MHC Associated Peptide ProteomicsIn Vitro System To Identify Presented Epitopes That Induce Cytotoxic CD8+ T Cell Responses

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MAPPs identi� es antigens and neo-antigens presented by MHC class I on tumours or infected cells • Discover new targets for immuno-oncology, vaccine development and diagnostics• Determine relative quanti� cation, i.e. protein/epitope over expression in tumour vs healthy tissue• Predict strength and frequency of CD8+ T cell response and potential generation of armed e� ector T cells against the

antigen of interest

BackgroundThe primary immune response to an antigen is initiated through antigen presenting cells such as dendritic cells (DC). Human leukocyte antigens (HLA) on the surface of these cells present protein-derived peptide fragments, where MHC class I associated HLA (i.e. HLA-A, B and C) present peptides to CD8+ T cells and MHC class II associated HLA (HLA-DR, DP and DQ) to CD4+ T cells. MHC-I presentation is fundamental for the development of immunity against tumours and viruses since endogenous proteins are normally presented to CD8+ T cells via this route. Presentation of exogenous antigens via MHC class I can also occur and trigger an immune response. Therefore, the identi� cation of these antigens presented via MHC class I creates huge potential for the generation of new immuno-oncology therapeutics, vaccines and diagnostics.

Vaccine DesignTo identify potential antigens for vaccine design putative antigens (i.e. viral proteins, tumour associated antigens) are expressed in human cell lines for a comparative analysis of the presented peptides. Unique viral peptides identi� ed can be synthesised for use in in vitro T cell assays to assess for CD8+ T cell activation.

Tumour Antigen Discovery & ValidationMAPPs can identify tumour associated antigens or neo-antigens based on comparisons with healthy tissue. Any variable presented protein peptides can be synthesised to check for CD8+ T cell activation. This has huge potential for the discovery of new targets for immunotherapeutics.

Applications of MAPPsBy comparing the MHC class I presented peptides of viral infected cells & tumour cells with equivalent healthy cells using MAPPs, utilising powerful mass spectrometry, allows for the identi� cation of antigens or neo-antigens for therapeutic targets. Combining in vitro T cell assays allows for con� rmation of whether identi� ed peptides isolated from MHC Class I molecules contain epitopes that can elicit CD8+ T cell response. Relative quanti� cation and a� nity of peptides that elicit T cell response can be measured by MAPPs.

DiagnosticsT Cell responses can be associated with disease outcome (i.e. chronic infection, cancer). Through MAPPs relevant T cell epitopes can be identi� ed from whole proteins and tumour antigens and used in assay design to monitor e� ective T cell responses ex vivo from patients.

TCR

KEISLSYSATEVETYVL

GILGFVFTL

Antigen processing

Mature APC

CD8+ T Cell

Activated CD8+ T Cell

Immature APC

Costimulatorymolecules

MHC Class I peptide presentation to CD8+ T Cells

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

T cell proliferation

IFN-γ production

LLLVFLFDGSMSLDD

DDNVDLVFLFDGGSL

Sample peptide

Endogenous peptides

Sample peptidescompete for MHC class I

Immature APC

Cytokines

Vira

l pro

tein

s pr

esen

ted

by M

HC

Clas

s I

Peptidomics via mass spectrometry

Evaluation of peptidomes to identify variable peptides

Presentation of peptide to antigen presenting cell

Synthesis of identi�ed viral peptides

Viral loaded cellNon-loaded cell

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MHC Class I presentationBoth viral and endogenous proteins are presented on the cell surface via MHC class I. Cells are lysed and MHC class I bound peptide complexes are captured by immunoprecipitation using a pan-MHC class I antibody. Naturally processed and presented peptides from the test sample, are eluted from the captured MHC molecules for subsequent analysis by nano-LC-MS/MS.

Case Study MAPPs for Vaccine Design

Working with AbzenaAbzena’s services are tailored for each project to ensure that the objectives are met or exceeded. Experienced project teams are assigned to each study focusing on progressing projects through to results in the minimum amount of time. Our clients uniformly judge us as professional and attentive partners who deliver quality results in a timely manner.

To identify MHC class I presented antigens derived from in� uenza strain H1N1, two sets of human epithelial cells are cultured, one is transfected with H1N1 antigen.

MHC Associated Peptide ProteomicsIn Vitro System To Identify Presented Epitopes That Induce T Cell Responses

Orbitrap-MS analysisEluted peptides are identi� ed using mass spectrometry, in-house search algorithms and databases. The range of peptides identi� ed from each set of epithelial cells (the peptidome) are evaluated and quanti� ed to identify new expression or di� erences in expression levels.

CD8+ T cell activationPeptides of interest are synthesised that match the core binding motifs. Peptides are then cultured with peripheral blood mononuclear cells (PBMCs) obtained from Abzena's HTA approved blood bank. Activation of a memory CD8+ T cell response is measured by IFN-γ release.

Sequences Affi nity HLA 02 (nM)*

Affi nity HLB 40 (nM)*

10μM 1μM 0.1μM 0.01μM

TEVETYVL 33573.5 119.5 0 5 0 0

KEISLSYSA 13998.8 732.7 5 0 0 0

GILGFVFTL 15.7 11037.2 35 29 29 23

ResultsThe MHC class I associated peptidome was succesfully isolated and analysed. Three unique virus derived peptides were identi� ed with high con� dence that bind HLA-A and -B alleles. Peptide GILGFVFTL was also shown to stimulate CD8+ T cells in 35% of donors.

KEISLSYSATEVETYVL

GILGFVFTL

Antigen processing

Mature APC

CD8+ T Cell

Activated CD8+ T Cell

Immature APC

Costimulatorymolecules

MHC Class I peptide presentation to CD8+ T Cells

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

T cell proliferation

IFN-γ production

LLLVFLFDGSMSLDD

DDNVDLVFLFDGGSL

Sample peptide

Endogenous peptides

Sample peptidescompete for MHC class I

Immature APC

Cytokines

Vira

l pro

tein

s pr

esen

ted

by M

HC

Clas

s I

Peptidomics via mass spectrometry

Evaluation of peptidomes to identify variable peptides

Presentation of peptide to antigen presenting cell

Synthesis of identi�ed viral peptides

Viral loaded cellNon-loaded cell

KEISLSYSATEVETYVL

GILGFVFTL

Antigen processing

Mature APC

CD8+ T Cell

Activated CD8+ T Cell

Immature APC

Costimulatorymolecules

MHC Class I peptide presentation to CD8+ T Cells

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

CD8+ T CellActivated CD8+ T Cell

T cell proliferation

IFN-γ production

LLLVFLFDGSMSLDD

DDNVDLVFLFDGGSL

Sample peptide

Endogenous peptides

Sample peptidescompete for MHC class I

Immature APC

Cytokines

Vira

l pro

tein

s pr

esen

ted

by M

HC

Clas

s I

Peptidomics via mass spectrometry

Evaluation of peptidomes to identify variable peptides

Presentation of peptide to antigen presenting cell

Synthesis of identi�ed viral peptides

Viral loaded cellNon-loaded cell

Mature APC

KEISLS

YSA

TEVETYVL

GILGFV

FTL

Antigen

pro

cess

ing

Mat

ure A

PCCD8+ T Cell

Activa

ted

CD8+ T Cell

Imm

ature

APC

Costim

ulatory

molec

ules

MHC C

lass I

pep

tide p

rese

ntatio

n

to C

D8+ T Cell

s

GNVDLVFL

FDGSM

SL

GNVDLVFL

FDGSM

SL

GNVDLVFL

FDGSM

SLGNVDLV

FLFD

GSMSL

T cell

pro

lifera

tion

IFN-γ

product

ion

LLLVFLFDGSMSLDD

DDNVDLVFL

FDGGSL

Sam

ple

peptid

e

Endogen

ous

peptid

es

Sam

ple pep

tides

com

pete f

or MHC cl

ass I

Imm

ature

APC

Cytokin

es

Viral proteins presented by MHC Class I

Peptid

omics

via m

ass

spec

trom

etry

Evalu

atio

n of pep

tidom

es to

iden

tify v

ariab

le pep

tides

Prese

ntatio

n of pep

tide t

o

antig

en p

rese

nting ce

ll

Synth

esis

of iden

ti�ed

viral

peptid

es

Viral lo

aded

cell

Non-load

ed ce

ll

KEISLSYSATEVETYVL

GILGFVFTL

Antigen processing

Mature APC

CD8+ T Cell

Activated CD8+ T Cell

Immature APC

Costimulatorymolecules

MHC Class I peptide presentation to CD8+ T Cells

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

T cell proliferation

IFN-γ production

LLLVFLFDGSMSLDD

DDNVDLVFLFDGGSL

Sample peptide

Endogenous peptides

Sample peptidescompete for MHC class I

Immature APC

Cytokines

Vira

l pro

tein

s pr

esen

ted

by M

HC

Clas

s I

Peptidomics via mass spectrometry

Evaluation of peptidomes to identify variable peptides

Presentation of peptide to antigen presenting cell

Synthesis of identi�ed viral peptides

Viral loaded cellNon-loaded cell

KEISLSYSATEVETYVL

GILGFVFTL

Antigen processing

Mature APC

CD8+ T Cell

Activated CD8+ T Cell

Immature APC

Costimulatorymolecules

MHC Class I peptide presentation to CD8+ T Cells

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

T cell proliferation

IFN-γ production

LLLVFLFDGSMSLDD

DDNVDLVFLFDGGSL

Sample peptide

Endogenous peptides

Sample peptidescompete for MHC class I

Immature APC

Cytokines

Vira

l pro

tein

s pr

esen

ted

by M

HC

Clas

s I

Peptidomics via mass spectrometry

Evaluation of peptidomes to identify variable peptides

Presentation of peptide to antigen presenting cell

Synthesis of identi�ed viral peptides

Viral loaded cellNon-loaded cell

KEISLSYSATEVETYVL

GILGFVFTL

Antigen processing

Mature APC

CD8+ T Cell

Activated CD8+ T Cell

Immature APC

Costimulatorymolecules

MHC Class I peptide presentation to CD8+ T Cells

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

GNVDLVFLFDGSMSL

T cell proliferation

IFN-γ production

LLLVFLFDGSMSLDD

DDNVDLVFLFDGGSL

Sample peptide

Endogenous peptides

Sample peptidescompete for MHC class I

Immature APC

Cytokines

Vira

l pro

tein

s pr

esen

ted

by M

HC

Clas

s I

Peptidomics via mass spectrometry

Evaluation of peptidomes to identify variable peptides

Presentation of peptide to antigen presenting cell

Synthesis of identi�ed viral peptides

Viral loaded cellNon-loaded cell

*database predicted a� nity