The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell ...immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.
Herein, we describe the design, synthesis, and biological evaluation of a series of DOTA-conjugated monomeric, dimeric, and tetrameric Tyr3octreotide-based analogues as a tool for tumor imaging ...and/or radionuclide therapy. These compounds were synthesized using a Cu(I)-catalyzed 1,3-dipolar cycloaddition (“click” reaction) between peptidic azides and dendrimer-derived alkynes and a subsequent metal-free introduction of DOTA via the thio acid/sulfonyl azide amidation (“sulfo-click” reaction). In a competitive binding assay using rat pancreatic AR42J tumor cells, the monomeric Tyr3octreotide conjugate displayed the highest binding affinity (IC50 = 1.32 nM) followed by dimeric Tyr3octreotide (2.45 nM), DOTA0,Tyr3octreotide (2.45 nM), and tetrameric Tyr3octreotide (14.0 nM). Biodistribution studies with BALB/c nude mice with subcutaneous AR42J tumors showed that the 111In-labeled monomeric Tyr3octreotide conjugate had the highest tumor uptake (42.3 ± 2.8 %ID/g) at 2 h p.i., which was better than 111In-DOTA0,Tyr3octreotide (19.5 ± 4.8 %ID/g). The 111In-labeled dimeric Tyr3octreotide conjugate showed a long tumor retention (25.3 ± 5.9 %ID/g at 2 h p.i. and 12.1 ± 1.3 %ID/g at 24 h p.i.). These promising results can be exploited for therapeutic applications.
We describe rapid and convenient access to smart libraries of protein surface discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, ...were conjugated to a triazacyclophane scaffold molecule via CuAAC. In this way protein mimics for use as synthetic vaccines and beyond will become available.
A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a ...wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC50 value of 1.7 μM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.
The highly amyloidogenic peptide sequence of amylin(20–29) was transformed into its corresponding peptoid and retropeptoid sequences to design a novel class of β-sheet breaker peptides as amyloid ...inhibitors. This report describes the synthesis of the chiral peptoid building block of
l-isoleucine, the solid phase synthesis of the peptoid and retropeptoid sequences of amylin(20–29), and the structural analysis of these amylin derivatives in solution by infrared spectroscopy, circular dichroism, and transmission electron microscopy. It was found that the peptoid sequence did not form amyloid fibrils or any other secondary structures and was able to inhibit amyloid formation of native amylin(20–29). Although the retropeptoid did not form amyloid fibrils it had only modest amyloid inhibitor properties since supramolecular tapes were formed.
Factors that determine the chirality of supramolecular helical tapes formed by a backbone-modified amylin(20–29) depsipeptide and inverso-depsipeptide, were studied by Fourier transform infrared ...spectroscopy, circular dichroism and transmission electron microscopy. Although β-sheet propensity was absent in both peptides, it was found that the
l-depsipeptide formed left-handed and the enantiomeric
d-depsipeptide right-handed helical tapes. Moreover, the backbone-modified depsipeptides, showed a certain degree of cross-recognition between both enantiomers, which might have implications in designing amyloid formation inhibitors.
We describe rapid and convenient access to smart libraries of protein surface discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, ...were conjugated to a triazacyclophane scaffold molecule
via
CuAAC. In this way protein mimics for use as synthetic vaccines and beyond will become available.
We describe rapid and convenient access to smart libraries of protein surface discontinuous epitope mimics.
A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a ...wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(
i
) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC
50
value of 1.7 μM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.
A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible.
Abstract
Monoclonal antibody (mAb) blockade of immune checkpoints such as PD-1 and CTLA-4 can stimulate therapeutic, T cell-dependent anti-tumor activity in mice and humans. We asked whether exome ...sequencing and epitope prediction algorithms could identify antigens in progressively growing tumors that render them susceptible to anti-PD-1- or anti-CTLA-4-induced immune elimination. Expressed mutations in progressively growing, T3 methylcholanthrene (MCA)-sarcomas were identified by exome sequencing and prioritized based primarily on potential MHC class I binding affinity and predicted peptide processing. The two best candidate tumor-specific mutant antigens (TSMA) were identified from mutations in Laminin α subunit 4 (Lama4) and a glucosyltransferase called Alg8. When tested in vitro, mutant Lama4 (mLama4) and mutant Alg8 (mAlg8) peptides were the only top predicted TSMA that stimulated T3-specific T cells. The relevance of these findings was validated by four in vivo studies: (i) mLama4 and mAlg8 peptides bound to H-2Kb were biochemically detected on the surface of T3 tumors, (ii) as shown by tetramer staining, CD8+ tumor infiltrating lymphocytes (TIL) expressing TCRs for mLama4 and mAlg8 accumulated over time in T3 tumors in checkpoint blockade treated, tumor-bearing mice, reaching maximum levels just prior to tumor rejection, (iii) vaccination of naïve wild type mice with mutant but not WT forms of Lama4 or Alg8 induced strong CD8+ T cell responses and (iv) prophylactic or therapeutic vaccination with mLama4 plus mAlg8 synthetic long peptides (SLP) resulted in rejection of T3 cells. The therapeutic protection provided by the SLP vaccine was equal to that afforded by checkpoint blockade therapy. Since we did not see evidence of T cell responses to any other TSMA, we then explored whether the immunodominant antigens masked responses to other TSMA. Using CRISPR/Cas9, we introduced point mutations to revert mLama4 and mAlg8 in T3 back to their nonimmunogenic wild type counterparts. T3 tumor cells with either mLama4 or mAlg8 reverted to wild type were still susceptible to anti-PD-1 or anti-CTLA-4 immune-mediated control with skewing of the T cell response in the TIL towards the remaining TSMA. Additionally, we have obtained T3 tumor cells with both TSMA reverted to wild type and are currently testing whether these “fixed” T3 tumor cells are still subject to anti-PD-1 or anti-CTLA-4 and whether SLP vaccines comprised of subdominant TSMA still present in fixed T3 tumor cells can protect mice against tumor growth. This system will allow us to address the many questions surrounding poorly understood phenomenon of immunodominance and how this may influence patient-specific personalized cancer immunotherapy directed against TSMA.
Citation Format: Matthew M. Gubin, Jeffrey P. Ward, Takuro Noguchi, Xiuli Zhang, Cora Arthur, Willem-Jan Krebber, Gwenn E. Mulder, Cornelis J.M. Melief, William E. Gillanders, Maxim Artyomov, Elaine R. Mardis, Robert D. Schreiber. Tumor-specific mutant antigens in cancer immunotherapy abstract. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A001.
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