Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed.

PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemiaassociated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)- IntroductionCD8 T cells specific for the human leukocyte antigen-A2 (HLA-A2)-restricted peptides WT1 and PR1, which are derived from the endogenous leukemia-associated antigens Wilms' tumor antigen [1][2][3] and proteinase 3 (P3), respectively, mediate cytotoxicity against acute myeloid leukemia (AML). PR1-specific T cells also contribute to cytogenetic remission of chronic myeloid leukemia (CML) in patients treated with interferon, 4,5 and vaccination with WT1 and PR1 6,7 can induce specific CD8 immunity in patients with myeloid malignancies. These results validate endogenous self-peptides as targets for immunotherapy, including vaccination, adoptive cell therapy, or antibodies that bind peptide/MHC. Such T-cell receptor (TCR)-like monoclonal antibodies (mAbs) may have selective activity against leukemia if target peptide/MHC complexes are aberrantly expressed on leukemia. Furthermore, mAbs are easy to administer and can be dosed frequently, which may increase their effectiveness against high leukemia burdens.Eliciting TCR-like mAbs has been technically challenging, 8 primarily because of the high immunogenicity of HLA molecules in mice. Phage-display libraries, 9 peptide/MHC immunization, 10,11 and the combination of both strategies 8,12 have been used to produce TCR-like mAbs targeting peptides derived from solid-tumor antigens (eg, MAGE, ␤-HCG, TARP, and NY-ESO-1) in the context of HLA-A1 or HLA-A2. [9][10][11]13,14 Although antibody activity against primary tumors has not been well studied, complement-dependent cytotoxicity (CDC) against tumor cell lines has been reported. 11 Some toxin-conjugated antibodies also show activity against tumor cells. 14-16 However, to eradicate cancer, these antibodies must be active against cancerinitiating cells, and TCR-like mAb-induced cytolysis of cancer stem cells has not been reported. Nevertheless, because PR1-specific CTLs suppress leukemia progenitor cells in vitro 17 and because Lin Ϫ CD34 ϩ CD38 Ϫ cells are enriched for leukemia stem cells (LSCs) 18 and can be easily studied, we hypothesized that if an anti-PR1/HLA-A2 antibody could be produced, it may be active against blasts and LSCs from HLA-A2 ϩ AML patients.We report the discovery of 8F4, a novel mAb that binds with high affinity to a conformational epitope of PR1/HLA-A2 and induces dose-dependent cytolysis of myeloid leukemia cells but not normal hematopoietic cells. 8F4 mediates CDC against Lin Ϫ CD34 ϩ CD38 Ϫ LSCs and preferentially inhibits the growth of leukemia progenitor cells. These results justify further study of TCR-like antibodies to verify the differential effects against normal stem cells and LSCs. Biologically significant differences may justify further study of a humanized form o...

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ∼2.35 × 10 6 motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/ annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications. To discover or analyze functional ligand-receptor interactions in blood vessels under disease conditions, we have used combinatorial screenings based on phage display, which has enabled the targeted delivery of agents to specific vascular beds (8,9). This approach allows the selection of homing peptides to specific organs in vivo after systemic administration of random peptide libraries (10, 11). We have isolated ligand peptides and identified their tissue-specific receptors in rodents and in a patient, and have developed a ligand-receptor in prostate cancer (12, 13) that serves as the basis for an ongoing first-in-man trial.Over the past few years we have developed a tripartite approach to enable serial combinatorial selections to humans. First, we established an ethical framework to ensure respectful research in patients who were brain-dead or whose families decided to terminate life support (14,15). Second, we adapted techniques that were validated in rodents (16) to enable synchronous selection of ligands to multiple organs. Third, we integrated genomic tools, in which recovery of 10 6 peptides is ∼1;000-fold faster and ∼250-fold cheaper (17). Here, these quantitative and qualitative methods enabled refined combinatorial selections in patients and identified unique ligand-receptors.

PR1 is a human leukocyte antigen (HLA)-A2 restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived antigen presenting cells, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific cytotoxic T lymphocytes (PR1-CTL) and the anti-PR1/HLA-A2 antibody 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an antigen become susceptible to therapies that target cross-presented antigens and suggest PR1 as a broadly expressed tumor antigen.

Background aims The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4- chimeric antigen receptor (CAR) that was transduced into T-cells to mediate anti-leukemia activity. Methods Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endodomain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. Results Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4-CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro. Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. Conclusions Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro. Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse.