Structural Basis of Egg Coat-Sperm Recognition at Fertilization.

Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

Ubiquitin Phosphorylation at Thr12 Modulates the DNA Damage Response.

The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.

tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

Enzymes known as PylRS offer the remarkable ability to expand the natural genetic code of a living cell with unnatural amino acids. Currently, over 200 unnatural amino acids can be genetically encoded with the help of PylRS and its partner tRNAPyl, enabling us to endow proteins with novel properties, or regulate protein activity using light or inducible cross-linking. One intriguing feature of PylRS enzymes is their ability to avoid cross-reactivity when two PylRS homologs from different organisms-such as those from the archaea Methanosarcina mazei and Methanomethylophilus alvus-are co-expressed in a single cell. This makes it possible to simultaneously encode two unnatural amino acids in a single protein. This study illuminates the elusive mechanism of PylRS specificity by using cryo-electron microscopy, biochemistry and molecular simulations. The interaction of PylRS from M. alvus with its tRNAPyl is best described as two pieces of a jigsaw puzzle; in which PylRS recognizes the unique shape of its cognate tRNA instead of specific nucleotides in the tRNA sequence like other tRNA-binding enzymes. This finding may streamline the rational design of tools for simultaneous genetic incorporation of multiple unnatural amino acids, thereby facilitating the development of valuable proteins for research, medicine, and biotechnology.

An engineered 4-1BBL fusion protein with "activity on demand".

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display "activity on demand" properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

Inference of molecular structure for characterization and improvement of clinical grade immunocytokines.

The use of immunomodulatory agents for the treatment of cancer is gaining a growing biopharmaceutical interest. Antibody-cytokine fusion proteins, namely immunocytokines, represent a promising solution for the regulation of the immune system at the site of disease. The three-dimensional arrangement of these molecules can profoundly influence their biological activity and pharmacokinetic properties. Structural techniques might provide important insight in the 3D arrangement of immunocytokines. Here, we performed structure investigations on clinical grade fusion proteins L19-IL2, IL12-L19L19 and L19L19-IL2 to elucidate their quaternary organization. Crystallographic characterization of the common L19 antibody fragment at a resolution of 2.0-Å was combined with low-resolution studies of the full-length chimeric molecules using small-angle synchrotron X-ray scattering (SAXS) and negative stain electron microscopy. Characterization of the full-length quaternary structures of the immunocytokines in solution by SAXS consistently supported the diabody structure in the L19-IL2 immunocytokine and allowed generation of low-resolution models of the chimeric proteins L19L19-IL2 and IL12-L19L19. Comparison with 3D reconstructions obtained from negative-stain electron microscopy revealed marked flexibility associated to the linker regions connecting the cytokine and the antibody components of the chimeric proteins. Collectively, our results indicate that low-resolution molecular structure characterizations provide useful complementary insights for the quality control of immunocytokines, constituting a powerful tool to guide the design and the subsequent optimization steps towards clinical enhancement of these chimeric protein reagents.

An Attenuated Targeted-TNF Localizes to Tumors In Vivo and Regains Activity at the Site of Disease.

Antibody-cytokine fusion proteins (immunocytokines) are gaining importance for cancer therapy, but those products are often limited by systemic toxicity related to the activity of the cytokine payload in circulation and in secondary lymphoid organs. Tumor necrosis factor (TNF) is used as a pro-inflammatory payload to trigger haemorrhagic necrosis and boost anti-cancer immunity at the tumor site. Here we describe a depotentiated version of TNF (carrying the single point mutation I97A), which displayed reduced binding affinity to its cognate receptor tumor necrosis factor receptor 1 (TNFR-1) and lower biocidal activity. The fusion of the TNF(I97A) mutant to the L19 antibody promoted restoration of anti-tumor activity upon accumulation on the cognate antigen, the alternatively spliced EDB domain of fibronectin. In vivo administration of high doses (375 µg/Kg) of the fusion protein showed a potent anti-tumor effect without apparent toxicity compared with the wild type protein. L19-TNFI97A holds promise for the targeted delivery of TNF activity to neoplastic lesions, helping spare normal tissues.

Therapeutic Evaluation of Antibody-Based Targeted Delivery of Interleukin 9 in Experimental Pulmonary Hypertension.

BACKGROUND AND AIMS: Pulmonary hypertension (PH) is a heterogeneous disorder associated with poor prognosis. For the majority of patients, only limited therapeutic options are available. Thus, there is great interest to develop novel treatment strategies focusing on pulmonary vascular and right ventricular remodeling. Interleukin 9 (IL9) is a pleiotropic cytokine with pro- and anti-inflammatory functions. The aim of this study was to evaluate the therapeutic activity of F8IL9F8 consisting of IL9 fused to the F8 antibody, specific to the alternatively-spliced EDA domain of fibronectin, which is abundantly expressed in pulmonary vasculature and right ventricular myocardium in PH. METHODS: The efficacy of F8IL9F8 in attenuating PH progression in the monocrotaline mouse model was evaluated in comparison to an endothelin receptor antagonist (ERA) or an IL9 based immunocytokine with irrelevant antibody specificity (KSFIL9KSF). Treatment effects were assessed by right heart catheterization, echocardiography as well as histological and immunohistochemical tissue analyses. RESULTS: Compared to controls, systolic right ventricular pressure (RVPsys) was significantly elevated and a variety of right ventricular echocardiographic parameters were significantly impaired in all MCT-induced PH groups except for the F8IL9F8 group. Both, F8IL9F8 and ERA treatments lead to a significant reduction in RVPsys and an improvement of echocardiographic parameters when compared to the MCT group not observable for the KSFIL9KSF group. Only F8IL9F8 significantly reduced lung tissue damage and displayed a significant decrease of leukocyte and macrophage accumulation in the lungs and right ventricles. CONCLUSIONS: Our study provides first pre-clinical evidence for the use of F8IL9F8 as a new therapeutic agent for PH in terms of a disease-modifying concept addressing cardiovascular remodeling.

The antibody-based delivery of interleukin-12 to solid tumors boosts NK and CD8+ T cell activity and synergizes with immune checkpoint inhibitors.

We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 antibody in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively spliced EDB domain of fibronectin), retained the activity of the parental cytokine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent antitumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with checkpoint blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case, cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anticancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.

Impact of Ligand Size and Conjugation Chemistry on the Performance of Universal Chimeric Antigen Receptor T-Cells for Tumor Killing.

All Universal Chimeric Antigen Receptor T-cells (UniCAR T-cells) are T-cells which have been engineered to recognize a haptenated ligand. Due to this feature, UniCAR T-cells have the potential to mediate a potent and selective tumor killing only in the presence of a haptenated tumor ligand, thus avoiding the long-lasting biocidal effects of conventional CAR T-cells. We have used fluorescein-labeled versions of small organic ligands and different antibody formats specific to carbonic anhydrase IX (a tumor-associated antigen) in order to assess whether the killing potential of UniCAR T-cells depended on the molecular features of the haptenated molecule. Both small molecule ligands and larger antibody fragments were potent in mediating tumor cell killing over a broad concentration range. Antibodies could be conveniently used both in IgG format and as smaller diabody fragments. Importantly, the use of site-specific chemical modification strategies for the antibody coupling to fluorescein led to a substantial improvement of tumor cell killing performance, compared to the random modification of primary amino groups on the antibody surface.

The immunocytokine L19-TNF eradicates sarcomas in combination with chemotherapy agents or with immune check-point inhibitors.

Antibody-cytokine fusion proteins (also called 'immunocytokines') represent an emerging class of biopharmaceutical products, which are being considered for cancer immunotherapy. When used as single agents, pro-inflammatory immunocytokines are rarely capable of inducing complete and durable cancer regression in mouse models and in patients. However, the combination treatment with conventional chemotherapy or with other immune-stimulatory agents typically increases the therapeutic efficacy of immunocytokines. In this article, we describe combination treatments of a tumor-targeting antibody-cytokine fusion protein based on the L19 antibody (specific to a splice isoform of fibronectin) fused to murine tumor necrosis factor with standard chemotherapy (dacarbazine, trabectedin or melphalan) or with an immune check-point inhibitor (anti-PD-1) in a BALB/c derived immunocompetent murine model of sarcoma (WEHI-164). All combination treatments led to improved tumor remission compared to single-agent treatments, suggesting that these combination partners may be suitable for further clinical development in sarcoma patients.

Axons Embedded in a Tissue May Withstand Larger Deformations Than Isolated Axons Before Mechanoporation Occurs.

Diffuse axonal injury (DAI) is the pathological consequence of traumatic brain injury (TBI) that most of all requires a multiscale approach in order to be, first, understood and then possibly prevented. While in fact the mechanical insult usually happens at the head (or macro) level, the consequences affect structures at the cellular (or microlevel). The quest for axonal injury tolerances has so far been addressed both with experimental and computational approaches. On one hand, the experimental approach presents challenges connected to both temporal and spatial resolution in the identification of a clear axonal injury trigger after the application of a mechanical load. On the other hand, computational approaches usually consider axons as homogeneous entities and therefore are unable to make inferences about their viability, which is thought to depend on subcellular damages. Here, we propose a computational multiscale approach to investigate the onset of axonal injury in two typical experimental scenarios. We simulated single-cell and tissue stretch injury using a composite finite element axonal model in isolation and embedded in a matrix, respectively. Inferences on axonal damage are based on the comparison between axolemma strains and previously established mechanoporation thresholds. Our results show that, axons embedded in a tissue could withstand higher deformations than isolated axons before mechanoporation occurred and this is exacerbated by the increase in strain rate from 1/s to 10/s.

Sequence dependency of canonical base pair opening in the DNA double helix.

The flipping-out of a DNA base from the double helical structure is a key step of many cellular processes, such as DNA replication, modification and repair. Base pair opening is the first step of base flipping and the exact mechanism is still not well understood. We investigate sequence effects on base pair opening using extensive classical molecular dynamics simulations targeting the opening of 11 different canonical base pairs in two DNA sequences. Two popular biomolecular force fields are applied. To enhance sampling and calculate free energies, we bias the simulation along a simple distance coordinate using a newly developed adaptive sampling algorithm. The simulation is guided back and forth along the coordinate, allowing for multiple opening pathways. We compare the calculated free energies with those from an NMR study and check assumptions of the model used for interpreting the NMR data. Our results further show that the neighboring sequence is an important factor for the opening free energy, but also indicates that other sequence effects may play a role. All base pairs are observed to have a propensity for opening toward the major groove. The preferred opening base is cytosine for GC base pairs, while for AT there is sequence dependent competition between the two bases. For AT opening, we identify two non-canonical base pair interactions contributing to a local minimum in the free energy profile. For both AT and CG we observe long-lived interactions with water and with sodium ions at specific sites on the open base pair.

Modeling p K Shift in DNA Triplexes Containing Locked Nucleic Acids.

The protonation states for nucleic acid bases are difficult to assess experimentally. In the context of DNA triplex, the protonation state of cytidine in the third strand is particularly important, because it needs to be protonated in order to form Hoogsteen hydrogen bonds. A sugar modification, locked nucleic acid (LNA), is widely used in triplex forming oligonucleotides to target sites in the human genome. In this study, the parameters for LNA are developed in line with the CHARMM nucleic acid force field and validated toward the available structural experimental data. In conjunction, two computational methods were used to calculate the protonation state of the third strand cytidine in various DNA triplex environments: λ-dynamics and multiple pH regime. Both approaches predict p K of this cytidine shifted above physiological pH when cytidine is in the third strand in a triplex environment. Both methods show an upshift due to cytidine methylation, and a small downshift when the sugar configuration is locked. The predicted p K values for cytidine in DNA triplex environment can inform the design of better-binding oligonucleotides.

The free energy of locking a ring: Changing a deoxyribonucleoside to a locked nucleic acid.

Locked nucleic acid (LNA), a modified nucleoside which contains a bridging group across the ribose ring, improves the stability of DNA/RNA duplexes significantly, and therefore is of interest in biotechnology and gene therapy applications. In this study, we investigate the free energy change between LNA and DNA nucleosides. The transformation requires the breaking of the bridging group across the ribose ring, a problematic transformation in free energy calculations. To address this, we have developed a 3-step (easy to implement) and a 1-step protocol (more efficient, but more complicated to setup), for single and dual topologies in classical molecular dynamics simulations, using the Bennett Acceptance Ratio method to calculate the free energy. We validate the approach on the solvation free energy difference for the nucleosides thymidine, cytosine, and 5-methyl-cytosine. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Triple helical DNA in a duplex context and base pair opening.

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson-Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson-Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region.

Loop-loop interaction in an adenine-sensing riboswitch: a molecular dynamics study.

Riboswitches are mRNA-based molecules capable of controlling the expression of genes. They undergo conformational changes upon ligand binding, and as a result, they inhibit or promote the expression of the associated gene. The close connection between structural rearrangement and function makes a detailed knowledge of the molecular interactions an important step to understand the riboswitch mechanism and efficiency. We have performed all-atom molecular dynamics simulations of the adenine-sensing add A-riboswitch to study the breaking of the kissing loop, one key tertiary element in the aptamer structure. We investigated the aptamer domain of the add A-riboswitch in complex with its cognate ligand and in the absence of the ligand. The opening of the hairpins was simulated using umbrella sampling using the distance between two loops as the reaction coordinate. A two-step process was observed in all the simulated systems. First, a general loss of stacking and hydrogen bond interactions is seen. The last interactions that break are the two base pairs G37-C61 and G38-C60, but the break does not affect the energy profile, indicating their pivotal role in the tertiary structure formation but not in the structure stabilization. The junction area is partially organized before the kissing loop formation and residue A24 anchors together the loop helices. Moreover, when the distance between the loops is increased, one of the hairpins showed more flexibility by changing its orientation in the structure, while the other conserved its coaxial arrangement with the rest of the structure.

Generation and in vivo characterization of a novel high-affinity human antibody targeting carcinoembryonic antigen.

There are no effective treatment options for most patients with metastatic colorectal cancer (mCRC). mCRC remains a leading cause of tumor-related death, with a five-year survival rate of only 15%, highlighting the urgent need for novel pharmacological products. Current standard drugs are based on cytotoxic chemotherapy, VEGF inhibitors, EGFR antibodies, and multikinase inhibitors. The antibody-based delivery of pro-inflammatory cytokines provides a promising and differentiated strategy to improve the treatment outcome for mCRC patients. Here, we describe the generation of a novel fully human monoclonal antibody (termed F4) targeting the carcinoembryonic antigen (CEA), a tumor-associated antigen overexpressed in colorectal cancer and other malignancies. The F4 antibody was selected by antibody phage display technology after two rounds of affinity maturation. F4 in single-chain variable fragment format bound to CEA in surface plasmon resonance with an affinity of 7.7 nM. Flow cytometry and immunofluorescence on human cancer specimens confirmed binding to CEA-expressing cells. F4 selectively accumulated in CEA-positive tumors, as evidenced by two orthogonal in vivo biodistribution studies. Encouraged by these results, we genetically fused murine interleukin (IL) 12 to F4 in the single-chain diabody format. F4-IL12 exhibited potent antitumor activity in two murine models of colon cancer. Treatment with F4-IL12 led to an increased density of tumor-infiltrating lymphocytes and an upregulation of interferon γ expression by tumor-homing lymphocytes. These data suggest that the F4 antibody is an attractive delivery vehicle for targeted cancer therapy.

Generation of a novel fully human non-superagonistic anti-CD28 antibody with efficient and safe T-cell co-stimulation properties.

Antibody-based therapeutics represent an important class of biopharmaceuticals in cancer immunotherapy. CD3 bispecific T-cell engagers activate cytotoxic T-cells and have shown remarkable clinical outcomes against several hematological malignancies. The absence of a costimulatory signal through CD28 typically leads to insufficient T-cell activation and early exhaustion. The combination of CD3 and CD28 targeting products offers an attractive strategy to boost T-cell activity. However, the development of CD28-targeting therapies ceased after TeGenero's Phase 1 trial in 2006 evaluating a superagonistic anti-CD28 antibody (TGN1412) resulted in severe life-threatening side effects. Here, we describe the generation of a novel fully human anti-CD28 antibody termed "E1P2" using phage display technology. E1P2 bound to human and mouse CD28 as shown by flow cytometry on primary human and mouse T-cells. Epitope mapping revealed a conformational binding epitope for E1P2 close to the apex of CD28, similar to its natural ligand and unlike the lateral epitope of TGN1412. E1P2, in contrast to TGN1412, showed no signs of in vitro superagonistic properties on human peripheral blood mononuclear cells (PBMCs) using different healthy donors. Importantly, an in vivo safety study in humanized NSG mice using E1P2, in direct comparison and contrast to TGN1412, did not cause cytokine release syndrome. In an in vitro activity assay using human PBMCs, the combination of E1P2 with CD3 bispecific antibodies enhanced tumor cell killing and T-cell proliferation. Collectively, these data demonstrate the therapeutic potential of E1P2 to improve the activity of T-cell receptor/CD3 activating constructs in targeted immunotherapeutic approaches against cancer or infectious diseases.

Selection of a PD-1 blocking antibody from a novel fully human phage display library.

Programmed cell death protein 1 (PD-1) is an immunoregulatory target which is recognized by different monoclonal antibodies, approved for the therapy of multiple types of cancer. Different anti-PD-1 antibodies display different therapeutic properties and there is a pharmaceutical interest to generate and characterize novel anti-PD-1 antibodies. We screened multiple human antibody phage display libraries to target novel epitopes on the PD-1 surface and we discovered a unique and previously undescribed binding specificity (termed D12) from a new antibody library (termed AMG). The library featured antibody fragments in single-chain fragment variable (scFv) format, based on the IGHV3-23*03 (VH ) and IGKV1-39*01 (Vκ) genes. The D12 antibody was characterized by surface plasmon resonance (SPR), cross-reacted with the Cynomolgus monkey antigen and bound to primary human T cells, as shown by flow cytometry. The antibody blocked the PD-1/PD-L1 interaction in vitro with an EC50 value which was comparable to the one of nivolumab, a clinically approved antibody. The fine details of the interaction between D12 and PD-1 were elucidated by x-ray crystallography of the complex at a 3.5 Å resolution, revealing an unprecedented conformational change at the N-terminus of PD-1 following D12 binding, as well as partial overlap with the binding site for the cognate PD-L1 and PD-L2 ligands which prevents their binding. The results of the study suggest that the expansion of antibody library repertoires may facilitate the discovery of novel binding specificities with unique properties that hold promises for the modulation of PD-1 activity in vitro and in vivo.

Complete eradication of human B-cell lymphoma xenografts using rituximab in combination with the immunocytokine L19-IL2.

The antibody-mediated delivery of therapeutic agents to sites of angiogenesis is an attractive strategy for anticancer therapy, but is largely unexplored in hematologic malignancies. In the present study, we show that the extra domain B (EDB) of fibronectin, a marker of angiogenesis, is expressed in B-cell non-Hodgkin lymphoma (NHL) and that the human monoclonal anti-EDB antibody L19 can selectively localize to the lymphoma-associated subendothelial extracellular matrix. In vivo, the preferential accumulation of the antibody at the tumor site was confirmed by quantitative biodistribution analyses with radioiodinated antibody preparations. The fusion protein L19-IL2, which mediates the delivery of interleukin-2 (IL-2) to the neovasculature, displayed a superior antilymphoma activity compared with unconjugated IL-2 in localized and systemic xenograft models of NHL. When coadministered with rituximab, L19-IL2 induced complete remissions of established localized lymphomas and provided long-lasting protection from disseminated lymphoma. The combined use of rituximab and L19-IL2, which dramatically increases the infiltration of immune effector cells in lymphomas, may deserve clinical investigations, facilitated by the fact that L19-IL2 is currently being studied in phase II clinical trials in patients with solid tumors.