T-cell Activating Tribodies as a Novel Approach for Efficient Killing of ErbB2-positive Cancer Cells.

The Tyrosine Kinase Receptor ErbB2 (HER2) when overexpressed in breast cancer (BC) is associated with poor prognosis. The monoclonal antibody Trastuzumab has become a standard treatment of ErbB2+BC. The antibody treatment has limited efficacy, often meets resistance and induces cardiotoxicity. T-cell recruiting bispecific antibody derivatives (TRBA) offer a more effective alternative to standard antibody therapy. We evaluated a panel of TRBAs targeting 3 different epitopes on the ErbB2 receptor either in a bivalent targeting tribody structure or as a monovalent scFv-fusion (BiTE format) for binding, cytotoxicity on Trastuzumab-resistant cell lines, and induction of cardiotoxicity. All three TRBAs bind with high affinity to the ErbB2 extracellular domain and a large panel of ErbB2-positive tumor cells. Tribodies had an increased in vitro cytotoxic potency as compared to BiTEs. It is interesting to note that, Tribodies targeting the epitopes on ErbB2 receptor domains I and II bind and activate lysis of mammary and gastric tumor cells more efficiently than a Tribody targeting the Trastuzumab epitope on domain IV. The first 2 are also active on Trastuzumab-resistant cancer cells lacking or masking the epitope recognized by Trastuzumab. None of the Tribodies studied showed significant toxicity on human cardiomyocytes. Altogether these results make these novel anti-ErbB2 bispecific Tribodies candidates for therapeutic development for treating ErbB2-positive Trastuzumab-resistant cancer patients.

Superior Suppression of ErbB2-positive Tumor Cells by a Novel Human Triparatopic Tribody.

Downregulation of the epidermal growth factor receptor family of receptors is improved by combining different antibodies to noncompetitive epitopes. For ErbB2/HER2 this has already been translated into clinical practice by using a combination of trastuzumab and pertuzumab. Moreover, cocktails of 2 or 3 anti-epidermal growth factor receptor antibodies show an enhanced downregulation of the receptor due to the induction of matrix formation. A more efficient method for inducing matrix formation and receptor downregulation might include the use of trispecific reagents. A triparatopic Tribody consisting of 3 noncompeting ErbB2 binders was compared with equivalent trivalent monoparatopic counterparts, as well as to a cocktail of 3 monoclonal antibodies for its effects on downregulation of the ErbB2 receptor's kinase activity and survival of several ErbB2-expressing cancer cell lines. The triparatopic Tribody was significantly more efficient in downregulating ErbB2 and inhibiting tumor cell growth than either the control monoparatope tribodies or the combinatorial treatment with the 3 different parental antibodies on all the tested tumor cells, including trastuzumab-resistant cell lines. The enhancement of effectivity was dependent on all 3 binding moieties. Because the novel Tribody allows reduction of the costs of production (as only 1 construct provides the antitumor effects of 3 antibodies) and has an intermediate molecular size (∼100 kDa) well suited for both tumor penetration and acceptable half-life, it has the potential to become a precious tool for therapeutic use particularly in trastuzumab-resistant cancer patients.

PH1-derived bivalent bibodies and trivalent tribodies bind differentially to shed and tumour cell-associated MUC1.

Most adenocarcinomas express altered MUC1 as a tumour-associated antigen. Due to suboptimal glycosylation in tumour-associated MUC1, the apomucin core is exposed, revealing new epitopes for antibody-directed immunotherapy. The human PH1 Fab binds specifically to this MUC1 apomucin. We describe the engineering and functional characterization of bi- and trivalent recombinant antibody derivatives from the PH1 Fab. Bi- and tribodies were made using the disulfide-stabilized Fab fragment as a heterodimerization scaffold with PH1 single-chain variable fragments fused to either one or both Fab-chain C-termini. Immunoassays revealed 27- and 165-fold improved dissociation constants (K(D) = 30 and 5 nM) of the PH1 bi- and tribodies compared with the parental Fab (K(D) = 820 nM). Unexpectedly, major differences were seen in the ability of the antibody constructs to bind shed and tumour cell-tethered MUC1. While the tribody did not discriminate between both MUC1 forms, the bibody demonstrated preferential interaction with membrane-bound MUC1 compared with shed MUC1. This preferential recognition of membrane-bound MUC1, along with the high serum stability of the bibody, its intermediate size and efficient internalization by MUC1(+) cells, makes the human PH1-derived bibody a valuable candidate as a cancer-targeting therapeutic.

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris.

BACKGROUND: Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs. RESULTS: Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli. CONCLUSION: Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.

Monoclonal antibody 14C5 targets integrin alphavbeta5.

This study identifies and characterizes the antigen recognized by monoclonal antibody (mAb) 14C5. We compared the expression of antigen 14C5 with the expression of eight integrin subunits (alpha1, alpha2, alpha3, alphav, beta1, beta2, beta3, and beta4) and three integrin heterodimers (alphavbeta3, alphavbeta5, and alpha5beta1) by flow cytometry. Antigen 14C5 showed a similar expression to alphavbeta5 in eight different epithelial cancer cell lines (A549, A2058, C32, Capan-2, Colo16, HT-1080, HT-29, and SKBR-3). Specific binding of P1F6, an anti-alphavbeta5 specific antibody, was blocked by mAb 14C5. After transient expression of alphavbeta5 in 14C5-negative Colo16 cells, mAb 14C5 was able to bind a subpopulation of alphavbeta5-positive cells. We evaluated the tissue distribution of the 14C5 antigen in colon (n = 20) and lung (n = 16) cancer tissues. The colon carcinoma cells stained positive for 14C5 in 50% of tumors analyzed, whereas bronchoalveolar lung carcinoma and typical carcinoid were not positive for the antigen. More common types of non-small cell lung cancer, i.e., squamous (n = 5) and adenocarcinoma (n = 3), stained positive in 2 of 5 squamous carcinomas and in 1 of 3 investigated adenocarcinoma. Colon (95%) and lung (50%) carcinoma tissues showed extensive expression of antigen 14C5 in the stroma surrounding the tumor cells and on the membrane of the adjacent fibroblasts. We show for the first time that mAb 14C5 binds the vascular integrin alphavbeta5, suggesting that mAb 14C5 can be used as a screening agent to select colon and lung cancer patients that are eligible for anti-alphavbeta5-based therapies.

Biodistribution and planar gamma camera imaging of (123)I- and (131)I-labeled F(ab')(2) and Fab fragments of monoclonal antibody 14C5 in nude mice bearing an A549 lung tumor.

UNLABELLED: Detection of antigen 14C5, involved in substrate adhesion and highly expressed on the membrane of many carcinomas, including lung cancer, provides important diagnostic information that can influence patient management. The aim of this study was to evaluate the biodistribution and planar gamma camera imaging characteristics of radioiodinated F(ab')(2) and Fab fragments of monoclonal antibody (mAb) 14C5 in tumor-bearing mice. METHODS: F(ab')(2) and Fab 14C5 fragments were radioiodinated using the Iodo-Gen method. In vitro stability, binding specificity and affinity of (125)I-labeled 14C5 fragments were studied in A549 lung carcinoma cells. Biodistribution, blood clearance and tumor-targeting characteristics of (131)I-labeled 14C5 fragments and intact mAb 14C5 were studied in Swiss nu/nu mice bearing A549 lung carcinoma tumors. Planar gamma imaging illustrated the potential use of these (123)I-labeled 14C5 fragments for radioimmunodetection (RID). RESULTS: Saturation binding experiments showed highest affinity for (125)I-labeled F(ab')(2) fragments (K(d)=0.37+/-0.10 nmol/L) and lowest affinity for (125)I-labeled Fab fragments (K(d)=2.25+/-0.44 nmol/L). Blood clearance studies showed that the alpha half-life (t(1/2)alpha) value for Fab, F(ab')(2) and mAb 14C5 was 14.9, 21 and 118 min, respectively. The beta half-life t(1/2)beta value for Fab, F(ab')(2) and mAb 14C5 was 439, 627 and 4067 min, respectively. (131)I-Fab fragments showed highest tumor uptake 3 h after injection (2.4+/-0.8 %ID/g), (131)I-labeled F(ab')(2) showed highest tumor uptake 6 h after injection (4.7+/-0.7 %ID/g) and for (131)I-labeled mAb highest tumor uptake was observed at 24 h (10.7+/-2.3 %ID/g). In planar gamma imaging, both labeled fragments gave better tumor-to-background contrast than (123)I-mAb 14C5. CONCLUSION: Fab and F(ab')(2) fragments derived from intact mAb 14C5 have significant potential for diagnostic and therapeutic applications and may provide new tools in mAb-based radiopharmaceuticals for targeting non-small cell lung cancer.

Angiogenesis enhances factor IX delivery and persistence from retrievable human bioengineered muscle implants.

Human muscle progenitor cells transduced with lentiviral vectors secreted high levels of blood clotting factor IX (FIX). When bioengineered into postmitotic myofibers as human bioartificial muscles (HBAMs) and subcutaneously implanted into immunodeficient mice, they secreted FIX into the circulation for >3 months. The HBAM-derived FIX was biologically active, consistent with the cells' ability to conduct the necessary posttranslational modifications. These bioengineered muscle implants are retrievable, an inherent safety feature that distinguishes this "reversible" gene therapy approach from most other gene therapy strategies. When myofibers were bioengineered from human myoblasts expressing FIX and vascular endothelial growth factor, circulating FIX levels were increased and maintained long term within the therapeutic range, consistent with the generation of a vascular network around the HBAM. The present study implicates an important role for angiogenesis in the efficient delivery of therapeutic proteins using tissue engineered stem cell-based gene therapies.

In vitro and in vivo targeting properties of iodine-123- or iodine-131-labeled monoclonal antibody 14C5 in a non-small cell lung cancer and colon carcinoma model.

PURPOSE: The monoclonal antibody (mAb) 14C5 is a murine IgG1 directed against a yet undefined molecule involved in cell substrate adhesion found on the surface of malignant breast cancer tissue. mAb 14C5 is able to inhibit cell substrate adhesion and invasion of breast cancer cells in vitro. In normal tissues as well as in the stroma surrounding in situ carcinomas of the breast, no expression of the antigen 14C5 occurs. The aim of this study was to investigate the in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 as a novel agent for radioimmunodetection and radioimmunotherapy. EXPERIMENTAL DESIGN: Internalization of mAb 14C5 was investigated with 125I-labeled mAb 14C5 and by confocal laser scanning microscopy. Biodistribution studies of 131I-labeled mAb 14C5 and planar gamma imaging were done in nude mice bearing an A549 (non-small cell lung carcinoma) or a LoVo (colon carcinoma) tumor. RESULTS: Internalization studies with both A549 and LoVo cells showed that 125I-labeled mAb 14C5 is slowly internalized with approximately 30% of the initially bound mAb 14C5 internalized after 2 hours at 37 degrees C. Internalization of mAb 14C5 could be visualized with confocal laser scanning microscopy. In vivo, radioisotope uptake peaked at 24 hours for both tumor models (n = 5) with no significant difference in percentage of injected dose/g tissue (A549 10.4 +/- 0.8 and LoVo 9.3 +/- 0.8). Via planar gamma camera imaging, A549 lung tumors as well as LoVo colon tumors could be clearly visualized. CONCLUSIONS: The in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 are promising and could provide a new antibody-based agent for radioimmunodetection and radioimmunotherapy of patients bearing antigen 14C5-expressing tumors.

CD3 x CD28 cross-interacting bispecific antibodies improve tumor cell dependent T-cell activation.

Bispecific antibodies (Bs-Abs) containing an anti-CD3 and an anti-TAA specificity can recruit T cells to the tumor for cancer immunotherapy. To be effective, efficient activation at the tumor site is a prerequisite. This can be achieved by triggering both the T-cell receptor and the co-stimulatory molecule CD28. We engineered two recombinant cross-interacting Bs-Abs (CriBs-Abs) by incorporating a peptide tag and its cognate single-chain variable fragment (scFv), respectively, into a pair of (tumor x CD3) and (tumor x CD28) binding Bs-Abs. A 30-fold lower concentration of the activating CriBs-Ab as compared to non interacting Bs-Ab was sufficient for strong T-cell activation in the presence of tumor cells. One thousand-fold higher concentrations of both CriBs-Abs were required for marginal T-cell activation (70-fold below maximal response) in the absence of tumor cells. An optimized stoichiometry (1 : 1000) of activating versus co-stimulating CriBs-Ab thus allowed low doses of activating CriBs-Ab to induce tumor-cell dependent T-cell activation when used in combination with high concentrations of the pre-targeted co-stimulating CriBs-Ab in vitro. This indicates a large window of operation in which only tumor cell dependent T-cell activation is induced and systemic tumor cell independent T-cell activation is avoided, while ensuring optimal activation with a low concentration of the activating CriBs-Ab, which has the highest potential to induce toxic effects in vivo.

Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS.

Neurotrophin treatment has so far failed to prolong the survival of individuals affected with amyotrophic lateral sclerosis (ALS), an incurable motoneuron degenerative disorder. Here we show that intracerebroventricular (i.c.v.) delivery of recombinant vascular endothelial growth factor (Vegf) in a SOD1(G93A) rat model of ALS delays onset of paralysis by 17 d, improves motor performance and prolongs survival by 22 d, representing the largest effects in animal models of ALS achieved by protein delivery. By protecting cervical motoneurons, i.c.v. delivery of Vegf is particularly effective in rats with the most severe form of ALS with forelimb onset. Vegf has direct neuroprotective effects on motoneurons in vivo, because neuronal expression of a transgene expressing the Vegf receptor prolongs the survival of SOD1(G93A) mice. On i.c.v. delivery, Vegf is anterogradely transported and preserves neuromuscular junctions in SOD1(G93A) rats. Our findings in preclinical rodent models of ALS may have implications for treatment of neurodegenerative disease in general.

Radiolabeled annexin-V for monitoring treatment response in oncology.

Because of its potential to allow for noninvasive, repetitive, and selective in vivo identification of the site and extent of apoptotic cell death and for monitoring cell death kinetics without the need for invasive biopsy, radiolabeled annexin-V is of major clinical relevance. This paper reviews available preclinical and clinical data on radiolabeled annexin-V pertaining to the domain of monitoring response to radiotherapy and chemotherapy, focusing especially on advantages and drawbacks of the different labeling procedures for the radiolabeling of annexin-V.

New strategies in polypeptide and antibody synthesis: an overview.

The synthesis of radioligands can benefit considerably from optimized recombinant protein production, both on the aspect of economy of production and on the level of improving the targeting and pharmacokinetics of the ligand. This paper first describes a general production optimization strategy, and then elaborates on a protein design strategy tailored to targeting applications. Production in Escherichia coli will benefit from economy of goods and time as compared to other organisms. In order to increase the chance of finding a successful production system in this host, we have assembled a large number of expression strategies in a single, uniform expression system (FastScreen). The system allows rapid optimization of direct production of native proteins or via a fusion protein strategy with subsequent recovery of the desired protein. As an example of recombinant radioligand synthesis for improved targeting and clearing, a manifold of intermediate molecular size was synthesized by fusing one Fab and two single-chain variable fragments (scFv) antibody binding fragments into a trifunctional molecule (Tribody). Due to the use of the specific heterodimerization of the Fab chains, trispecific, bispecific, or trivalent antibody derived targeting reagents can easily be obtained. Recombinant production techniques also allow for specific incorporation of amino acids favoring a site specific labeling (labeling tags).

Optimizing expression and purification from cell culture medium of trispecific recombinant antibody derivatives.

Antibody fragments offer the possibility to build multifunctional manifolds tailored to meet a large variety of needs. We optimized the production of a manifold consisting of one (bibody) or two (tribody) single-chain variable fragments coupled to the C-terminus of Fab chains. Different strong mammalian promoters were compared and the influence of expression media on production and recovery was investigated. Since the physical and chemical nature of these molecules largely depends on the nature of the antibody building blocks incorporated, a generally applicable process for the purification of recombinant antibody derivatives from serum containing mammalian cell culture medium was designed. To this end we compared protein L, hydroxyapatite, immobilized metal affinity chromatography, cation-exchange chromatography and hydrophobic charge induction chromatography.