13 negative samples over 161 peptides were analyzed and plotted as a heat map over the most prominent peptide binder [0-1]; each rectangle corresponds to a single patient sample reactivity.Two main epitopes were identified between the peptides 156-161.(E) Single-amino acid mapping resolves the two minimal motifs.A 15-mer library revealed a shortest motif for E1 (KTANCTLA) and E2 (ANTNYVNITK).(F) Phosphorylation dependency of the two autoantibody epitopes.Here, two different samples (left: sera 11; right: sera 1) were tested on unphosphorylated and phosphorylated Y-489 peptides.E2 from patient 11 is negatively affected by phosphorylation; in contrast, E1 from patient 1 is not affected.(G) Visualization of the identified core motifs.Cartoon model of the K v 1.2 tetramer highlighting the two intracellular epitopes.
FIGURE 2 (A) K v 1.2 autoantibody detection within CSF by CBA and 20-mer peptides in array format.Top: Cerebrospinal fluid autoantibody binding of six positive samples on 161 K v 1.2 peptides.E1 has been detected between the overlapping peptides 156-157.Bottom: Comparison of HEK293 cell K v 1.2 binding assay and 20-mer peptide array for CSF 1 and 2. (B) K v 1.2 autoantibody detection within CSF by CBA and 15-mer peptides in array format.Core binding motif resolved for seven additional CSF-positive patients, binding observed between the K v 1.2 peptides 55-62.Comparison of HEK293 cell K v 1.2 binding assay and 15-mer peptide array for patients 25 and 36.(C) Overview of the K v 1.2 autoantibody detection within CSF.CSFs from patients 2, 25, and 36 tested positive in microarray but negative in CBA under the conditions tested.Patient 12 was tested positive for K v 1.1 and K v 1.2 autoantibodies in serum CBA.n.d., non detected.

4 Anti-K v 1 . 2
FIGURE 4 Anti-K v 1.2 Autoantibody Neutralization excludes the presence of additional epitopes.(A) Scheme of the identified K v 1.2 epitope landscape.(B) Onchip neutralization.Mapped peptides were synthesized and applied for pre-absorption experiments with patient sera.Upon incubation with peptide epitope, the microarray intensity has been neutralized.(C) HEK293 cell neutralization confirms E1 as solely mediator of the K v binding.Transfected K v 1.2 cells were permeabilized and incubated with either commercial anti-K v 1.2 or patient sera, IgG binding was visualized with either anti-mouse IgG or anti-human.Peptides were designed based on the previous mappings (Figures 1, 2).Pre-incubation with the mapped peptide epitope results in a complete autoantibody neutralization, whereas the non-neutralizing peptide epitope did not affect the binding.Neutralizing peptide #6 and #7: EDFREENLKTANCTLANTNY and ENLKTANCTLANTNYVNITK. Non-neutralizing peptide #5: ANCTLANTNYVNITKMLTDV. (D) HEK293 cell neutralization of E2 patient.Neutralizing peptide #5 and #7: ANCTLANTNYVNITKMLTDV and ENLKTANCTLANTNYVNITK. Non-neutralizing peptide #6: EDFREENLKTANCTLANTNY. Scale bar, 10 µm.

TABLE 1
DNA oligonucleotide primers for PCR amplification of K v 1.2, K v 1.1, and K v 1.6.

TABLE 2
Serum and cerebrospinal fluid peptide microarray samples used in this study.