Ulla Ravn

The Binding Properties and Biological Activities of Bcl-2 and Bax in Cells Exposed to Apoptotic Stimuli

The oncogene product Bcl-2 protects cells from apoptosis whereas its homolog Bax functions to kill cells. Several binding partners of Bcl-2 and Bax have been isolated, but none of them has yet provided clues as to exactly how Bcl-2 and Bax work. According to one view, Bcl-2 and Bax interact with survival and death effector molecules, respectively, and neutralize each other through heterodimerization. Alternatively, Bcl-2 requires Bax for death protection, and additional proteins bind to the heterodimer to regulate its activity. Here we used a co-immunoprecipitation strategy to distinguish between these two possibilities. We show that the Bcl-2-Bax heterodimer is maintained, and no other protein associates stably in detectable amounts with Bcl-2, Bax, or the heterodimer in anti-Bcl-2 and anti-Bax immunoprecipitates from normal cells and cells exposed to apoptotic stimuli. Analysis of cells expressing various levels of Bcl-2 and Bax, however, revealed that the degree of protection against apoptosis does not correlate with the number of Bcl-2-Bax heterodimers but the amount of Bcl-2 that is free of Bax. In addition, the survival activity of Bcl-2 is unaffected when Bax expression is ablated by an antisense strategy. Our findings suggest that the Bcl-2-Bax heterodimer is a negative regulator of death protection, and that Bcl-2 requires neither Bax nor major, stable interactions with other cellular proteins to exert its survival function. We therefore propose that Bcl-2 acts as an enzyme (capturing substrates in a transient way), as a homodi- or multimer, or through the interaction with non-proteaceous targets (lipids, ions).

Deep sequencing of phage display libraries to support antibody discovery.

The use of next generation sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.

Specificity tuning of antibody fragments to neutralize two human chemokines with a single agent

Chemokines are important mediators of the immune response that are responsible for the trafficking of immune cells between lymphoid organs and migration towards sites of inflammation. Using phage display selection and a functional screening approach, we have isolated a panel of single-chain fragment variable (scFv) capable of neutralizing the activity of the human chemokine CXCL10 (hCXCL10). One of the isolated scFv was weakly cross-reactive against another human chemokine CXCL9, but was unable to block its biological activity. We diversified the complementarity determining region 3 (CDR3) of the light chain variable domain (VL) of this scFv and combined phage display with high throughput antibody array screening to identify variants capable of neutralizing both chemokines. Using this approach it is therefore possible to engineer pan-specific antibodies that could prove very useful to antagonize redundant signaling pathways such as the chemokine signaling network.

Transferring the Characteristics of Naturally Occurring and Biased Antibody Repertoires to Human Antibody Libraries by Trapping CDRH3 Sequences

Antibody repertoires are characterized by diversity as they vary not only amongst individuals and post antigen exposure but also differ significantly between vertebrate species. Such plasticity can be exploited to generate human antibody libraries featuring hallmarks of these diverse repertoires. In this study, the focus was to capture CDRH3 sequences, as this region generally accounts for most of the interaction energy with antigen. Sequences from human as well as non-human sources were successfully integrated into human antibody libraries. Next generation sequencing of these libraries proved that the CDRH3 lengths and amino acid composition corresponded to the species of origin. Specific CDRH3 sequences, biased towards the recognition of a model antigen either by immunizing mice or by selecting with phage display, were then integrated into another set of libraries. From these antigen biased libraries, highly potent antibodies were more frequently isolated, indicating that the characteristics of an immune repertoire is transferrable via CDRH3 sequences into a human antibody library. Taken together, these data demonstrate that the properties of naturally or experimentally biased repertoires can be effectively harnessed for the generation of targeted human antibody libraries, substantially increasing the probability of isolating antibodies suitable for therapeutic and diagnostic applications.

Rapid, simple and high yield production of recombinant proteins in mammalian cells using a versatile episomal system

Many research projects in life sciences require purified biologically active recombinant protein. In addition, different formats of a given protein may be needed at different steps of experimental studies. Thus, the number of protein variants to be expressed and purified in short periods of time can expand very quickly. We have therefore developed a rapid and flexible expression system based on described episomal vector replication to generate semi-stable cell pools that secrete recombinant proteins. We cultured these pools in serum-containing medium to avoid time-consuming adaptation of cells to serum-free conditions, maintain cell viability and reuse the cultures for multiple rounds of protein production. As such, an efficient single step affinity process to purify recombinant proteins from serum-containing medium was optimized. Furthermore, a series of multi-cistronic vectors were designed to enable simultaneous expression of proteins and their biotinylation in vivo as well as fast selection of protein-expressing cell pools. Combining these improved procedures and innovative steps, exemplified with seven cytokines and cytokine receptors, we were able to produce biologically active recombinant endotoxin free protein at the milligram scale in 4-6weeks from molecular cloning to protein purification.

Dual Specificity of Anti-CXCL10-CXCL9 Antibodies Is Governed by Structural Mimicry

Background: Dual-specific antibodies can bind to several antigens via different mechanisms. Results: Mutagenesis of CXCL9/CXCL10 from different species identifies serine 13 as a key residue targeted by anti-human CXCL9/CXCL10 dual-specific scFvs. Conclusion: Structural mimicry between human CXCL9 and CXCL10 governs dual-specific scFv binding. Significance: Dissecting the binding mechanism of dual-specific antibodies is important for the development of this class of antibodies. Dual-specific antibodies are characterized by an antigen-combining site mediating specific interactions with two different antigens. We have generated five dual-specific single chain variable fragments (scFv) that neutralize the activity of the two chemokines, CXCL9 and CXCL10, to bind to their receptor CXCR3. To better understand how these dual-specific scFvs bind these two chemokines that only share a 37% sequence identity, we mapped their epitopes on human CXCL9 and CXCL10 and identified serine 13 (Ser13) as a critical residue. It is conserved between the two chemokines but not in the third ligand for CXCR3, CXCL11. Furthermore, Ser13 is exposed in the tetrameric structure of CXCL10, which is consistent with our finding that the scFvs are able to bind to CXCL9 and CXCL10 immobilized on glycosaminoglycans. Overall, the data indicate that these dual-specific scFvs bind to a conserved surface involved in CXCR3 receptor interaction for CXCL10 and CXCL9. Thus, structural mimicry between the two targets is likely to be responsible for the observed dual specificity of these antibody fragments.

Robust Antibody-Antigen Complexes Prediction Generated by Combining Sequence Analyses, Mutagenesis, In Vitro Evolution, X-ray Crystallography and In Silico Docking.

Hu 15C1 is a potent anti-human Toll-like receptor 4 (TLR4) neutralizing antibody. To better understand the molecular basis of its biological activity, we used a multidisciplinary approach to generate an accurate model of the Hu 15C1-TLR4 complex. By combining site-directed mutagenesis, in vitro antibody evolution, affinity measurements and X-ray crystallography of Fab fragments, we identified key interactions across the Hu 15C1-TLR4 interface. These contact points were used as restraints to predict the structure of the Fab region of Hu 15C1 bound to TLR4 using computational molecular docking. This model was further evaluated and validated by additional site-directed mutagenesis studies. The predicted structure of the Hu 15C1-TLR4 complex indicates that the antibody antagonizes the receptor dimerization necessary for its activation. This study exemplifies how iterative cycles of antibody engineering can facilitate the discovery of components of antibody-target interactions.

Abstract B54: Antagonizing CD47-SIRP alpha interaction with a bispecific antibody: A novel cancer immunotherapy approach

CD47 is a ubiquitous cell surface glycoprotein that serves as a negative regulator of numerous innate immune functions such as phagocytosis, neutrophil inflammatory responses or dendritic cell maturation and activation. In particular, the interaction of CD47 with Signal Regulatory Protein Alpha (SIRP alpha) on myeloid cells is a mechanism of self-recognition, a ‘don9t eat me’ signal used by healthy cells to impede their elimination by phagocytes. Cancer cells often up-regulate CD47 expression, which helps them to evade immune surveillance and killing. What is more, increased CD47 levels are generally predictive of poor clinical outcome. Inhibiting CD47-SIRP alpha interaction represents therefore an attractive, generally applicable therapeutic strategy. Yet, achieving that goal with an anti-CD47 monoclonal antibody (Mab) may be challenging in practice, given the ubiquitous expression of the target. The “drug sink” represented by erythrocytes, platelets and other CD47-expressing cells may lead to a rapid elimination of an anti-CD47 Mab through target-mediated drug disposition; another likely consequence of ubiquitous CD47 expression are on-target toxicities like, e.g., anemia. To overcome these limitations, we have developed dual-targeting bispecific antibodies to CD47 and different Tumor-Associated Antigens (TAAs). Dual targeting CD47/TAA bispecific antibodies bind preferentially to TAA-expressing cancer cells and enable selective CD47 neutralization at the same time sparing healthy TAA-negative cells. Various TAAs are currently being pursued at Novimmune (e.g., CD19, mesothelin, or glypican 3). Such dual targeting CD47/TAA bispecific antibodies have the kappa/lambda body format i.e., they are fully human bispecific IgGs composed of a high-affinity anti-TAA arm, a CD47-neutralizing arm, and an unmodified IgG1 Fc. So far, only CD47/CD19 kappa/lambda bodies have been thoroughly characterized in vitro and in vivo. Various human B cell leukemia and lymphoma lines were used to demonstrate selective, CD19-dependent binding of these CD47/CD19 kappa/lambda bodies. As anticipated, the neutralization of the CD47-SIRP alpha interaction was also CD19-dependent, and led to the enhancement of Fc-mediated cancer cell killing through ADCP (antibody mediated cellular phagocytosis) or ADCC (antibody mediated cellular cytotoxicity). Efficient killing of CD19-positive cancer cells was also observed in vivo using a localized tumor xenograft model with NOD/SCID mice implanted with Raji Burkitt lymphoma cells. A dose dependent inhibition of tumor growth or tumor regression was observed, depending on the potency of the CD47-neutralizing arm used to generate the kappa/lambda body. In parallel, a single dose PK study in cynomolgus demonstrated favorable elimination kinetics, comparable to human IgG Mab. Moreover, no effects on hematological parameters were apparent at either of the doses tested (0.5 and 10 mg/kg). The example of CD47/CD19 kappa/lambda bodies illustrates the power of the dual-targeting approach: A favorable safety profile and pharmacokinetics could be achieved in parallel to potent inhibition of the CD47-SIRP alpha interaction and effective cancer cell killing. Antagonizing CD47-SIRP alpha interaction with bispecific antibodies is therefore an attractive immune-potentiating strategy. Because of its distinct mechanism of action and the ability to that harnesses the innate immunity to fight cancer, CD47/TAA bispecific antibodies would be good candidates for combination therapies with immune checkpoint inhibitors or other immunotherapeutics. Citation Format: Krzysztof Masternak, Zoe Johnson, Vanessa Buatois, Francois Rousseau, Giovanni Magistrelli, Valery Moine, Ulla Ravn, Franck Gueneau, Lucile Broyer, Susana Salgado Pires, Maureen Deehan, Nicolas Fischer, Walter Ferlin, Marie Kosco-Vilbois. Antagonizing CD47-SIRP alpha interaction with a bispecific antibody: A novel cancer immunotherapy approach. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B54.

Dual display: phage selection driven by co-engagement of two targets by two different antibody fragments

Antibody phage display technology has supported the emergence of numerous therapeutic antibodies. The development of bispecific antibodies, a promising new frontier in antibody therapy, could be facilitated by new phage display approaches that enable pairs of antibodies to be co-selected based on co-engagement of their respective targets. We describe such an approach, making use of two complementary leucine zipper domains that heterodimerize with high affinity. Phagemids encoding a first antibody fragment (scFv) fused to phage coat protein via the first leucine zipper are rescued in bacteria expressing a second scFv fused to the second leucine zipper as a soluble periplasmic protein, so that it is acquired by phage during assembly. Using a soluble scFv specific for a human CD3-derived peptide, we show that its acquisition by phage displaying an irrelevant antibody is sufficiently robust to drive selection of rare phage (1 in 10(5)) over three rounds of panning. We then set up a model selection experiment using a cell line expressing the chemokine receptor CCR5 fused to the CD3 peptide together with a panel of phage clones capable displaying either an anti-CCR5 scFv or an irrelevant antibody, with or without the capacity to acquire the soluble anti-CD3 scFv. In this experiment we showed that rare phage (1 in 10(5)) capable of displaying the two different scFvs can be specifically enriched over four rounds of panning. This approach has the potential to be applied to the identification of pairs of ligands capable of co-engaging two different user-defined targets, which would facilitate the discovery of novel bispecific antibodies.

Abstract 1495: Neutralization of CD47 in cancer cells with bispecific antibodies harnesses the phagocytic potential of tumor-infiltrating macrophages

The inhibitory “don9t eat me” signal of phagocytosis, CD47, is commonly overexpressed in cancer cells, a feature generally associated with poor prognosis. CD47 overexpression in cancer is believed to promote immune evasion by allowing tumor cells to “hide” from innate immune phagocytes like macrophages or dendritic cells. CD47 is therefore a new type of immune checkpoint and an attractive target for cancer immunotherapy. However, as CD47 is also universally expressed on healthy cells, clinical development of anti-CD47 monoclonal antibodies is inevitably limited by toxicity and/or pharmacokinetic issues. To overcome these liabilities, we engineered dual-targeting bispecific antibodies (biAbs) for selective blockade of CD47 in malignant cells. By tethering the biAbs strongly to cells expressing a tumor-associated antigen (TAA), such as CD19 or mesothelin, CD47 is blocked selectively on the target cell. In contrast, as these biAbs will lose the avidity effect with TAA-negative cells, they will bind with very low affinity to healthy cells which express CD47. In this manner, dual-targeting should help to sidestep safety and pharmacokinetic “sink” problems resulting from ubiquitous CD47 expression. Studies in non-human primates performed with the CD47/CD19 therapeutic candidate NI-1701 confirmed this prediction, demonstrating normal IgG1 pharmacokinetics and absence of toxicity, even at high antibody doses (100 mg/kg per week). Hence, the mechanism of action of CD47/TAA dual-targeting antibodies is heavily contingent upon target co-engagement. In vitro, CD19-positive or mesothelin-positive cancer cells are efficiently killed through antibody dependent cellular phagocytosis (ADCP) and/or antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of effector cells, such as macrophages or natural killer cells, and the corresponding dual-targeting CD47/TAA antibodies. Their enhanced ability to induce tumor cell phagocytosis was also demonstrated in vivo, in xenograft models: Mice implanted with subcutaneous human B cell lymphoma xenografts controlled tumor growth following therapy with NI-1701, contrary to mice treated with an anti-CD19 mAb. Importantly, tumor microenvironment (TME) studies revealed that mouse macrophages infiltrating human tumors engulfed tumor cells more frequently—and at a significantly higher rate—in animals treated with NI-1701 as compared to controls. Moreover, the observed superior phagocytic activity of tumor-infiltrating macrophages was associated with a reduction of granulocytic myeloid-derived suppressor cell infiltrates, suggesting that NI-1701 may favor the establishment of a tumor-hostile, immunostimulatory TME. We conclude that dual-targeting CD47/TAA bispecific antibodies may open the way to the safe and efficacious therapeutic neutralization of CD47, the universal ‘don9t eat me’ signal hijacked by cancer cells. Citation Format: Krzysztof Masternak, Valery Moine, Lucile Broyer, Xavier Chauchet, Vanessa Buatois, Elie Dheilly, Stefano Majocchi, Giovanni Magistrelli, Yves Poitevin, Ulla Ravn, Eric Hatterer, Susana Salgado Pires, Limin Shang, Zoe Johnson, Walter Ferlin, Marie Kosco-Vilbois, Nicolas Fischer. Neutralization of CD47 in cancer cells with bispecific antibodies harnesses the phagocytic potential of tumor-infiltrating macrophages. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1495.