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The Challenge of Complexity: Peptide Tools for the Development of Immunotherapies K. Schnatbaum, T. Knaute, J. Zerweck, M. Eckey, P. Holenya, F. Kern, H. Wenschuh, U. Reimer* JPT Peptide Technologies GmbH, 12489 Berlin, Germany. * Correspondence should be addressed to Ulf Reimer ([email protected]) Introduction Immunotherapy is gaining attention as promising approach to fight cancer as well as infectious diseases. 1 A major challenge in immunotherapy is the selection of optimal antigen sequences to derive efficient therapeutic agents. Among others, this task is hampered by sequence diversity in the target organisms caused by isoforms, splice variants, polymorphisms, mutations, and PTMs. An example is the Human Immunodeficiency Virus (HIV), for which thousands of different sequences are known just for the envelope protein alone (Figure 1). Figure 1: Sequence variability in the HIV Nef protein (aa20-67 in reference strain HXB2). One representative sequence per frequent clade is shown. Methods We address the challenge of sequence diversity by a peptide library based workflow that combines: Improved bioinformatic algorithms: New algorithms for library design were developed. These are based on the scoring of all possible peptides according to their frequency of occurrence across all sequences to provide the most homogenous overall coverage. 2 The result of the so-called Ultra Concept is illustrated in Figure 2 for the HIV Nef protein. The majority of the 3903 known sequences is covered by only 150 peptides (for antigen specific T-cell stimulation with peptide pools) or 667 peptides (for humoral immune monitoring with peptide microarrays). Figure 2: Sequence coverage by different HIV Nef libraries. Red/orange: Sequence parts which are covered by the respective peptide library. Figure 3: HIV Env-specific antibody responses using different dosing regimens (IPCAVD 001 trial). Cellular immune response: To increase stimulating efficiency for antigen specific T- cell responses (HLA independence, reduction of assay numbers, sample volume requirements), antigen- spanning overlapping peptide pools were developed. Examples include pools for the ex vivo generation of T cells for HIV 6 (Figure 4) and broad-spectrum antiviral (AdV, EBV, CMV, BKV, HHV6) 7 treatment, where a 94% virological and clinical response rate was achieved. Figure 4: HIV-specific T cell responses after ex vivo expansion of T cells from seven HIV+ donors with Ultra peptide libraries. 6 Summary To address the challenge of sequence diversity in immunotherapy, a peptide based workflow was established that combines bioinformatic algorithms, high throughput peptide synthesis, innovative peptide presentation approaches and synergistic assay formats. High throughput peptide synthesis, peptide presentation and synergistic assay formats: For B-cell epitope discovery and humoral immune monitoring high density peptide microarrays represent an efficient technology that accommodates vast numbers of sequence variants and PTMs. Peptides are synthesized by SPOT synthesis 3 and re-immobilized on microarrays 4 in a clean-room environment (Scheme 2). Readout is usually performed by fluorescently labeled secondary antibodies. Scheme 1: Preparation of peptide microarrays to study humoral immunity. For T-cell epitope discovery and cellular immune monitoring peptides selected by the Ultra Concept can be synthesized and presented as individual peptides, matrix pools or antigen spanning pools for application in T-cell assays such as Elispot (Scheme 2). Scheme 2: Preparation of peptide pools to study cellular immunity. Application Examples Humoral immune response: To provide guidance for vaccine development, plasma samples from several HIV/SIV vaccination studies were examined with peptide microarrays. 2,5 The analysis of serum samples from the first successful HIV vaccination trial (RV144 trial) with peptide microarrays showed that plasma levels of IgG directed towards the V2 loop of gp120 correlated with a reduced risk of infection. 5a In a follow-up study, an Ad26 vector-based vaccine stimu- lated a dose dependent response against the V2 loop (Figure 3). 5b Clade A B C D G CRF1 CRF2 other Conventional Scan through Protein (49 Peptides) Peptide Pool Ultra (150 Peptides) Peptide Microarray Ultra (667 Peptides) Coverage: 9.5 % 51.8 % 56.3 % Fluorescence Intensity Peptide Number Fluorescence Intensity Peptide Number Group 2 (10 10 vp – 3 doses) Group 3 (10 11 vp – 3 doses) A1.UG B.US C.ZA D.UG G.SE CRF1.HK CRF2.CM REF.HXB2 References (1) e.g. (a) Vormehr, M. et al. J. Immunol. Res. 2015, 595363. (b) Kranz, L. M. et al. Nature 2016, 534, 396-401. (2) Stephenson, K. et al. J. Immunol. Meth. 2015, 416, 105-123. (3) Wenschuh, H. et al. Biopolymers 2000, 55, 188-206. (4) (a) Schutkowski, M. et al. Angew. Chem. Int. Ed. Engl. 2004, 43, 2671-2674. (b) Masch, A. et al. Methods Mol. Biol. 2010, 669, 161-172. (5) (a) Gottardo, R. et al. PLoS ONE 2013, 8, e75665. (b) Barouch, D. H. et al. J. Infect. Dis. 2013, 207, 248-256. (c) Barouch, D. H. et al. Science 2015, 349, 320-324. (6) Lam, S. et al. Mol. Ther. 2015, 23, 387-395. (7) Papadopoulou, A. et al. Sci. Transl. Med. 2014, 6, 242ra83. Spatially addressed SPOT Synthesis Functionalization Membrane Cutting of Peptide Spots and Cleavage from Membrane Peptide Microarrays Incubation, Readout, Data Analysis Visualized and Raw Data Chemoselective Immobilization Antigen Sequence ID Synthesis of Overlapping Peptides Pooling & Aliquotation Peptide Pool Ag I Peptide Pool Collections (all immunodominant Ag‘s of HIV, CMV, EBV, cancers, …) Peptide Pool Ag II Peptide Pool Ag III Collection & Plating Antigen I Antigen II Antigen III
Transcript of The Challenge of Complexity: Peptide Tools for the ... · The Challenge of Complexity: Peptide...
The Challenge of Complexity: Peptide Tools for the Development of Immunotherapies
K. Schnatbaum, T. Knaute, J. Zerweck, M. Eckey, P. Holenya, F. Kern, H. Wenschuh, U. Reimer*
JPT Peptide Technologies GmbH, 12489 Berlin, Germany.
* Correspondence should be addressed to Ulf Reimer ([email protected])
Introduction
Immunotherapy is gaining attention as promisingapproach to fight cancer as well as infectious diseases.1
A major challenge in immunotherapy is the selection ofoptimal antigen sequences to derive efficient therapeuticagents. Among others, this task is hampered bysequence diversity in the target organisms caused byisoforms, splice variants, polymorphisms, mutations,and PTMs. An example is the Human ImmunodeficiencyVirus (HIV), for which thousands of different sequencesare known just for the envelope protein alone(Figure 1).
Figure 1: Sequence variability in the HIV Nef protein (aa20-67 inreference strain HXB2). One representative sequence perfrequent clade is shown.
Methods
We address the challenge of sequence diversity by apeptide library based workflow that combines:
Improved bioinformatic algorithms:New algorithms for library design were developed.These are based on the scoring of all possible peptidesaccording to their frequency of occurrence across allsequences to provide the most homogenous overallcoverage.2 The result of the so-called Ultra Concept isillustrated in Figure 2 for the HIV Nef protein. Themajority of the 3903 known sequences is covered byonly 150 peptides (for antigen specific T-cell stimulationwith peptide pools) or 667 peptides (for humoralimmune monitoring with peptide microarrays).
Figure 2: Sequence coverage by different HIV Nef libraries.Red/orange: Sequence parts which are covered by the respectivepeptide library.
Figure 3: HIV Env-specific antibody responses using differentdosing regimens (IPCAVD 001 trial).
Cellular immune response:
To increase stimulating efficiency for antigen specific T-cell responses (HLA independence, reduction of assaynumbers, sample volume requirements), antigen-spanning overlapping peptide pools were developed.Examples include pools for the ex vivo generation ofT cells for HIV6 (Figure 4) and broad-spectrum antiviral(AdV, EBV, CMV, BKV, HHV6)7 treatment, where a 94%virological and clinical response rate was achieved.
Figure 4: HIV-specific T cell responses after ex vivo expansion ofT cells from seven HIV+ donors with Ultra peptide libraries.6
Summary
To address the challenge of sequence diversity inimmunotherapy, a peptide based workflow wasestablished that combines bioinformatic algorithms, highthroughput peptide synthesis, innovative peptidepresentation approaches and synergistic assay formats.
High throughput peptide synthesis, peptide
presentation and synergistic assay formats:
For B-cell epitope discovery and humoral immunemonitoring high density peptide microarrays representan efficient technology that accommodates vastnumbers of sequence variants and PTMs. Peptides aresynthesized by SPOT synthesis3 and re-immobilized onmicroarrays4 in a clean-room environment (Scheme 2).Readout is usually performed by fluorescently labeledsecondary antibodies.
Scheme 1: Preparation of peptide microarrays to study humoralimmunity.
For T-cell epitope discovery and cellular immunemonitoring peptides selected by the Ultra Concept canbe synthesized and presented as individual peptides,matrix pools or antigen spanning pools for application inT-cell assays such as Elispot (Scheme 2).
Scheme 2: Preparation of peptide pools to study cellularimmunity.
Application Examples
Humoral immune response:To provide guidance for vaccine development, plasmasamples from several HIV/SIV vaccination studies wereexamined with peptide microarrays.2,5 The analysis ofserum samples from the first successful HIV vaccinationtrial (RV144 trial) with peptide microarrays showed thatplasma levels of IgG directed towards the V2 loop ofgp120 correlated with a reduced risk of infection.5a In afollow-up study, an Ad26 vector-based vaccine stimu-lated a dose dependent response against the V2 loop(Figure 3).5b
Clade
A
B
C
D
G
CRF1
CRF2
other
Conventional Scan through Protein (49 Peptides)
Peptide Pool Ultra (150 Peptides)
Peptide Microarray Ultra (667 Peptides)
Coverage: 9.5 % 51.8 % 56.3 %
Flu
ore
scen
ce In
ten
sit
y
Peptide Number
Flu
ore
scen
ce In
ten
sit
y
Peptide Number
Group 2 (1010 vp – 3 doses) Group 3 (1011 vp – 3 doses)
A1.UG
B.US
C.ZA
D.UG
G.SE
CRF1.HK
CRF2.CM
REF.HXB2
References(1) e.g. (a) Vormehr, M. et al. J. Immunol. Res. 2015, 595363. (b)Kranz, L. M. et al. Nature 2016, 534, 396-401.(2) Stephenson, K. et al. J. Immunol. Meth. 2015, 416, 105-123.(3) Wenschuh, H. et al. Biopolymers 2000, 55, 188-206.(4) (a) Schutkowski, M. et al. Angew. Chem. Int. Ed. Engl. 2004,43, 2671-2674. (b) Masch, A. et al. Methods Mol. Biol. 2010, 669,161-172.(5) (a) Gottardo, R. et al. PLoS ONE 2013, 8, e75665. (b)Barouch, D. H. et al. J. Infect. Dis. 2013, 207, 248-256. (c)Barouch, D. H. et al. Science 2015, 349, 320-324.(6) Lam, S. et al. Mol. Ther. 2015, 23, 387-395.(7) Papadopoulou, A. et al. Sci. Transl. Med. 2014, 6, 242ra83.
Spatially addressed SPOT SynthesisFunctionalization
Membrane
Cutting of Peptide Spots and Cleavage
from Membrane
Peptide Microarrays
Incubation, Readout,
Data Analysis
Visualized and Raw Data
ChemoselectiveImmobilization
Antigen
Sequence ID
Synthesis ofOverlapping Peptides
Pooling & Aliquotation Peptide Pool Ag I
Peptide Pool Collections(all immunodominant Ag‘s of HIV, CMV, EBV, cancers, …)
Peptide Pool Ag II Peptide Pool Ag III
Collection & Plating
Antigen I Antigen II Antigen III
