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 New Anti-CXCR4 Antibody That Blocks the CXCR4/SDF-1 Axis and Mobilizes Effector Cells.

The type IV C-X-C-motif chemokine receptor (CXCR4) is expressed in a large variety of human cancers, including hematologic malignancies, and this receptor and its ligand, stromal cell-derived factor-1 (SDF-1), play a crucial role in cancer progression. We generated a humanized immunoglobulin G1 mAb, hz515H7, which binds human CXCR4, efficiently competes for SDF-1 binding, and induces a conformational change in CXCR4 homodimers. Furthermore, it inhibits both CXCR4 receptor-mediated G-protein activation and ß-arrestin-2 recruitment following CXCR4 activation. The binding of the hz515H7 antibody to CXCR4 inhibits the SDF-1-induced signaling pathway, resulting in reduced phosphorylation of downstream effectors, such as Akt, Erk1/2, p38, and GSK3ß. Hz515H7 also strongly inhibits cell migration and proliferation and, while preserving normal blood cells, induces both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against neoplastic cells. In mouse xenograft models, hz515H7 displays antitumor activities with multiple hematologic tumor cell lines, with its Fc-mediated effector functions proving essential in this context. Furthermore, hz515H7 binds to primary tumor cells from acute myeloid leukemia and multiple myeloma patients. Collectively, our results demonstrate two major mechanisms of action, making hz515H7 unique in this regard. Its potential as a best-in-class molecule is currently under investigation in a phase I clinical trial. Mol Cancer Ther; 15(8); 1890-9. ©2016 AACR.

A novel antagonist anti-cMet antibody with antitumor activities targeting both ligand-dependent and ligand-independent c-Met receptors.

c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers.

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

BACKGROUND: c-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. METHOD: We 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). RESULTS: ABT-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. CONCLUSIONS: The 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.

Evaluation of complement-dependent cytotoxicity using ATP measurement and C1q/C4b binding.

One of the major issues for antibody treatment is enhancement of efficacy. Recent studies have highlighted the important role of effector functions in improvement of antibody therapy. Among effector functions, complement-dependent cytotoxicity (CDC), which induces cell lysis by a cascade of activation triggered by the binding of C1q subunits to the Fc regions of antibodies bound to the cell surface, is part of the mechanism of several antibody therapies. CDC can be modulated by either Fc isotype engineering or Fc genetic mutations or Fc glycosylation profile modifications. To evaluate the impact of such modifications on CDC, we describe a luminescence method based on ATP measurement to estimate tumor damaged cells and a flow cytometry method to evaluate the binding of C1q on the Fc region and the binding of C4b on cell surface. The luminescence method coupled with complement protein analysis by flow cytometry encompasses all needed methods to evaluate antibody ability to trigger CDC.

Biological significance and targeting of c-Met tyrosine kinase receptor in cancer.

c-Met is a tyrosine kinase receptor largely described to be involved in cancer progression and metastasis. In such pathologic situation, many alterations of this receptor were noticed that include transcriptional overexpression, gene amplification, somatic or germline mutations and/or ligand dependent autocrine/paracrine loops. More recently it has also been suggested that c-Met would be involved in resistance to targeted therapies directed towards EGFR or angiogenesis. Major efforts from a large number of pharmaceutical companies are invested dedicated to evaluate the efficacy of either small molecule inhibitors or monoclonal antibodies directed against c-Met or its unique ligand HGF. A series of promising results from the first completed clinical trials indicated that compounds targeting c-Met have an acceptable toxicity profile and that efficacy was noticed in some treated patients. Non squamous NSCLC patients that express more often high levels of c-Met seemed to represent a most sensitive subset for and anti-c-Met/erlotinib therapy. Many Phase III trials are currently recruiting and a particular effort was performed in order to discover biomarkers associated with efficacy and patient selection. This review will provide an overview of the current knowledge on the c-Met axis for development of novel therapeutics in Oncology.

Insulin-like growth factor receptor type I as a target for cancer therapy.

After more than 20 years of extensive work, insulin-like growth factor receptor 1 (IGF-IR) is still an attractive target for drug development. Due to its close homology to insulin receptor, IGF-IR is of interest for antibody design while antibody great specificity allows to discriminate between the two receptors. Major efforts from a large number of pharmaceutical companies are invested to evaluate the efficacy of such molecules in human without so far an obvious success. Discovery of biomarkers associated with efficacy and patient selection is one of the main challenges that we will have to deal with in order to target the appropriate patient population that will most benefit anti-IGF-IR monoclonal antibody (Mab) and combined treatments. This review will provide an overview of the current knowledge on IGF-IR axis for development of novel therapeutics in Oncology.

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.

7th cancer scientific forum of theCancéropôle Lyon Auvergne Rhône-Alpes: March 20-21, 2012, Lyon, France.

The Innovative Approaches in Anti-Cancer Monoclonal Antibodies meeting, held on March 20, 2012 in Lyon, was organized by Cancéropôle Lyon Auvergne-Rhône-Alps in partnership with the French competitiveness cluster Lyonbiopôle. CLARA is one of the seven cancer research clusters within France in charge of facilitating Translational Oncology Research by taking into account the objectives of the French National Cancer Plans I and II and, in coordination with the French National Cancer Institute and local authorities (mainly Grand Lyon, Rhône County and Rhône-Alpes Region), to perform economic development of research findings. The contribution of lectures by outstanding speakers as described in this report, the organization of two-round tables: "Antibody treatment in cancer: Unmet needs in solid tumors and hematological malignancies," and "From chimeric to more than human antibodies," together with face-to-face meetings, was shared by over 230 participants. The lectures provided an overview of the commercial pipeline of monoclonal antibody (mAb) therapeutics for cancer; discussion of the distinction between biosimilar, biobetter and next generation therapeutic antibodies for cancer; updates on obinutuzumab and the use of mAbs in lymphoma; and discussion of antibody-drug conjugates.

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 α3ß1 or α6ß4. 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.

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

We 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.

The next generation of antibody-drug conjugates comes of age.

Monoclonal antibodies (mAbs) and derivatives are currently the fastest growing class of therapeutic molecules. More than 30 G-type immunoglobulins (IgG) and related agents have been approved over the past 25 years mainly for cancers and inflammatory diseases. In oncology, mAbs are often combined with cytotoxic drugs to enhance their therapeutic efficacy. Alternatively, small anti-neoplastic molecules can be chemically conjugated to mAbs, used both as carriers (increased half-life) and as targeting agents (selectivity). Potential benefits of antibody-drug conjugates (ADCs), strategies, and development challenges are discussed in this review. Several examples of ADCs are presented with emphasis on three major molecules currently in late clinical development as well as next generation thio-mAbs conjugates with improved therapeutic index.

Selection criteria for c-Met-targeted therapies: emerging evidence for biomarkers.

Extensive development of targeted therapies emphasize the critical need for biomarkers and major efforts have been engaged to identify screening, prognostic, stratification and therapy-monitoring markers. One of the challenges in translating preclinical studies into effective clinical therapies remains the accurate identification of a responsive subsets of patients. Studies on trastuzumab demonstrated that patient response could be specifically correlated with the amplification of the Her2 gene. However, for the EGF receptor, it has been more difficult to find the right stratification biomarker and recent data demonstrate that genetic alterations for the EGF receptor have to be considered. Taken together, these data underline the need for a deeper understanding of both targeted receptor and human disease to determine pathways that might be investigated during early clinical trials in order to define relevant biomarkers for patient selection. This article, dealing with the c-Met tyrosine kinase receptor, provides an overview of c-Met alterations observed in cancer and proposes approaches for stratification biomarker selection.

[The potential of monoclonal antibodies in cancer : established trastuzumab and cetuximab and promising targets IGF-1R and c-MET]. / Anticorps et tumeurs solides : cibles établies et pistes prometteuses.

Recent biotechnological advances allowed the development of a novel class of anti-cancer drugs called monoclonal antibodies (mAb). To illustrate the potential of these new agents, two mAbs used in daily practice (i.e., trastuzumab and cetuximab) and two promising targets (i.e., IGF-1R and c-Met) for which mAbs should be available in a near future are discussed here. Trastuzumab and cetuximab deeply changed treatment strategies for breast, colon, and head and neck cancers. However their efficacy is observed in a fraction of patients only and is often time limited. Thus, current challenges are to better understand the mechanisms of action of mAbs, to identify mechanisms of resistance, to exploit the synergy between mAbs and chemotherapy drugs, and to better select patients with a potential benefit. Resolving these issues should pave the way for tailored treatment according to tumor and patient characteristics.

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 alpha- and beta-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.

Insulin-like growth factor receptor type I as a target for cancer therapy.

In recent years, improvements in the understanding of oncogenesis has permitted the identification of new molecular targets for cancer therapy. Among all the different approaches, inhibition of tyrosine kinase receptor activity using small molecules or biomolecules for controlling cancer growth has been successful and has brought new therapeutic opportunities to the medical community. After more than 20 years of extensive work, insulin-like growth factor receptor I (IGF-IR) is becoming an attractive target for drug development. Owing to its close homology to insulin receptor, IGF-IR is of interest for antibody design while its specificity allows us to discriminate between the two receptors. Major efforts from a large number of pharmaceutical companies are invested in evaluating the efficacy of such molecules in humans. Discovery of biomarkers associated with efficacy and patient selection are the main challenges that we will have to deal with in order to target the appropriate patient population that will most benefit from anti-IGF-IR monoclonal antibodies and combined treatments. This review will provide an overview of the current knowledge on IGF-IR and ongoing clinical trials.

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.

Peptides as tools and drugs for immunotherapies.

Peptides are essential tools for discovery and pre-clinical and pharmaceutical development of viral and cancer vaccines ('active immunotherapies') as well as for therapeutic antibodies ('passive immunotherapies'). They help to trigger and analyze immune responses at a molecular level (B-cell, T-helper and CTL epitopes). They contribute largely to the design of new vaccine candidates and to the generation of monoclonal antibodies. They are also valuable analytical reference compounds for the structural characterisation by liquid chromatography and mass spectrometry of recombinant proteins used as biopharmaceuticals. As for other therapeutic applications, formulation, solubilisation, batch consistency and stability, issues have to be addressed to allow the pre-clinical and clinical development of this class of compounds as immunotherapeutic drugs. In the present review, three case studies dealing with (i) the design and the characterisation of Respiratory Syntycial Virus subunit vaccines, (ii) peptide-based melanoma vaccines, and (iii) therapeutic monoclonal antibodies, all investigated in clinical trials, are reported and discussed.

Functional responses and in vivo anti-tumour activity of h7C10: a humanised monoclonal antibody with neutralising activity against the insulin-like growth factor-1 (IGF-1) receptor and insulin/IGF-1 hybrid receptors.

A novel humanised monoclonal antibody (Mab, h7C10) was raised against the human insulin-like growth factor-1 receptor (IGF-1R); it exhibited potent inhibition of tumour growth in animal models. Further evaluation of its inhibitory activity at hybrid receptors (Hybrid-Rs) composed of the association between IGF-1R and insulin receptor (IR) was performed. Selective, potent and efficacious inhibition of [(125)I]IGF-1 binding as well as IGF-1- and IGF-2-mediated receptor phosphorylation was demonstrated at both IGF-1R and Hybrid-Rs, without activity at IR. Ligand-independent down-regulation of both IGF-1R and Hybrid-Rs was obtained upon long-term association with h7C10. In vivo evaluation was performed in a MDA-MB-231 xenograft mouse model, showing a 14-fold higher level of Hybrid-Rs as compared to IGF-1R. A more potent anti-tumoural response was obtained for h7C10 as compared to Mabs targeting solely IGF-1R or Hybrid-Rs. The herewith described neutralising properties of h7C10 as potent inhibitor of both IGF-1R and Hybrid-Rs are likely to participate in its anti-tumoural activities and maybe of interest for therapeutic applications.