Antibody Fragment scFv Research Articles

Tumor Radioimmunolocalization in Nude Mice by Mono- and Divalent- Single-Chain Fv Antiplacental Alkaline Phosphatase Antibodies

One single-chain Fv antibody fragment (scFv) and a new recombinant covalently linked dimeric scFv antibody (sc(Fv)(2)) against placental alkaline phosphatase (PLAP) were investigated for selective tumor targeting. The biological behavior of these new antibodies was compared to that of the original native antibody, H7 MAb. The sc(Fv)(2)) antibody displayed convincing tumor localization properties with a rapid excretion pattern comparable to the scFv, but with a longer retention time in the tumor, and higher tumor-to-nontumor ratio (27:1), compared to the scFv (15:1), at 48 hours. For the sc(Fv)(2) antibody, more than 50% of the remaining activity in the mouse was present in the tumor between 24 and 48 hours after the injection. With this antibody, scintigraphic visualization of the tumor was also possible 1 week after the injection. It is concluded that this sc(Fv)(2) antibody fragment, with two binding sites, displays properties suitable for in vivo targeting of PLAP expressing tumors.

Production of recombinant antibody fragments in Bacillus megaterium.

BackgroundRecombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments.ResultsThe lysozyme specific single chain Fv (scFv) fragment D1.3 was succesfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41°C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 μg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli.ConclusionHigh yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli.

Monovalent antibody scFv fragments selected to modulate T-cell activation by inhibition of CD86–CD28 interaction

Beside the interaction of the antigen-presenting major histocompatibility complex with the T-cell receptor, a co-stimulatory signal is required for T-cell activation in an immune response. To reduce immune-mediated graft rejection in corneal transplantation, where topical application of drugs in ointments or eye-drops may be possible, we selected single-chain antibody fragments (scFv) with binding affinity to rat CD86 (B7.2) that inhibit the co-stimulatory signal. We produced the IgV-like domain of rat CD86 as a fusion protein in Escherichia coli by refolding from inclusion bodies. This protein was used as a target for phage display selection of scFv from HuCAL-1, a fully artificial human antibody library. Selected binding molecules were shown to specifically bind to rat CD86 and inhibit the interaction of CD86 with CD28 and CTLA4 (CD152) in flow cytometry experiments. In an assay for CD86-dependent co-stimulation, the selected scFv fragment successfully inhibited the proliferation of T-cells induced by CD86-expressing P815 cells.

Characterization of a Human Monoclonal Antibody Directed Against the Spacer Domain of ADAMTS13; a Prototype of Antibodies Present in Patients with Acquired Thrombotic Thrombocytopenic Purpura.

Acquired thrombotic thrombocytopenic purpura (TTP) is often accompanied by the presence of (auto-) antibodies directed against the plasma metalloprotease ADAMTS13. The antibodies prevent efficient proteolytic cleavage of unusually large von Willebrand factor multimers by ADAMTS13, which is highly reactive with platelets. This may result in the formation of platelet aggregates that occlude the microvasculature of patients with TTP. In all presently characterized patients with acquired TTP and detectable anti-ADAMTS13 antibodies, antibodies directed to the (cysteine-rich) spacer domain were present. In order to study anti-ADAMTS13 antibodies at the molecular level, we have isolated single-chain Fv antibody fragments (scFv) from an immunoglobulin V-gene phage-display library derived from CD19+ B-cells of a patient with acquired TTP. From this library scFv I-9 was isolated, of which the variable heavy chain segment is homologous to members of the VH1-69 family. Using ELISA and immunoprecipitation analysis we confirmed that antibody I-9 also binds to the spacer domain. We converted the scFv antibody fragment into a full-length human antibody to better study its effect on proteolytic cleavage of VWF and substrate binding. Antibody I-9 was included into a modified FRETS-VWF73 assay and a dose-dependent inhibition of VWF73 proteolysis by ADAMTS13 was observed. In addition, antibody I-9 was able to inhibit the proteolysis of plasma-derived VWF multimers by ADAMTS13. In both assays inhibition was not very efficient; which is most likely due to the low affinity of antibody I-9 for ADAMTS13. Chimeric ADAMTS13 variants in which 5–10 amino acid residues of the ADAMTS13 spacer domain have been exchanged for the corresponding sequence of ADAMTS1 were used to determine the binding site for antibody I-9 in the spacer domain. Our findings show that amino acid regions 572–579 and 657–666 of the spacer domain are required for binding of antibody I-9, although several other regions are also involved in the interaction. We have previously shown that these amino acid regions are also crucial for binding of (polyclonal) antibodies present in plasma of patients with acquired TTP. Additionally, competition experiments were performed to address whether antibodies with similar properties as antibody I-9 are indeed present in plasma from patients with acquired TTP during acute disease. Plasma from 6 patients with acquired TTP was able to compete in a dose-dependent manner with antibody I-9 for binding to immobilized ADAMTS13, including plasma from the patient whose lymphocytes were used for construction of the phage-display library. These results show that antibodies with similar epitope-specificity as antibody I-9 are present in plasma of patients with acquired TTP. Since anti-spacer domain antibodies have been found in all presently characterized patients with anti-ADAMTS13 antibodies, the current data suggest that antibody I-9 is representative of the pathogenic antibodies present in plasma of the majority of patients with acquired TTP.

121 POSTER Isolation and characterisation of anti-idiotypic scFv antibody fragments and llama VHH domains used as a surrogate tumour antigen to elicit an anti-HER-2 humoral response in mice

121 POSTER Isolation and characterisation of anti-idiotypic scFv antibody fragments and llama VHH domains used as a surrogate tumour antigen to elicit an anti-HER-2 humoral response in mice

Selective antibody‐mediated targeting of class I MHC to EGFR‐expressing tumor cells induces potent antitumor CTL activity in vitro and in vivo

Epidermal growth factor receptor (EGFR) is highly overexpressed in many tumor types. We present a new fusion molecule that can target solid tumors that express EGFR. The fusion molecule combines the advantage(s) of the well-established tumor targeting capabilities of high affinity recombinant fragments of antibodies with the known efficient, specific and potent killing ability of CD8 T lymphocytes directed against highly antigenic MHC/peptide complexes. A recombinant chimeric molecule was created by the genetic fusion of the scFv antibody fragment derived from the anti-EGFR monoclonal antibody C225, to monomeric single-chain HLA-A2 complexes containing immunodominant tumor or viral-specific peptides. The fusion protein can induce very efficiently CTL-dependent lysis of EGFR-expressing tumor cells regardless of the expression of self peptide-MHC complexes. Moreover, the molecule exhibited very potent antitumor activity in vivo in nude mice bearing preestablished human tumor xenografts. These in vitro and in vivo results indicate that recombinant scFv-MHC-peptide fusion molecules might represent a novel and powerful approach to immunotherapy of solid tumors, bridging antibody and T lymphocyte attack on cancer cells.

In vitro evolution of single-chain antibodies using mRNA display

Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations. Kinetic analysis of the mutant scFvs revealed that the off-rates have been decreased by more than one order of magnitude and the dissociation constants were improved ∼30-fold. The antigen-specificity was not improved by affinity maturation, but remained similar to that of the wild type. Although the five consensus mutations of the high-affinity mutants were scattered over the scFv sequence, analysis by site-directed mutagenesis demonstrated that the critical mutations for improving affinity were the two that lay within the complementarity determining regions (CDRs). Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.

Adsorption and Activity of a Domoic Acid Binding Antibody Fragment on Mesoporous Silicates

The adsorption of an anti-domoic acid single-chain Fv (scFv) antibody fragment onto a range of mesoporous silicate supports was investigated. The scFv fragment adsorbed to all materials investigated, and pI had an apparently large effect on coating, with the greatest-and fastest-adsorption found on the most negatively charged silicates. Maximal coating levels attainable did not reflect the pore diameters of the materials. The immobilized antibody was functional on all materials and bound its antigen, a naturally occurring neurotoxin produced by shellfish, in a rapidly saturating manner that suggested the antibody adsorbed in a multilayer on the mesoporous particles. The antigen:antibody ratio decreased from 1:1.3 to <1:10 with increasing concentration of immobilized antibody, and the immobilized scFv exhibited no detectable reduction in domoic acid binding over a 42-day incubation period.

Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support

Domoic acid is a potent neurotoxin that can lead to amnesic shellfish poisoning in humans through ingestion of contaminated shellfish. We have produced and purified an anti-domoic acid single-chain Fragment variable (scFv) antibody fragment from the Escherichia coli periplasm. Yields of functional protein were increased by up to 100-fold upon co-production of E. coli DnaKJE molecular chaperones but co-overproduction of GroESL led to a reduction in solubility of the scFv. Co-production of the peptidyl-prolyl isomerase trigger factor resulted in accumulation of unprocessed scFv in the E. coli cytoplasm. This was due to an apparent bottleneck in translocation of the cytoplasmic membrane by the recombinant polypeptide. Co-expression of the E. coli disulfide bond isomerase dsbC increased scFv yields by delaying lysis of the host bacterial cells though this effect was not synergistic with molecular chaperone co-production. Meanwhile, use of a cold-shock promoter for protein production led to accumulation of greater amounts of scFv polypeptide which was predominantly in insoluble form and could not be rescued by chaperones. Purification of the scFv was achieved using an optimised metal affinity chromatography procedure and the purified protein bound domoic acid when immobilised on a mesoporous silicate support. The work outlines the potential benefit of applying a molecular chaperone/folding catalyst screening approach to improve antibody fragment production for applications such as sensor development.

An engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting properties

The antibody-mediated targeted delivery of interleukin-12 (IL12) to the EDB domain of fibronectin, a marker of angiogenesis, is a promising avenue for enhancing the therapeutic index of this anti-cancer cytokine. Previous experiments, based on sequential fusion of a single-chain IL12 derivative to the anti-EDB antibody fragment scFv(L19) had yielded a therapeutic fusion protein [IL12-scFv(L19)-FLAG], which displayed an impressive therapeutic activity in murine models of cancer, in spite of a tumor uptake, which was less efficient compared to the parental unmodified scFv(L19). In this article, we describe the comparative analysis of 3 recombinant fusion proteins comprising the scFv(L19) and IL12 moieties. One of them, in which the p40 and p35 form a covalent heterodimer and in which each subunit is fused to a molecule of scFv(L19), displays an excellent tumor targeting performance in vivo, as assessed by quantitative biodistribution analysis, and a potent anti-tumor effect, superior to the one of IL12-scFv(L19)-FLAG. These results may have a clinical impact, considering the fact that the tumor targeting ability of scFv(L19) in patients with cancer has been demonstrated using scintigraphic methods, and that 2 scFv(L19)-based antibody-cytokine fusion are currently entering clinical trials.

Her2-targeted pegylated stealth liposomal doxorubicin (PLD) retains its specific targeting ability to Her2-expressing tumor cells after in vivo circulation and extravasation to mouse malignant ascites

13097 Background: Receptor-directed targeting of ligand-bearing liposomes to tumor cells may enhance therapeutic efficacy by intracellular delivery of a concentrated payload of liposomal drug. The goal of this study was to assess whether Her2-targeted PLD retains its binding ability to Her2-expressing target cells through circulation in the blood and extravasation to the ascitic fluid of mice with malignant ascites. Methods: PLD was grafted with a pegylated lipophilic conjugate of an anti-Her2 scFv antibody fragment (F5) at a ratio of 15 ligands per liposome. BALB/c mice were injected with J6456 lymphoma cells into the peritoneal cavity to generate malignant ascites. When abdominal swelling developed, Her2-targeted PLD and nontargeted PLD were injected into the mice i.v. at a dose of 15 mg/kg. The ascitic fluid was collected 48 hr later, ascitic tumor cells were removed, and the doxorubicin levels in the cell-free ascitic fluid and plasma were determined. Binding of the liposome-containing ascitic fluid was tested in vitro against Her2-expressing human tumor cell lines (N87, SKBR-3) and compared to the binding of shelf formulations (not passaged in vivo) of Her2-PLD and PLD, using as parameter the amount of cell-associated doxorubicin. Results: Plasma and ascitic fluid levels of Her2-PLD were only slightly below those of PLD indicating that the Her2 ligand did not cause any significant change in the clearance rate of PLD. Her2-PLD and PLD bound to an equal extent to J6456 cells in vivo. The in vitro binding of Her2-PLD from ascites to Her2- expressing cells was increased 10 to 20-fold above that of PLD from ascites, similarly to the 20-fold difference in binding between shelf Her2-PLD and PLD. Conclusions: Her2-targeted PLD demonstrates similar circulation time to that of nontargeted PLD. After in vivo passage, the targeted liposome retains most of its original binding capacity to Her2 expressing cells indicating that the ligand is stably maintained in association with the doxorubicin liposomal carrier. Targeting of PLD using this Her2 antibody fragment should provide an important means of selective drug delivery to tumors expressing the Her2 receptor. [Table: see text]

Evaluation of Mut+ and MutS Pichia pastoris Phenotypes for High Level Extracellular scFv Expression under Feedback Control of the Methanol Concentration

Extracellular secretion of over 4 g x L(-1) of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut+ and MutS phenotypes were compared in a bioreactor under closed-loop PID control of the methanol level. As expected, the MutS phenotype has a growth rate lower than that of the Mut+ (0.37 vs 1.05 d(-1)) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut+ (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g x L(-1) for the MutS also has a positive effect on the final product concentration. Both Mut+ and MutS phenotypes reach a maximum biomass density around 450 g x L(-1) wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOX promoters, without prior knowledge of the culture growth parameters.

Rapid induction of apoptosis in B‐cell lymphoma by functionally isolated human antibodies

Novel panning and screening methodology was devised to isolate high affinity human recombinant scFv antibody fragments with functionally associated properties in B lymphoma cells. The approach was used to generate a panel of apoptosis-inducing antibodies specific for antigens differentially expressed in B lymphoma vs. T leukaemia cells. The selections resulted in an antibody pool with near perfect selectivity (>99%) for the B lymphoma target cells. Randomly picked clones (72) revealed 7 unique antibody genotypes. Six of these rapidly induced apoptosis in target cells. Following the conversion to full IgGs, the antibodies were shown to be specific for HLA-DR/DP, the B-cell receptor mu chain and for CD54/ICAM-1. The latter receptor was not previously associated with apoptotic properties in B-cell lymphomas. Anti-ICAM-1 IgG induced apoptosis in a broad range of B lymphoma cell lines and were shown by immunohistochemistry to bind strongly to B lymphoma tissue obtained from 5 different B lymphoma patients. The recombinant IgG antibodies had affinities in the subnanomolar (0.3 nM) to nanomolar (3 nM) range. The described technology is generally applicable for the rapid isolation of high affinity human antibodies with specificity for differentially expressed cell surface receptors with intrinsic negative or positive signalling properties from naïve phage libraries.

CD7-restricted activation of Fas-mediated apoptosis: a novel therapeutic approach for acute T-cell leukemia

AbstractAgonistic anti-Fas antibodies and multimeric recombinant Fas ligand (FasL) preparations show high tumoricidal activity against leukemic cells, but are unsuitable for clinical application due to unacceptable systemic toxicity. Consequently, new antileukemia strategies based on Fas activation have to meet the criterion of strictly localized action at the tumor-cell surface. Recent insight into the FasL/Fas system has revealed that soluble homotrimeric FasL (sFasL) is in fact nontoxic to normal cells, but also lacks tumoricidal activity. We report on a novel fusion protein, designated scFvCD7:sFasL, that is designed to have leukemia-restricted activity. ScFvCD7:sFasL consists of sFasL genetically linked to a high-affinity single-chain fragment of variable regions (scFv) antibody fragment specific for the T-cell leukemia-associated antigen CD7. Soluble homotrimeric scFvCD7:sFasL is inactive and acquires tumoricidal activity only after specific binding to tumor cell-surface-expressed CD7. Treatment of T-cell acute lymphoblastic leukemia (T-ALL) cell lines and patient-derived T-ALL, peripheral T-cell lymphoma (PTCL), and CD7-positive acute myeloid leukemia (AML) cells with homotrimeric scFvCD7:sFasL revealed potent CD7-restricted induction of apoptosis that was augmented by conventional drugs, farnesyl transferase inhibitor L-744832, and the proteasome inhibitor bortezomib (Velcade; Millenium, Cambridge, MA). Importantly, identical treatment did not affect normal human peripheral-blood lymphocytes (PBLs) and endothelial cells, with only moderate apoptosis in interleukin-2 (IL-2)/CD3-activated T cells. CD7-restricted activation of Fas in T-cell leukemic cells by scFvCD7:sFasL revitalizes interest in the applicability of Fas signaling in leukemia therapy.

ATP Hydrolysis Is Essential for the Function of the Uup ATP-binding Cassette ATPase in Precise Excision of Transposons

In Escherichia coli K-12, the RecA- and transposase-independent precise excision of transposons is thought to be mediated by the slippage of the DNA polymerase between the two short direct repeats that flank the transposon. Inactivation of the uup gene, encoding an ATP-binding cassette (ABC) ATPase, led to an important increase in the frequency of precise excision of transposons Tn10 and Tn5 and a defective growth of bacteriophage Mu. To provide insight into the mechanism of Uup in transposon excision, we purified this protein, and we demonstrated that it is a cytosolic ABC protein. Purified recombinant Uup binds and hydrolyzes ATP and undergoes a large conformational change in the presence of this nucleotide. This change affects a carboxyl-terminal domain of the protein that displays predicted structural homology with the socalled little finger domain of Y family DNA polymerases. In these enzymes, this domain is involved in DNA binding and in the processivity of replication. We show that Uup binds to DNA and that this binding is in part dependent on its carboxyl-terminal domain. Analysis of Walker motif B mutants suggests that ATP hydrolysis at the two ABC domains is strictly coordinated and is essential for the function of Uup in vivo.

A CD33‐specific single‐chain immunotoxin mediates potent apoptosis of cultured human myeloid leukaemia cells

A novel single-chain immunotoxin was constructed by combining a CD33-specific single chain Fv (scFv) antibody fragment with an engineered variant of Pseudomonas exotoxin A (ETA). The variant toxin carries the KDEL peptide at its C-terminus, a cellular peptide mediating improved retrograde transport to the endoplasmic reticulum. The purified recombinant fusion protein induced potent apoptosis of the human myeloid cell lines U937, HL-60 and THP-1. Up to 98% of U937 cells were eliminated after treatment for 72 h with a single dose of 500 ng/ml (c. 7 nmol/l). Killing was antigen-specific and occurred by apoptosis. A control protein, consisting of a CD19-specific scFv antibody fragment fused to the ETA-KDEL toxin, failed to induce death of the CD19-negative cell lines U937, HL-60 and THP-1. The CD33-ETA toxin also mediated apoptosis of fresh patient-derived acute myeloid leukaemia cells from bone marrow and peripheral blood. The pronounced antigen-restricted cytotoxicity of the novel fusion protein makes it a candidate for further evaluation of its therapeutic potential.

Direct Selection of Antibodies from Complex Libraries with the Protein Fragment Complementation Assay

The aim of the present study was to develop the protein fragment complementation assay (PCA) for the intracellular selection of specific binding molecules from the fully synthetic HuCAL antibody library. Here, we describe the first successful selections of specific antibodies by PCA, and we discuss the opportunities and limitations of this approach. First, we enriched an antibody specific for the capsid protein D of bacteriophage lambda (gpD) by ten successive rounds of competitive liquid culture selection. In an independent approach, we selected a specific antibody for the c-Jun N-terminal kinase 2 (JNK2) in a single-step selection setup. In order to obtain specific antibodies in only a single PCA selection round, the selection system was thoroughly investigated and several strategies to reduce the amount of false positives were evaluated. When expressed in the cytoplasm of Escherichia coli, the PCA-selected scFv antibody fragments could be purified as soluble and monomeric proteins. Denaturant-induced unfolding experiments showed that both antibody fragments are stable molecules, even when the disulfide bonds are reduced. Furthermore, antigen-specificity of the PCA-selected antibody fragments is demonstrated by in vivo and in vitro experiments. As antigen binding is retained regardless of the antibody redox state, both PCA-selected antibody fragments can tolerate the loss of disulfide bridge formation. Our results illustrate that it is possible to select well-expressed, stable, antigen-specific, and intracellular functional antibodies by PCA directly.

Cytolytic and Cytotoxic Activity of a Human Natural Killer Cell Line Genetically Modified to Specifically Recognize HER-2/neu Overexpressing Tumor Cells

NK92 cells genetically engineered to recognize the HER-2/neu oncoprotein have been previously reported to lyse HER-2/neu positive tumor cell lines through direct cell to cell contact. In the present study we have transduced NK92 cells with a chimeric receptor gene composed of the HER-/neu specific scFv (FRP5) antibody fragment, joined to the peptide CD8 hinge region and the signaling CD3 ζ chain. NK92 cells expressing this chimeric receptor (NK92.HER-2/neu/ζ) specifically recognized and lysed HER-2/neu overexpressing tumor cell lines both in vitro and in preclinical tumor models in vivo. More important we demonstrate that NK92.HER-2/neu/ζ cells constitutively secrete high levels of soluble scFv which mediate strong tumor cytostatic effects by directly binding on cell surface HER-2/neu. Our data uncover an additional mechanism through which NK92.HER-2/neu/ζ cells mediate antitumor effects and further support their use in cell based therapeutics for the treatment of HER-2/neu expressing cancers.

A multivalent recombinant antibody fragment specific for carcinoembryonic antigen

In the present paper we report the development of a bivalent scFv (single-chain Fv) antibody fragment, starting from a mouse mAb (monoclonal antibody) specific for CEA (carcinoembryonic antigen) that has received approval for in vivo radioimmunodiagnosis in humans. The diabody is well expressed in Escherichia coli, is easily purified by a combination of immobilized metal ion affinity chromatography and gel filtration and exhibits high affinity and specificity for CEA, comparable with those of the original mAb. Biodistribution experiments in athymic nude mice transplanted with human CEA+ cancer cells showed that the 125I-labelled diabody preferentially localizes in the tumour tissue and that retention is still high 48 h after injection. The diabody can be advantageous for some in vivo tumour targeting applications, due to the faster clearance derived from its smaller molecular mass.

Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae

In this study, secretory processing of cell-surface displayed Aga2p fusions to bovine pancreatic trypsin inhibitor (BPTI) and the single chain Fv (scFv) antibody fragment D1.3 are examined. BPTI is more efficiently processed than D1.3 both when secreted and surface-displayed, and D1.3 expression imparts a greater amount of secretory stress on the cell as assayed by a reporter of the unfolded protein response (UPR). Surprisingly, simultaneous expression of the two proteins in the same cell somewhat improves BPTI surface display while decreasing D1.3 surface display with minimal effect on UPR activation. Furthermore, co-expression leads to the accumulation of punctate vacuolar aggregates of D1.3 and increased secretion of the D1.3-Aga2p fusion into the supernatant. Overexpression of the folding chaperones protein disulfide isomerase (PDI) and BiP largely mitigates the D1.3 surface expression decrease, suggesting that changes in vacuolar and cell surface targeting may be due, in part, to folding inefficiency. Titration of constitutive UPR expression across a broad range progressively decreases surface display of both proteins as UPR increases. D1.3-Aga2p traffic through the late secretory pathway appears to be strongly affected by overall secretory load as well as folding conditions in the ER.