Targeting of palpable B16-F10 melanoma tumors with polyclonal antibodies on white blood cells.

BACKGROUND: Antibodies and other recognition molecules direct cancer cell death by multiple types of immune cells. Therapy directed at only one target typically results in tumor regrowth because of tumor heterogeneity. Our goal is to direct therapy to multiple targets simultaneously. Our previous studies showed that multiple antibodies targeting mutated tumor proteins inhibited tumor growth when injected subcutaneously near the time of cancer cell implantation. METHODS: A cocktail of rabbit antibodies against B16-F10 cell surface related mutated proteins were generated. Implanted B16-F10 cells were allowed to grow to palpable size before treatment. Antibodies were administered using different routes of exposure. Free antibody was compared to antibody armed on mouse splenic white blood cells (WBCs). Binding of the antibody cocktail was determined for mouse and human WBCs. RESULTS: The antibody cocktail inhibited tumor growth and prolonged survival when administered as free antibody or armed on WBCs. The antibody cocktail armed on WBCs achieved similar tumor inhibition as free antibody but at a dose 1000-fold less. Armed WBCs achieved tumor inhibition by intravenous and subcutaneous administration. The antibody cocktail bound well to human WBCs and saturation dose was defined. Binding was stable under simulated in vivo condition in human plasma at 37 °C. CONCLUSIONS: Antibodies targeting multiple tumor mutated proteins inhibited tumor growth and prolonged survival. Effective antibody dose was reduced 1000-fold by arming WBCs. Rabbit antibodies saturated human WBCs using <1 mg per billion cells. A phase I trial in cancer patients using this strategy has been approved by the FDA.

Development of Clinical-Grade Antibodies against Tumor-Specific Mutations to Target Neuroblastoma.

OBJECTIVE: Tumor heterogeneity is a fundamental problem in treating cancer with monotargeting therapy, including chemical, antibody, and T cell therapies. Our goal is to target multiple mutated peptides found in a patient's cancer to increase antibody therapy effectiveness. METHODS: Tumor samples were derived from patients with neuroblastoma. Whole-exome sequencing was performed of tumor and normal cells. Mutated proteins with missense mutations were selected from the patient tumor. These mutated proteins were further selected for the presence of missense mutations in the outer cell surface. Peptides representing a mutated section of the proteins were used for vaccinating rabbits and generating anti-peptide antibodies. The binding of individual polyclonal antibodies (pAbs) and the mixtures of pAbs were determined against the patient's tumor as cultured neuroblastoma cells and in a murine xenograft model. Antibodies were prepared according to FDA requirements of a phase I clinical protocol. RESULTS: All of the generated rabbit pAbs bound with high affinity to the corresponding peptide used for vaccination. The pAbs also bound to low passage neuroblastoma cells. Mixed as cocktails, the pAbs had substantially increased binding to cells and bound well to the xenograft tissue. No binding was observed to the panel of normal human tissues. Preparation of pAbs by an academic lab to clinical-grade was approved by FDA for phase I clinical trial. CONCLUSION: We describe a new strategy to make customized antibodies for individual cancer patients and present the data required to meet FDA specifications to begin a phase I clinical trial.

Preparation of clinical-grade WBCs using leukocyte reduction filters.

OBJECTIVE: Our goal was to develop a simpler and less expensive method of obtaining human clinical-grade WBCs using an alternative method to continuous leukapheresis. Our purpose for the WBCs is to arm them with rabbit anticancer antibodies for a phase I clinical trial. METHODS: Using leukocyte reduction filters (LRFs) discarded from the blood bank, we evaluated multiple variables to maximize recovery of WBCs with the lowest contamination of RBCs. Using an optimized protocol, full-scale runs according to FDA current Good Manufacturing Practice (cGMP) standards were completed with immediate filtration of blood obtained from donors participating in our study. RESULTS: Forward flushing of the filter removed 85% to 95% of residual RBCs and platelets. When backward flushed with 800 mL, 95% of the WBCs recovered were contained in the first 400 mL. The number of recovered WBCs was in the range of 166-211 million/100 mL filtered blood. Subpopulations of WBCs recovered from the LRFs were in the same proportion as the donors' whole blood. Viability of recovered WBCs was 96-99%. Exogenous rabbit antibodies bound well to the recovered WBCs and were retained for at least 5 h without significant reduction. Three full scale runs of WBCs recovered from donor blood filtered through the LRF met all FDA specification of sterility, endotoxin levels, viability and stability. CONCLUSION: Using LRFs, high quality clinical grade WBCs are readily obtained in quantities of 0.2 to 1.2 billion cells from 100 mL to 450 mL (1 unit) of whole blood.

A cocktail of polyclonal affinity enriched antibodies against melanoma mutations increases binding and inhibits tumor growth.

BACKGROUND: Antibodies that target a single tumor antigen fail to cure stage IV cancer patients due to tumor heterogeneity and variable expression of antigen. Tumor cells with insufficient binding of antibody will not undergo antibody induced cytotoxicity. We describe targeting multiple tumor-specific antigens that resulted in homogeneous dense binding to mouse melanoma cells and significant tumor growth inhibition. METHODS: Surface-related tumor-specific mutations on B16-F10 cells were identified. Peptides containing the single amino acid mutation were synthesized for 9 different neoantigens. Rabbits were vaccinated with each of these peptides and high affinity polyclonal antibodies to each peptide were obtained. The 9 antibodies were combined as a cocktail and mice with implanted B16-F10 cells were treated with and without PD1 inhibitor. RESULTS: Even a single dose of the antibody cocktail inhibited tumor growth and prolonged survival. PD1 inhibitor alone had little effect on tumor growth. The antibody cocktail plus PD1 inhibition increased tumor response and 4 doses of the cocktail completely prevented tumor growth in 50% of the mice. Complete responses were durable. The complete responders were highly resistant to tumor re-challenge at 6 months. No adverse events were identified in the antibody treated mice. CONCLUSIONS: Multiple tumor-specific cell surface-related neoantigens were abundant in B16-F10 cells. Antibodies to 9 of these neoantigens had variable binding but when combined had dense homogeneous binding. Even one dose of this cocktail of 9 antibodies improved survival and when multiple doses were combined with PD1 inhibition 50% of the mice were rendered permanently tumor free.

Immunization with tumor neoantigens displayed on T7 phage nanoparticles elicits plasma antibody and vaccine-draining lymph node B cell responses.

The aim of this preclinical study was to evaluate T7 bacteriophage as a nanoparticle platform for expression of neoantigens that could allow rapid generation of vaccines for potential studies in human cancer patients. We have generated recombinant T7 phage vaccines carrying neoepitopes derived from mutated proteins of B16-F10 melanoma tumor cells. With the single mutated amino acid (AA) centered, peptides were expressed on the outer coat of T7 phage. All peptides with 11 and 34 AAs were successfully expressed. Trimers of the 11-AA peptides were successfully expressed in only 3 of 8 peptides. The 11-AA peptide was better in stimulating antibodies selective for the mutated region than the longer 34-AA peptide. We observed a dose response for vaccines which provides an initial framework of the minimum phage required for vaccination. A single injection with phage-peptide vaccines in both monomer and trimer formats produced significant immune responses in mice on day 21, as assessed by lymph node cell counts, next generation sequencing (NGS), and plasma titers against T7 phage and vaccine peptides. A trimer provided no additional serum response to the monomer format. Immunization of mice with a mixture of 8 different peptide vaccines resulted in antibodies to most of the peptides. It was encouraging that induced antibodies had higher binding to the mutated peptides compared to the corresponding normal peptides. The NGS of lymph node cells demonstrated a low B cell receptor diversity and clonal hyperpolarization in vaccine-draining lymph nodes in comparison to those in unvaccinated mice nodes. The NGS data also revealed phenomenal increase in IgG and other class-switched antibodies following vaccination. These results agree with the higher plasma titers of IgG antibodies against T7 phage and vaccine peptides. Antibodies bound whole B16-F10 cells, lysates and multiple bands on Western blot. This indicates that these vaccine peptides successfully induced antibodies that bind full proteins from which the vaccine peptides were derived. We demonstrate a preclinical platform for rapid production of vaccines that can deliver mutated peptides and stimulate an appropriate B cell response. We anticipate further research in utilizing the cells from a tumor or vaccine draining lymph node as a resource for therapeutic anticancer reagents.

Identification of tumor-reactive B cells and systemic IgG in breast cancer based on clonal frequency in the sentinel lymph node.

A better understanding of antitumor immune responses is the key to advancing the field of cancer immunotherapy. Endogenous immunity in cancer patients, such as circulating anticancer antibodies or tumor-reactive B cells, has been historically yet incompletely described. Here, we demonstrate that tumor-draining (sentinel) lymph node (SN) is a rich source for tumor-reactive B cells that give rise to systemic IgG anticancer antibodies circulating in the bloodstream of breast cancer patients. Using a synergistic combination of high-throughput B-cell sequencing and quantitative immunoproteomics, we describe the prospective identification of tumor-reactive SN B cells (based on clonal frequency) and also demonstrate an unequivocal link between affinity-matured expanded B-cell clones in the SN and antitumor IgG in the blood. This technology could facilitate the discovery of antitumor antibody therapeutics and conceivably identify novel tumor antigens. Lastly, these findings highlight the unique and specialized niche the SN can fill in the advancement of cancer immunotherapy.

Characterization of sentinel node-derived antibodies from breast cancer patients.

Autoantibodies to breast and other cancers are commonly present in cancer patients. A method to rapidly produce these anti-cancer autoantibodies in the lab would be valuable for understanding immune events and to generate candidate reagents for therapy and diagnostics. The purpose of this report is to evaluate sentinel nodes (SNs) of breast cancer patients as a source of anti-cancer antibodies. Radiotracer lymphatic mapping in 29 patients with breast cancer confirmed the identity of the SNs which provided source cells for this study. Flow cytometry demonstrated ~28% of the MNCs were B cells and ~44% of the B cells were class switched memory B cells. EBV-induced proliferation of B cells yielded tumor binding antibodies from 3 wells per 1000 but cultures were too unstable for detailed evaluations. Hybridomas generated by electrofusion produced IgG (48%), IgM (34%) and IgA (18%) antibody isotypes which were screened for binding to a panel of breast cancer cells of the major molecular subtypes. Tumor lysate binding was observed in 28% of the hybridoma clones and 10% of these bound whole tumor cells with unique binding patterns. More detailed evaluation of selected clones showed binding to the patient's own tumor. SNs are removed from more than 100,000 breast cancer patients in the US per year. Samples from these lymph nodes represent a substantial opportunity to generate anticancer antibodies.

Vaccine-draining lymph nodes of cancer patients for generating anti-cancer antibodies.

BACKGROUND: Our research is focused on using the vaccine draining lymph node to better understand the immune response to cancer vaccines and as a possible source of anti-cancer reagents. We evaluated vaccine draining lymph nodes archived from a clinical study in melanoma patients and determined the reaction of B cells to the vaccine peptides. METHODS: Mononuclear cells (MNCs) were recovered from cryopreserved lymph nodes that were directly receiving drainage from multi-peptide melanoma vaccine. The patients were enrolled on a vaccine study (NCT00089219, FDA, BB-IND No. 10825). B cell responses in the vaccine-draining lymph nodes were studied under both stimulated and un-stimulated conditions. Cryopreserved cells were stimulated with CD40L, stained with multiple human cell-surface markers (CD19, CD27, IgM) to identify different categories of B cell sub populations with flow cytometry. Hybridomas were generated from the lymph node cells after CD40L-stimulation. Cells were fused to murine plasmacytoma P3X63.Ag8.653 using Helix electrofusion chamber. ELISA was used to evaluate hybridoma derived antibody binding to vaccine peptides. RESULTS: Viable MNCs were satisfactorily recovered from lymph nodes cryopreserved from six vaccine study patients 8-14 years previously. B cell ELISPOT demonstrated responses for each patient to multiple vaccine peptides. CD40L stimulation of lymph node cells increased the proportion of CD19+ CD27+ cells from 12 to 65% of the sample and increased the proportion of class-switched cells. Screening of IgG secreting clones demonstrated binding to melanoma vaccine peptides. CONCLUSIONS: B cells were successfully recovered and expanded from human cryopreserved vaccine-draining lymph nodes. Individual B cells were identified that secreted antibodies that bound to cancer vaccine peptides. The ability to reliably generate in vitro the same antibodies observed in the blood of vaccinated patients will facilitate research to understand mechanisms of human antibody activity and possibly lead to therapeutic antibodies.

Vaccine draining lymph nodes are a source of antigen-specific B cells.

PURPOSE: Our research is focused on using vaccine draining lymph nodes as a source of immune cells to better understand the immune response and to attempt to generate new anti-cancer reagents. Following a vaccine, harvesting the lymph node can only be done once. We endeavored to determine the range of times that B cells secreting anti-KLH antibodies were present in the node of KLH-vaccinated mice. RESULTS: Following vaccination the total number of mononuclear cells (MNCs) increased in the vaccine-draining lymph node (VDN). The percentage of MNCs that were B cells nearly doubled. B cells recovered from the node that secreted anti-KLH antibodies were evident by day 7. The number continued to increase and then slowly decreased over the observed time range to 28days after vaccination. The VDN, compared to the spleen, the bone marrow and the nonVDN, contained a higher percentage of B cells that secreted anti-KLH antibodies. CONCLUSIONS: After a vaccine, there is a multi-week window of time when an increasing number of B cells are present in a VDN that secrete anti-KLH antibodies. These results support using the VDN as a source for B cells that secrete anti-vaccine antibodies.

Autologous antibodies that bind neuroblastoma cells.

Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

Intravenous infusion of phage-displayed antibody library in human cancer patients: enrichment and cancer-specificity of tumor-homing phage-antibodies.

Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage-antibodies and a Stage IV cancer status. All patients were intravenously administered 1 × 10(11) TUs/kg of an scFv library 1 to 4 h before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0-5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor-specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2,355) that was found to share a 12-amino-acid-long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis.

Structural basis of binding by cyclic nonphosphorylated peptide antagonists of Grb7 implicated in breast cancer progression.

Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7.

Cancer cell-specific internalizing ligands from phage displayed beta-lactamase-peptide fusion libraries.

The present study was focused on identifying cancer cell-specific internalizing ligands using a new kind of phage display library in which the linear or cysteine-constrained random peptides were at amino-terminus fusion to catalytically active P99 beta-lactamase molecules. The size and quality of libraries were comparable to other reported phage display systems. Several cancer cell-specific binding and internalizing beta-lactamase-peptide fusion ligands were isolated by selecting these libraries on the live BT-474 human breast cancer cells. The identified ligands shared several significant motifs, which showed their selectivity and possible binding to some common cancer cell targets. The beta-lactamase fusion made the whole process of clone screening efficient and simple. The ligands selected from such libraries do not require peptide synthesis and modifications, and can be used directly for applications that require ligand tracking. In addition, the selected beta-lactamase-peptide ligands have a potential for their direct use in targeted enzyme prodrug therapy. The cancer-specific peptides can also be adopted for other kinds of targeted delivery protocols requiring cell-specific affinity reagents. This is first report on the selection of cell-internalized enzyme conjugates using phage display technology, which opens the possibility for new fusion libraries with other relevant enzymes.

Phage-displayed combinatorial peptide libraries in fusion to beta-lactamase as reporter for an accelerated clone screening: Potential uses of selected enzyme-linked affinity reagents in downstream applications.

Phage-display selection of combinatorial libraries is a powerful technique for identifying binding ligands against desired targets. Evaluation of target binding capacity of multiple clones recovered from phage display selection to a specific target is laborious, time-consuming, and a rate-limiting step. We constructed phage-display combinatorial peptide libraries in fusion with a beta-lactamase enzyme, which acts as a reporter. Linear dodecapeptide and cysteine-constrained decapeptide libraries were created at the amino-terminus of the Enterobacter cloacae P99 cephalosporinase molecule (P99 beta-lactamase). The overall and positional diversity of amino acids in both libraries was similar to other phage-display systems. The libraries were selected against the extracellular domain of ErbB2 receptor (ErbB2(ECD)). The target-selected clones were already conjugated to an enzyme reporter, therefore, did not require subcloning or any other post-panning modifications. We used beta-lactamase enzyme activity-based assays for sample normalizations and clone binding evaluation. Clones were identified that bound to purified ErbB2(ECD) and ErbB2-overexpressing cell-lines. The peptide sequences of the selected binding clones shared significant motifs with several rationally designed peptide mimetics and phage-display derived peptides that have been reported to bind ErbB2(ECD). beta-Lactamase fusion to peptides saved time and resources otherwise required by the phage-ELISA of a typical phage display screening protocol. The beta-lactamase enzyme assay protocols is a one-step process that does not require secondary proteins, several steps of lengthy incubations, or washings and can be finished in a few minutes instead of hours. The clone screening protocol can be adopted for a high throughput platform. Target-specific beta-lactamase-linked affinity reagents selected by this procedure can be produced in bulk, purified, and used, without any modification, for a variety of downstream applications, including targeted prodrug therapy.

Novel beta-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy.

Novel phage-displayed random linear dodecapeptide (X(12)) and cysteine-constrained decapeptide (CX(10)C) libraries constructed in fusion to the amino-terminus of P99 beta-lactamase molecules were used for identifying beta-lactamase-linked cancer cell-specific ligands. The size and quality of both libraries were comparable to the standards of other reported phage display systems. Using the single-round panning method based on phage DNA recovery, we identified several beta-lactamase fusion peptides that specifically bind to live human breast cancer MDA-MB-361 cells. The beta-lactamase fusion to the peptides helped in conducting the enzyme activity-based clone normalization and cell-binding screening in a very time- and cost-efficient manner. The methods were suitable for 96-well readout as well as microscopic imaging. The success of the biopanning was indicated by the presence of approximately 40% cancer cell-specific clones among recovered phages. One of the binding clones appeared multiple times. The cancer cell-binding fusion peptides also shared several significant motifs. This opens a new way of preparing and selecting phage display libraries. The cancer cell-specific beta-lactamase-linked affinity reagents selected from these libraries can be used for any application that requires a reporter for tracking the ligand molecules. Furthermore, these affinity reagents have also a potential for their direct use in the targeted enzyme prodrug therapy of cancer.

Phage-display selection on tumor histological specimens with laser capture microdissection.

A method was developed to obtain phage-display ligands that bind to a select population of cells in histological specimens of freshly harvested solid human cancers. It combines phage-display panning with laser capture microdissection (LCM). This method allows selection of phage ligands bound to subpopulations of specific cells contained in tumor tissue on histological sections. Naïve phage scFv library was incubated directly on a histological section of human breast cancer that was snap frozen immediately after surgical resection. Tumor and stromal cells were captured by LCM and bound phages were recovered by bacterial infection. Individual phage clones selected after panning were evaluated for their binding ability by immunofluorescence staining on tumor tissue from the same patient. One phage-display antibody clone selected on tumor stroma showed selective binding on tumor stroma but did not bind to malignant cell population. The expressed scFv of this clone showed no significant binding to normal tissue, or 13 other breast cancers, or 4 colon cancer samples. Using the same method, phage display antibody clones were selected on tumor cells which showed binding to tumor cells and normal tissue. This method is applicable for selection of ligands to virtually any portion of a histological specimen amenable to LCM. This may speed the process of generating ligands to any subset of cells or noncellular feature present on histological specimens.

Developing bifunctional beta-lactamase molecules with built-in target-recognizing module for prodrug therapy: identification of Enterobacter Cloacae P99 cephalosporinase loops suitable for randomization and phage-display selection.

This study was focused on developing catalytically active beta-lactamase enzyme molecules that have target-recognizing sites built within their scaffold. Using phage-display approach, nine libraries were constructed by inserting the randomized linear or cysteine-constrained heptapeptides in the five different loops on the outer surface of P99 beta-lactamase molecule. The pIII signal peptide of Sec-pathway was employed for a periplasmic translocation of the beta-lactamase fusion protein, which we found more efficient than the DsbA signal peptide of SRP-pathway. The randomized heptapeptide loops replaced native amino acids between positions (34)Y-(37)K, (238)M-(246)A, (275)N-(280)A, (305)A-(311)S, or (329)I-(334)I of the P99 beta-lactamase molecules for generating the loop-1 to -5 libraries, respectively. The diversity of each loop library was judged by counting the primary and beta-lactamase-active clones. The linear peptide inserts in the loop-2 library showed the maximum number of the beta-lactamase-active clones, followed by the loop-5, loop-3, and loop-4. The insertion of the cysteine-constrained loops exhibited a dramatic loss of the enzyme-active beta-lactamase clones. The complexity of the loop-2 linear library, as determined by the frequency and diversity of amino acid distributions in the randomized region, appears consistent with the standards of other types of phage display library systems. The selection of the loop-2 linear library on streptavidin protein as a test target identified several beta-lactamase clones that specifically bound to streptavidin. In conclusion, this study identified the suitability of the loop-2 of P99 beta-lactamase for constructing a phage-display library of the beta-lactamase enzyme-active molecules that can be selected against a target. This is an enabling step in our long-term goal of developing bifunctional beta-lactamase molecules against cancer-specific targets for enzyme prodrug therapy of cancer.

Biopanning Phage-Display Libraries on Small Tissue Sections Captured by Laser Capture Microdissection.

Phage-display technology has been widely used for developing tumor-targeting agents. Laser capture microdissection (LCM) has proven to be an accurate method to select specific cells from histological sections. Our goal was to develop a method to combine phage-display with LCM to obtain phage-displayed ligands that bind to selected cells in human solid tumors. Two panning strategies were evaluated and optimized. The first strategy was to pan on patient tissue mounted to LCM slides before LCM occurred. The poor panning output showed that phage did not tolerate the drying conditions during LCM. The second strategy was to pan on tumor cells from the patient tumor tissue that were isolated by LCM. The catapulted tumor cells were transferred to a filter unit which retained cells but allowed rinsing of unbound phage. Six commercially available filter units were evaluated and the one with the lowest nonspecific binding to phage was selected for the panning steps. The smallest number of cells (500) in which panning could be successfully accomplished was also determined. A micropipette system was developed to further decrease background by removing catapulted cells from the filter unit after panning was complete. This left behind nearly all background binding phage in the filter unit. This strategy led to the selection of individual phage antibody clones (5 out of 79 tested) specific for tumor cells of the patient's cancer tissue. Immunofluorescence staining on tumor tissues from the same patient showed that these clones have selective signals on tumor island cells, while the scFv library only showed low nonspecific signals on tumor tissues. We established a method of panning on a small number of LCM-captured solid tumor specimens. The quick identification of specific phage-displayed antibodies in the cancer tissue of human patients will greatly enhance the therapy and diagnosis of cancer.

Towards a ligand targeted enzyme prodrug therapy: single round panning of a beta-lactamase scaffold library on human cancer cells.

A novel beta-lactamase scaffold library in which the target-binding moiety is built into the enzyme was generated using phage display technology. The binding element is composed of a fully randomized 8 amino acid loop inserted at position between Y34 and K37 on the outer surface of Enterobacter cloacae P99 cephalosporinase (beta-lactamase, E.C. 3.5.2.6) with all library members retaining catalytic activity. The frequency and diversity of amino acids distributions in peptide inserts from library clones were analyzed. The complexity of the randomized loop appears consistent with standards of other types of phage display library systems. The library was panned against SKBR3 human breast cancer cells in 1 round using rolling circle amplification of phage DNA to recover bound phage. Individual beta-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound beta-lactamase activity. SKBR3 cell-binding beta-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected beta-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor cell-lines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted beta-lactamase ligands that may have a potential for their use in enzyme prodrug therapy with cephalosporin-based prodrugs. It is expected that a similar approach will be useful in developing tumor-targeting molecules of several other enzyme candidates of cancer prodrug therapy.

Selection of tumor-binding ligands in cancer patients with phage display libraries.

Phage display has been used extensively in vitro and in animal models to generate ligands and to identify cancer-relevant targets. We report here the use of phage-display libraries in cancer patients to identify tumor-targeting ligands. Eight patients with stage IV cancer, including breast, melanoma, and pancreas, had phage-displayed random peptide or scFv library (1.6 x 10(8)-1 x 10(11) transducing units/kg) administered i.v.; tumors were excised after 30 minutes; and tumor-homing phage were recovered. In three patients, repeat panning was possible using phage recovered and amplified from that same patient's tumor. No serious side effects, including allergic reactions, were observed with up to three infusions. Patients developed antiphage antibodies that reached a submaximal level within the 10-day protocol window for serial phage administration. Tumor phage were recoverable from all the patients. Using a filter-based ELISA, several clones from a subset of the patients were identified that bound to a tumor from the same patient in which clones were recovered. The clone-binding to tumor was confirmed by immunostaining, bioassay, and real-time PCR-based methods. Binding studies with noncancer and cancer cell lines of the same histology showed specificity of the tumor-binding clones. Analysis of insert sequences of tumor-homing peptide clones showed several motifs, indicating nonrandom accumulation of clones in human tumors. This is the first reported series of cancer patients to receive phage library for serial panning of tumor targeting ligands. The lack of toxicity and the ability to recover clones with favorable characteristics are a first step for further research with this technology in cancer patients.