B and T cell epitopes in PLA2R: development of novel treatment strategies in membranous nephropathy

Transcription

1 B and T cell epitopes in PLA2R: development of novel treatment strategies in membranous nephropathy Paul Brenchley Director of Renal Research Labs Manchester University Hospital Foundation Trust and University of Manchester, UK Nephrology Workshop, Papendal, Dec 12th 2018

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3 Pathobiology Genetics

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5 Combined effect of genetic risk variants of DQA1 and PLA2R Sekula P et al 2017

6 THSD7A another IMN target antigen THSD7A: another target antigen in MN Tomas N et al 2014

7 Why are PLA2R/THSD7A autoantigens? What s special about these autoantigens? PLA2R THSD7A Location Podocyte membrane Podocyte Membrane Form Transmembrane receptor Transmembrane receptor Soluble receptor Yes Yes Size 180kDa 250kDa Glycosylation Heavily N-glycoslyated Heavily N-glycosylated Domain structure Multi-domain disulphide bonded Epitope(s) Dominant epitope in N- term cys-rich domain. Sequence known Minor epitopes in CTLD1, 5, 7, 8 (epitope spreading) Multi-domain disulphide bonded unknown Autoantibody Predominantly IgG4 Predominantly IgG4

8 Structure, Sequence, Shape PLA2R sequence 10 E MLLSPSLLLL LLLGAPRGCA EGVAAALTPE RLLEWQDKGI FVIQSESLKK CIQAGKSVLT LENCKQANKH MLWKWVSNHG LFNIGGSGCL GLNFSAPEQP E2 200 LSLYECDSTL VSLRWRCNRK MITGPLQYSV QVAHDNTVVA SRKYIHKWIS YGSGGGDICE YLHKDLHTIK GNTHGMPCMF PFQYNHQWHH ECTREGREDD 210 E3 250 E4 300 LLWCATTSRY ERDEKWGFCP DPTSAEVGCD TIWEKDLNSH ICYQFNLLSS LSWSEAHSSC QMQGGTLLSI TDETEENFIR EHMSSKTVEV WMGLNQLDEH 310 E5 350 E6 400 AGWQWSDGTP LNYLNWSPEV NFEPFVEDHC GTFSSFMPSA WRSRDCESTL PYICKKYLNH IDHEIVEKDA WKYYATHCEP GWNPYNRNCY KLQKEEKTWH 410 E7 450 E8 500 EALRSCQADN SALIDITSLA EVEFLVTLLG DENASETWIG LSSNKIPVSF EWSNDSSVIF TNWHTLEPHI FPNRSQLCVS AEQSEGHWKV KNCEERLFYI 510 E9 550 E10 proline endopeptidase 600 CKKAGHVLSD AESGCQEGWE RHGGFCYKID TVLRSFDQAS SGYYCPPALV TITNRFEQAF ITSLISSVVK MKDSYFWIAL QDQNDTGEYT WKPVGQKPEP CyS-rich FIBII CTLD1 CTLD2 CTLD3 610E E VQYTHWNTHQ PRYSGGCVAM RGRHPLGRWE VKHCRHFKAM SLCKQPVENQ EKAEYEERWP FHPCYLDWES EPGLASCFKV FHSEKVLMKR TWREAEAFCE 710 E E EFGAHLASFA HIEEENFVNE LLHSKFNWTE ERQFWIGFNK RNPLNAGSWE WSDRTPVVSS FLDNTYFGED ARNCAVYKAN KTLLPLHCGS KREWICKIPR E15 810E E17 E DVKPKIPFWY QYDVPWLFYQ DAEYLFHTFA SEWLNFEFVC SWLHSDLLTI HSAHEQEFIH SKIKALSKYG ASWWIGLQEE RANDEFRWRD GTPVIYQNWD 910 E E TGRERTVNNQ SQRCGFISSI TGLWGSEECS VSMPSICKRK KVWLIEKKKD TPKQHGTCPK GWLYFNYKCL LLNIPKDPSS WKNWTHAQHF CAEEGGTLVA E E IESEVEQAFI TMNLFGQTTS VWIGLQNDDY ETWLNGKPVV YSNWSPFDII NIPSHNTTEV QKHIPLCALL SSNPNFHFTG KWYFEDCGKE GYGFVCEKMQ E E24 Caspase DTSGHGVNTS DMYPMPNTLE YGNRTYKIIN ANMTWYAAIK TCLMHKAQLV SITDQYHQSF LTVVLNRLGY AHWIGLFTTD NGLNFDWSDG TKSSFTFWKD 1210 E E EESSLLGDCV FADSNGRWHS TACESFLQGA ICHVPPETRQ SEHPELCSET SIPWIKFKSN CYSFSTVLDS MSFEAAHEFC KKEGSNLLTI KDEAENAFLL 1310 E E28 E EELFAFGSSV QMVWLNAQFD GNNETIKWFD GTPTDQSNWG IRKPDTDYFK PHHCVALRIP EGLWQLSPCQ EKKGFICKME ADIHTAEALP EKGPSHSIIP LAVVLTLIVI VAICTLSFCI YKHNGGFFRR LAGFRNPYYP ATNFSTVYLE ENILISDLEK SDQ E30 CTLD4 CTLD5 CTLD6 CTLD7 CTLD8 Autoantibody epitope epitopes recognised by commercial antibodies exon boundaries E1-E30 N potential N glycosylation sites

9 Best fit model of PLA2R domain Structure (20Å) from Single Molecule Negative Stain EM Relative orientation of domains unknown Cryo-EM study of PLA2R interactions Need PLA2R crystals for X- ray crystallography and definitive structure Fresquet M et al 2015

10 Crystallisation of PLA2R fragment (NC2) for X ray Diffraction

11 Characteristics of antibody response to antigens

12 Quantity of antibody Normal antibody response to antigen Transient low affinity IgM Vaccine/infection Class switch to IgG1, IgG3 and affinity maturation requires T cell help (class II presentation of peptides) Class switch to predominant IgG4 and high affinity Increased frequency of circulating antigen-specific B cells Memory for antigen established IgM IgG1 IgG3 Time (months) IgG4>IgG1, IgG3, IgG2

13 Quantity of antibody Time (months) Does the autoantibody response to PLA2R follow this pattern? Environmental trigger? Mimicry from natural immunity? Could B cells bind PLA2R and process, present it to T cells? Absolute requirement for MN pathological Class II alleles Class switch to predominant IgG4 and high affinity Increased frequency of circulating antigen-specific B cells Memory for antigen established IgM IgG1 IgG3 IgG4>IgG1, IgG3, IgG2

14 Anti-PLA2R is a high affinity autoantibody Table 2 - Affinity of purified human anti-pla2r to captured N-C8 and N-C3 This table summarizes the kinetic constants of 4 separate runs for the binding of purified human α-pla2r to N-C8 and N-C3. Results were obtained after reference subtraction. The association (k a ), dissociation (k d ) and equilibrium (K D ) constants for each runs were similar and revealed an overall high binding affinity with an apparent K D of ~0.1 nm.

15 Defining the important structural sequences in PLA2R that interact with the immune system A mature IgG response is likely to react to several epitopes in PLA2R (B cell or antibody binding sequences) Question: is there an initial dominant epitope with more epitopes involved over time? Class switched, high affinity IgG implies significant T cell help for B cell differentiation Question: what are the T cell peptides of PLA2R that bind to Class II receptor to stimulate T cells

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18 Characteristics of the Cys-Rich epitope in PLA2R N terminal domain: linear and conformation epitopes PLA 2 R GIFVIQSESLKKCIQAGKSVLTLENCKQA 1) Two linear sequences contain key amino acids required for optimum binding 2) The distance between these sequences is important for optimum binding 3) The optimum 3D orientation of these sequences is maintained by the disulphide bond This epitope sequence has a biological function in PLA2R. Collaborating on making crystals of NC2 (first four domains) for X ray crystallography to determine the precise structure and orientation in the protein.

19 K S V L V L G A T L T L Q I N E N E K C C K C K PLA2R epitope Clostridium S11 carboxypeptidase Blast search of the epitope sequence shows half of the cyclic peptide ring is displayed on a cell wall carboxypeptidase. Could natural immunity to this infectious agent -prime B cells to make a cross reacting antibody? -increase the frequency of B cells able to process and present PLA2R peptides to T cells?

20 BLAST search of SVLTLENCK peptide from PLA2R epitope Organism Score Expect Identities Positives Gaps Sequence Peptidase S11, Clostridium sp CAG245 Mds3p, Saccharomyces cerevisiae HrcC Type III outer ring membrane protein Pseudomonas 26.9 bits (56) 24.4 Bits (50) 24.4 bits (50) 182 7/8 (88%) 8/8 (100%) 0/8 (0%) Query 2 VLTLENCK 9 +LTLENCK Sbjct 110 ILTLENCK /7 (100%) 7/7 (100%) 0/7 (0%) Query 1 SVLTLEN 7 SVLTLEN Sbjct1418 SVLTLEN /7 (100%) 7/7 (100%) 0/7 (0%) Query 1 SVLTLEN 7 SVLTLEN Sbjct 407 SVLTLEN 413

21 Position specific iterated (PSI) BLAST search of UniProtKB/Swissprot database using PLA2R motif (Query 1) identifies only THSD7A PLA 2 R (P28mer) THSD7A (T28mer) GIFVIQSESLKKCIQAGKSVLTL ENCKQA GPGGIQTRAVW CAHVEGWT TLHTNCKQA

22 Structure of modelled P28mer (red) and T28mer (blue) peptides and their overlay P28mer T28mer PLA2R and THSD7A sequences were subject to 3D de novo structure prediction using PEP-FOLD and the best structures output as PDB coordinates. overlay

23 Developing new therapy for specific control of anti-pla2r? This requires new knowledge of the autoimmune mechanism using the dominant B cell epitope may allow immunoadsorption of autoantibody?

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25 Dr Patrick Hamilton Glomerular Diseases and Vasculitis Fellow Manchester Institute of Nephrology and Transplantation

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27 Progress 12 patients consented 12 have completed treatment 8 have completed 1 year follow up Patient 1 Antibody negative (at 15 months) Partial remission by proteinuria Patient 2 Failed treatment and started Cyclophosphamide therapy Last patient follow up April 2019

28 PRISM Patient 1

29 PRISM Study (Immunoadsorption of anti-pla2r) 12 month follow up on all patients in April 2019 Study to be submitted for publication next summer

30 Developing new therapy for specific control of anti-pla2r? This requires new knowledge of the autoimmune mechanism using the dominant B cell epitope may allow immunoadsorption of autoantibody? If we can demonstrate benefit from IA would using a PLA2R peptide column be more effective? nature of the T cell peptides in PLA2R may allow Antigen specific immunotherapy to restore tolerance?

31 Restoring T cell tolerance to PLA2R? Genetic Restriction: Need the pathological Class II alleles Restoring tolerance to PLA2R How to identify the PLA2R peptides that can restore T cell tolerance and increase Treg cells to block antibody production? APC X Tc Y Bc Y B cell proliferation T cell help X Plasma cell secretion of antibody Anti-PLA2R autoantibody Autoimmune Membranous Nephropathy

32 Class II restricted B cells could capture PLA2R and present PLA2R peptides to T cells How to identify peptides to block T cell activation? X

33 Class II receptor groove selects PLA2R peptides Solvent P2 P3 P5 P8 PLA2R Peptide chain P4 P6 P7 P1 Anchor pockets in Class II groove P9 W, F N, I L, D D, E E, Y, D E, D A, P, E M, F, W

34 MHC Class II Proimmune REVEAL Assay signal Peptide class II receptor detection ab

35 Developing an antigen specific immunotherapy using T cell peptides 1) PLA2R peptide binding expts to recombinant Class II receptors to identify high and stable binders 2) PLA2R antigen presentation expts with genetically relevant normal and MN cells in vitro

36 Summary T & B cell peptides from PLA2R provide tools to interogate the autoimmune state in MN patients and develop new therapies Is immunoadsorption treatment suitable for some MN patients and which ones? Treat early when the antibody response only recognises the dominant epitope? Can we develop a tolerogenic strategy using delivery of T cell peptides to switch off anti-pla2r production and reset normal tolerance to PLA2R?

37 MN Manchester Team NHS Clinical team: Patrick Hamilton Durga Kanigicherla Sandip Mitra Shelly Harris Manchester University Team from left to right: Edward Mckenzie Rachel Lennon Maryline Fresquet Paul Brenchley Thomas Jowitt Sam Rhoden (absent) Supported by: