Liliane Goetsch

Strategies and challenges for the next generation of antibody–drug conjugates

Antibody–drug conjugates (ADCs) are one of the fastest growing classes of oncology therapeutics. After half a century of research, the approvals of brentuximab vedotin (in 2011) and trastuzumab emtansine (in 2013) have paved the way for ongoing clinical trials that are evaluating more than 60 further ADC candidates. The limited success of first-generation ADCs (developed in the early 2000s) informed strategies to bring second-generation ADCs to the market, which have higher levels of cytotoxic drug conjugation, lower levels of naked antibodies and more-stable linkers between the drug and the antibody. Furthermore, lessons learned during the past decade are now being used in the development of third-generation ADCs. In this Review, we discuss strategies to select the best target antigens as well as suitable cytotoxic drugs; the design of optimized linkers; the discovery of bioorthogonal conjugation chemistries; and toxicity issues. The selection and engineering of antibodies for site-specific drug conjugation, which will result in higher homogeneity and increased stability, as well as the quest for new conjugation chemistries and mechanisms of action, are priorities in ADC research.

A recombinant humanized anti-insulin-like growth factor receptor type I antibody (h7C10) enhances the antitumor activity of vinorelbine and anti-epidermal growth factor receptor therapy against human cancer xenografts.

Interaction of insulin‐like growth factor receptor I (IGF‐IR) with its ligands has been reported to induce cell proliferation, transformation and blockade of cell apoptotic functions. IGF‐IR is overexpressed on numerous tumor cell types and its blockade could be of importance for anti‐cancer therapy. We have generated a humanized anti‐IGF‐IR antibody h7C10 that blocks in vitro IGF‐I and IGF‐II‐induced cell proliferation of MCF‐7 breast cancer cells. Analysis of the IGF‐I transduction cascade demonstrated that the humanized anti‐IGF‐IR antibody and its murine parental form block IGF‐I‐induced tyrosine phosphorylation, both its β‐chain and IRS‐1 tyrosine phosphorylation. This presumably leads to cell cycle arrest and, consequently, growth inhibition. Treatment of nude mice bearing either human breast cancer cells (MCF‐7) or non small lung cancer cells (A549) with h7C10, or its murine parental form 7C10, inhibited significantly tumor growth. An almost complete inhibition of A549 tumor growth was observed when mice were treated with the anti‐IGF‐IR antibody combined with either a chemotherapeutic agent, Vinorelbine or an anti‐epidermal growth factor receptor (EGFR) antibody, 225. Combined therapy prolonged significantly the life span of mice in an orthotopic in vivo model of A549; the combination of the anti‐IGF‐IR antibody with an anti‐EGFR antibody was superior to the Vinorelbine combination. The present results indicate that the humanized anti‐IGF‐IR antibody h7C10 has a great potential for cancer therapy when combined with either a chemotherapeutic agent or an antibody that targets other growth factor receptors, such as the epidermal growth factor receptor.

Trends in Glycosylation, Glycoanalysis and Glycoengineering of Therapeutic Antibodies and Fc-Fusion Proteins

Monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. More than 20 MAbs have been approved and several hundreds are in clinical trials in various therapeutic indications including oncology, inflammatory diseases, organ transplantation, cardiology, viral infection, allergy, and tissue growth and repair. Most of the current therapeutic antibodies are humanized or human Immunoglobulins (IgGs) and are produced as recombinant glycoproteins in eukaryotic cells. Many alternative production systems and improved constructs are also being actively investigated. IgGs glycans represent only an average of around 3% of the total mass of the molecule. Despite this low percentage, particular glycoforms are involved in essential immune effector functions. On the other hand, glycoforms that are not commonly biosynthesized in human may be allergenic, immunogenic and accelerate the plasmatic clearance of the linked antibody. These glyco-variants have to be identified, controlled and limited for therapeutic uses. Glycosylation depends on multiple factors like production system, selected clonal population, manufacturing process and may be genetically or chemically engineered. The present account reviews the glycosylation patterns observed for the current approved therapeutic antibodies produced in mammalian cell lines, details classical and state-of-the-art analytical methods used for the characterization of glycoforms and discusses the expected benefits of manipulating the carbohydrate components of antibodies by bio- or chemical engineering as well as the expected advantages of alternative biotechnological production systems developed for new generation of therapeutic antibodies and Fc-fusion proteins.

Preclinical activity of F14512, designed to target tumors expressing an active polyamine transport system

SummaryWe have exploited the polyamine transport system (PTS) to deliver selectively a spermine-drug conjugate, F14512 to cancer cells. This study was aimed to define F14512 anticancer efficacy against tumor models and to investigate whether fluorophor-labeled polyamine probes could be used to identify tumors expressing a highly active PTS and that might be sensitive to F14512 treatments. Eighteen tumor models were used to assess F14512 antitumor activity. Cellular uptake of spermine-based fluorescent probes was measured by flow cytometry in cells sampled from tumor xenografts by needle biopsy. The accumulation of the fluorescent probe within B16 tumors in vivo was assessed using infrared fluorescence imaging. This study has provided evidence of a major antitumor activity for F14512. Significant responses were obtained in 67% of the tumor models evaluated, with a high level of activity recorded in 33% of the responsive models. Complete tumor regressions were observed after i.v., i.p. or oral administrations of F14512 and its antitumor activity was demonstrated over a range of 2–5 dose levels, providing evidence of its good tolerance. The level of cellular fluorescence emitted by the fluorescent probes was higher in cells sampled from tumors sensitive to F14512 treatments than from F14512-refractory tumors. We suggest that these probes could be used to identify tumors expressing a highly active PTS and guide the selection of patients that might be treated with F14512. These results emphasize the preclinical interest of this novel molecule and support its further clinical development.

Tetraspanin CD151 as a target for antibody-based cancer immunotherapy

CD151 is a plasma membrane protein belonging to the tetraspanin superfamily which is expressed on normal cells such as endothelial cells and platelets and frequently overexpressed on cancer cells. It is known to be functionally linked to cancer metastasis. In humans, increased expression of CD151 is indicative of a poor prognosis in different cancer types. Whereas its mechanism of action remains obscure, CD151 was shown to regulate cell motility and adhesion through association with laminin-binding integrins such as α3β1 or α6β4. Several anti-CD151 mAbs (monoclonal antibodies) have been shown to display anti-metastatic activity in vivo. Inhibition of metastasis was not attributed to any effect of these mAbs on tumour cell growth, but was essentially attributed to inhibition of cell motility. We have generated anti-CD151 mAbs which can inhibit the tumoral growth in different xenograft cancer models. As expected, these mAbs were also able to inhibit metastasis in orthotopic cancer models. These data suggest that CD151 could function at multiple cancer stages, including not only metastasis cascade steps, but also earlier steps of primary tumour growth, thus reinforcing the interest of this innovative target in oncology. mAbs targeting CD151 may be of significant interest for cancer biotherapy.

Molecular mechanisms involved in activity of h7C10, a humanized monoclonal antibody, to IGF-1 receptor.

IGF‐1 receptor (IGF‐1R) plays a key role in the development of numerous tumors. Blockade of IGF‐1R axis using monoclonal antibodies constitutes an interesting approach to inhibit tumor growth. We have previously shown that h7C10, a humanized anti–IGF‐1R Mab, exhibited potent antitumor activity in vivo. However, mechanisms of action of h7C10 are still unknown. Here, we showed that h7C10 inhibited IGF‐1–induced IGF‐1R phosphorylation in a dose‐dependent manner. Also, h7C10 abolished IGF‐1–induced activation of PI3K/AKT and MAPK pathways. Cell cycle progression and colony formation were affected in the presence of h7C10 probably because of the inhibition of IGF‐1–induced cyclin D1 and E expression. In addition, we demonstrated that h7C10 induced a rapid IGF‐1R internalization leading to an accumulation into cytoplasm resulting in receptor degradation. Using lysosome and proteasome inhibitors, we observed that the IGF‐1R α‐ and β‐chains could follow different degradation routes. Thus, we demonstrated that antitumoral properties of h7C10 are the result of IGF‐1–induced cell signaling inhibition and down‐regulation of IGF‐1R level suggesting that h7C10 could be a candidate for therapeutic applications.

Development of Non-Pathogenic Staphylococci as Vaccine Delivery Vehicles

Among the bacteria being considered as live recombinant vaccine vehicles, the most well studied during the past decade are attenuated Salmonella species1 and mycobacterial bacille Calmette-Guerin (BCG) due to their capacity to colonize mucosal surfaces and invade macrophages in the liver, spleen and lymph nodes of the host.2,3 Surface-display of the foreign antigens to be delivered, has in both these systems proven to be beneficial in eliciting an immune response.4–7 The risk of reversion to a virulent phenotype and the potential side-effects in immunocompromised individuals and infants have, however, raised concern of the use of Salmonella or BCG-based recombinant vaccines in humans.8

A novel role for junctional adhesion molecule‐A in tumor proliferation: Modulation by an anti‐JAM‐A monoclonal antibody

To identify new potential targets in oncology, functional approaches were developed using tumor cells as immunogens to select monoclonal antibodies targeting membrane receptors involved in cell proliferation. For that purpose cancer cells were injected into mice and resulting hybridomas were screened for their ability to inhibit cell proliferation in vitro. Based on this functional approach coupled to proteomic analysis, a monoclonal antibody specifically recognizing the human junctional adhesion molecule‐A (JAM‐A) was defined. Interestingly, compared to both normal and tumor tissues, we observed that JAM‐A was mainly overexpressed on breast, lung and kidney tumor tissues. In vivo experiments demonstrated that injections of anti‐JAM‐A antibody resulted in a significant tumor growth inhibition of xenograft human tumors. Treatment with monoclonal antibody induced a decrease of the Ki67 expression and downregulated JAM‐A levels. All together, our results show for the first time that JAM‐A can interfere with tumor proliferation and suggest that JAM‐A is a potential novel target in oncology. The results also demonstrate that a functional approach coupled to a robust proteomic analysis can be successful to identify new antibody target molecules that lead to promising new antibody‐based therapies against cancers.

Anti-c-Met monoclonal antibody ABT-700 breaks oncogene addiction in tumors with MET amplification.

Backgroundc-Met is the receptor tyrosine kinase for hepatocyte growth factor (HGF) encoded by the MET proto-oncogene. Aberrant activation of c-Met resulting from MET amplification and c-Met overexpression is associated with poor clinical outcome in multiple malignancies underscoring the importance of c-Met signaling in cancer progression. Several c-Met inhibitors have advanced to the clinic; however, the development of inhibitory c-Met-directed therapeutic antibodies has been hampered by inherent agonistic activity.MethodWe generated and tested a bivalent anti-c-Met monoclonal antibody ABT-700 in vitro for binding potency and antagonistic activity and in vivo for antitumor efficacy in human tumor xenografts. Human cancer cell lines and gastric cancer tissue microarrays were examined for MET amplification by fluorescence in situ hybridization (FISH).ResultsABT-700 exhibits a distinctive ability to block both HGF-independent constitutive c-Met signaling and HGF-dependent activation of c-Met. Cancer cells addicted to the constitutively activated c-Met signaling driven by MET amplification undergo apoptosis upon exposure to ABT-700. ABT-700 induces tumor regression and tumor growth delay in preclinical tumor models of gastric and lung cancers harboring amplified MET. ABT-700 in combination with chemotherapeutics also shows additive antitumor effect. Amplification of MET in human cancer tissues can be identified by FISH.ConclusionsThe preclinical attributes of ABT-700 in blocking c-Met signaling, inducing apoptosis and suppressing tumor growth in cancers with amplified MET provide rationale for examining its potential clinical utility for the treatment of cancers harboring MET amplification.

Evaluation of Antibody-Dependent Cell Cytotoxicity Using Lactate Dehydrogenase (LDH) Measurement

In order to improve therapeutic antibodies efficacy in cancer patients, several strategies were developed. One of these strategies consists in the enhancement of effector functions. Antibody-dependent cellular cytotoxicity (ADCC) was shown to mediate the activity of several therapeutic antibodies through interaction of the constant fragment (Fc) with immune cells. The interactions of Fc fragment can be modulated by engineering through modifications of the carbohydrate moieties or through modifications of some critical amino acids for its binding. Such modifications have to be studied in an in vitro assay to evaluate their impact on the regulation of effector functions. Here, we described a method to evaluate ADCC using a nonradioactive assay based on the measurement of lactate dehydrogenase (LDH) release. NK cells were purified by negative immunomagnetic selection and used as effector cells to trigger ADCC against specific target tumor cells. The LDH release measurement from lysed cells is performed after 4 h incubation. This method can replace the (51)Cr release assay since it is less restrictive and highly sensitive.