Search

Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. They combine monoclonal antibody specificity for over-expressed tumor antigens and the high cytoxicity of small molecular drugs (SMDs) and can therefore selectively kill tumor cells while minimizing toxicity to normal cells. Nevertheless, the premature deconjugation of ADCs in the circulation may trigger off target toxicity in patients. The released free drug level must be low in circulation for an extended period of time as well as the de-conjugation rate to ensure an acceptable therapeutic window. As a result, the assessment of the stability of the linker between payload and mAb in the systemic circulation is of paramount importance before entering in clinical trial. Here we report a new universal method to immunocapture and analyze by LC-MS the stability and distribution of ADCs in sera from relevant preclinical species (mouse, rat and cynomolgus monkey). Furthermore we demonstrated that this workflow can be applied to both ADCs with cleavable and non cleavable linkers. Last but not least, the results obtained in cynomolgus serum using immunoprecipitation and LC-MS analysis were cross validated using an ELISA orthogonal method. As the ligand used for immunoprecipitation is targeting the Fc part of mAb (CaptureSelect™ Human IgG-Fc PK Biotin), this protocol can be applied to analyze the stability of virtually all ADCs in sera for preclinical studies without the need to prepare specific molecular tools.

Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. Because ADCs combine the monoclonal antibody specificity with the high toxicity of a drug, they can selectively kill tumor cells while minimizing toxicity to normal cells. Most of the current ADCs in clinical trials are controlled, but heterogeneous mixtures of isomers and isoforms. Very few protocols on ADC characterization at the peptide level have been published to date. Here, we report on the improvement of an ADC peptide mapping protocol to characterize the drug-loaded peptides by LC-MS analysis. These methods were developed on brentuximab vedotin (Adcetris), a commercial ADC with an average of four drugs linked to interchain cysteine residues of its antibody component. Because of the drug hydrophobicity, all the steps of this protocol including enzymatic digestion were improved to maintain the hydrophobic drug-loaded peptides in solution, allowing their unambiguous identification by LC-MS. For the first time, the payloads positional isomers observed by RP-HPLC after IdeS-digestion and reduction of the ADC were also characterized.

The World Antibody-Drug Conjugate (WADC) Summits organized by Hanson Wade are currently the largest meetings fully dedicated to ADCs. The first global ADC Summit was organized in Boston in October 2010. Since 2011, two WADC are held every year in Frankfurt and San Francisco, respectively. The 2013 WADC San Francisco event was structured around plenary sessions with keynote speakers from AbbVie, Agensys, ImmunoGen, Immunomedics, Genentech, Pfizer and Seattle Genetics. Parallel tracks were also organized addressing ADC discovery, development and optimization of chemistry, manufacturing and control (CMC) issues. Discovery and process scientists, regulatory experts (US Food and Drug Administration), academics and clinicians were present, including representatives from biotechnology firms (Concortis, CytomX Therapeutics, Glykos, Evonik, Igenica, Innate Pharma, Mersana Therapeutics, Polytherics, Quanta Biodesign, Redwood Bioscience, Sutro Biopharma, SynAffix), pharmaceutical companies (Amgen, Genmab, Johnson and Johnson, MedImmune, Novartis, Progenics, Takeda) and contract research or manufacturing organizations (Baxter, Bayer, BSP Pharmaceuticals, Fujifilm/Diosynth, Lonza, Pierre Fabre Contract Manufacturing, Piramal, SAFC, SafeBridge).

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 β-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 GSK3β. 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.

Monoclonal antibodies (mAb) are attractive biologic drugs because of their specificity and well understood mechanisms of action. So far, most mAb have been developed for treating cancers or immunological disorders. However, the antibiotic resistance crisis, emerging viral diseases and bioterrorism have increased the development of anti-infectious mAb, for which more than twenty clinical trials are in progress to evaluate their safety and efficacy. The synergies obtained using combinations of anti-infectious mAb and small molecule drugs will certainly offer new opportunities for the treatment of infectious diseases.

The 101-residue long Tat protein of primary isolate 133 of the human immunodeficiency virus type 1 (HIV-1), wt-Tat(133) displays a high transactivation activity in vitro, whereas the mutant thereof, STLA-Tat(133), a vaccine candidate for HIV-1, has none. These two proteins were chemically synthesized and their biological activity was validated. Their structural properties were characterized using circular dichroism (CD), fluorescence emission, gel filtration, dynamic light scattering, and small angle X-ray scattering (SAXS) techniques. SAXS studies revealed that both proteins were extended and belong to the family of intrinsically unstructured proteins. CD measurements showed that wt-Tat(133) or STLA-Tat(133) underwent limited structural rearrangements when complexed with specific fragments of antibodies. Crystallization trials have been performed on the two forms, assuming that the Tat(133) proteins might have a better propensity to fold in supersaturated conditions, and small crystals have been obtained. These results suggest that biologically active Tat protein is natively unfolded and requires only a limited gain of structure for its function.

Despite technological advances, detection of deamidation in large proteins remains a challenge and the use of orthogonal methods is needed for unequivocal assignment. By a combination of cation-exchange separation, papain digestion, and a panel of mass spectrometry techniques we identified asparagine deamidation in light chain complementarity determining region 1 (CDR1) of a humanized IgG1 monoclonal antibody. The reaction yields both Asp and isoAsp, which were assigned by Edman degradation and by isoAsp detection using protein isoaspartate methyltransferase. The deamidated antibody variants were less potent in antigen binding compared to the nondegraded antibody. Changes in near-UV CD spectra, susceptibility to papain cleavage in an adjacent CDR2 loop, and the tendency of the newly formed isoAsp to undergo isomerization suggest local perturbations in the structure of the isoAsp-containing antibody.

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. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

Background: In preclinical studies, monoclonal antibodies (mAbs) are traditionally assayed by ligand-binding-assays. Recently, quantitative liquid chromatography mass spectrometry (MS)-based assays have emerged which circumvent a number of challenges. These assays may also be multiplex, making them potentially compatible with pharmacokinetic assays for combined antibody therapies. Materials & methods: We combined a quantitative MS-based approach with the protein standard for absolute quantification (PSAQ™) strategy to simultaneously quantify three mAb variants presenting minor sequence differences. Stable isotopically labeled mAbs were produced and used as quantification standards. Titration curves were performed to assess the analytical performances of the method. LC-MS/MS and ELISA data were cross-compared. Results: The approach presented provides similar accuracy and precision than ELISA, while being multiplex and faster to develop. It has applications at all stages of the pharmaceutical development.

The design of new antigens with both high immunogenic and safety properties is of particular interest to vaccine against infectious diseases. In the present study, we describe the synthesis and the refolding of peptide G20 derived from the Human Respiratory Syncytial Virus (hRSV) G-protein. G20 (MEF G140–190 G144–158) is a peptide of 69 amino acids with two disulfide bridges, which comprises multiple protective B-cell epitopes. It was deleted of the T helper cell epitope 184–198 of the RSV G-protein, which was found to induce pulmonary pathology after RSV challenge in mice. Interestingly, we showed in the present study that G20 generated a highly protective antibody response against RSV challenge in Balb/c mice. Therefore, G20 represents a new potential antigen for an RSV vaccine.

The protective efficacy of the influenza matrix protein epitope 58–66 (called M1), recognized in the context of human HLA-A2 molecules, was evaluated in a HLA-A2/Kb transgenic mouse model of lethal influenza infection. Repeated subcutaneous immunizations with M1 increased the percentage of survival. This effect was mediated by T cells since protection was abolished following in vivo depletion of all T lymphocytes, CD8+, or CD4+ T cells. The survival correlated with the detection of memory CD8+ splenocytes able to proliferate in vitro upon stimulation with M1 and to bind M1-loaded HLA-A2 dimers, as well as with M1-specific T cells in the lungs, which were directly cytotoxic to influenza-infected cells following influenza challenge. These results demonstrated for the first time that HLA-A2-restricted cytotoxic T cells specific for the major immunodominant influenza matrix epitope are protective against the infection. They encourage further in vivo evaluation of T cell epitopes recognized in the context of human MHC molecules.

Human respiratory syncytial virus (hRSV) is one of the most common causes of respiratory infection in infants and the elderly. Previous attempts to vaccinate children against RSV failed and the induction of an aberrant Th2-type immune response was shown to induce severe to fatal pulmonary disease characterised in part by eosinophilia. BBG2Na is a promising human RSV subunit vaccine candidate which successfully passed phase II clinical trials in adults in association with Adju-Phos ® . However, this formulation is not the most suitable for use in children since aluminium salts are known to induce a Th2-based immune response. In this study, we describe a potent and safe adjuvant formulation for BBG2Na in dimethyldioctadecylammonium bromide (DDA) that induces a mixed Th1/Th2 immune response in BALB/c mice. Furthermore, BBG2Na showed the same protective efficacy against RSV challenge when formulated either in DDA or in alum in mice and cotton rats.

Several cytotoxic T lymphocyte peptide-based vaccines against hepatitis B, human immunodeficiency virus and melanoma were recently studied in clinical trials. One interesting melanoma vaccine candidate alone or in combination with other tumor antigens, is the decapeptide ELA. This peptide is a Melan-A/MART-1 antigen immunodominant peptide analog, with an N-terminal glutamic acid. It has been reported that the amino group and gamma-carboxylic group of glutamic acids, as well as the amino group and gamma-carboxamide group of glutamines, condense easily to form pyroglutamic derivatives. To overcome this stability problem, several peptides of pharmaceutical interest have been developed with a pyroglutamic acid instead of N-terminal glutamine or glutamic acid, without loss of pharmacological properties. Unfortunately compared with ELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminal acetyl-capped derivative (AcELA) failed to elicit cytotoxic T lymphocyte (CTL) activity. Despite the apparent minor modifications introduced in PyrELA and AcELA, these two derivatives probably have lower affinity than ELA for the specific class I major histocompatibility complex. Consequently, in order to conserve full activity of ELA, the formation of PyrELA must be avoided. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride salt, shows higher stability than the acetate salt and may be suitable for use in man. Similar stability data were also obtained for MAGE-3, another N-terminal glutamic acid containing CTL peptide in clinical development, leading us to suggest that all N-terminal glutamic acid and probably glutamine-containing CTL peptide epitopes may be stabilized as hydrochloride salts.

Antibodies and related products represent one of the fastest growing areas of new drug development within the pharmaceutical industry. Monoclonal antibodies (mAbs) undergo many posttranslational modifications (PTMs) that must be extensively characterized. Here we described a rapid mass spectrometry (MS) method for the characterization of cetuximab glycosylation. The reported analytical technique is based on the use of a cystein protease, immunoglobulin-degrading enzyme of Streptococcus pyogenes that allows a fast limited proteolysis of the mAb with low material consumption. The resulting large fragments are analyzed by ultrahigh-performance liquid chromatography combined to an electrospray ionization mass spectrometer and a time-of-flight analyzer (ESI-TOF). Cetuximab is a potent chimeric mouse/human antibody worldwide approved for the treatment of colon and head and neck cancers. This antibody, produced by SP2/0 murine myeloma cells, is N-glycosylated both in the Fc and Fab moieties, which have been shown to impact on safety and PK/PD and considered as a critical quality attribute. The method can also be applied for biosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.

Antibodies and related products represent one of the fastest growing areas of new drug development within the pharmaceutical industry. Monoclonal antibodies (mAbs) undergo many posttranslational modifications (PTMs) that must be extensively characterized. Here we described a rapid mass spectrometry (MS) method for the characterization of cetuximab glycosylation. The reported analytical technique is based on the use of a cystein protease, immunoglobulin-degrading enzyme of Streptococcus pyogenes that allows a fast limited proteolysis of the mAb with low material consumption. The resulting large fragments are analyzed by ultrahigh-performance liquid chromatography combined to an electrospray ionization mass spectrometer and a time-of-flight analyzer (ESI-TOF). Cetuximab is a potent chimeric mouse/human antibody worldwide approved for the treatment of colon and head and neck cancers. This antibody, produced by SP2/0 murine myeloma cells, is N-glycosylated both in the Fc and Fab moieties, which have been shown to impact on safety and PK/PD and considered as a critical quality attribute. The method can also be applied for biosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.

Glycosylation is one of the most important posttranslational protein modificationsand has essential roles in antibody effectors functions, immunogenicity, plasmaticclearance (Jefferis 2005; Beck et al. 2008a), and resistance toward proteases (Rajuand Scallon 2006).Like for natural IgGs, all recombinant antibodies contain an Asn-Xxx-Ser/Thr(Xxx is not Pro) consensus sequence for N-glycosylation in their C

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.

7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI–TOF, ES–TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine 297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a mixture of three families of antibodies corresponding (i) to the expected structure (17%; 149 297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 152 878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 154 459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also identified by peptide mapping, mass spectrometry and microsequencing.

The decapeptide ELA (ELAGIGILTV), a Melan-A/MART-1 antigen immunodominant peptide analogue, is an interesting melanoma vaccine candidate alone or in combination with other tumour antigens. P40, the recombinant outer membrane protein A of Klebsiella pneumoniae (kpOmpA), was recently shown to target dendritic cells and to induce peptide-specific CTLs. Here we investigated the adjuvant role of P40 mixed or chemically conjugated to ELA. This compound is an N-terminal glutamic acid-containing peptide. However, it has been reported that the amino group and the gamma-carboxylic group of glutamic acids easily condense to form pyroglutamic derivatives. Usually, to overcome this stability problem, peptides of pharmaceutical interest were developed with a pyroglutamic acid instead of N-terminal glutamic acid, without loss of pharmacological properties. Unfortunately, the pyroglutamic acid derivative (PyrELA) as well as the N-terminal acetyl capped derivative (AcELA) failed to elicit CTL activity when mixed with P40 adjuvant protein. Despite the apparent minor modifications introduced by PyrELA and AcELA, these two derivatives have probably lower affinity than ELA for the class I Major Histocompatibility Complex. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride shows a higher stability than the acetate and may be suitable for use in man.

BBG2Na, a well-defined recombinant protein produced in Escherichia coli, is a promising human respiratory syncytial virus subunit vaccine candidate. This study describes the quantification of residual DNA in large scale batches used in phase I to III clinical trials. Two different analytical methods were developed and applied on five different final bulks of Drug Substance and their associated in process control samples, namely a chemiluminescent hybridisation assay and the total DNA Threshold System assay. These two complementary methods demonstrated the clearance of residual DNA during the downstream purification process. The amount of residual DNA found in the final bulks was below 20 pg of DNA per 300 microg BBG2Na, the highest tested clinical dose of antigen. This is very low level of residual DNA for a recombinant subunit vaccine produced in a bacteria and contribute to make for BBG2Na a well-characterised biopharmaceutical. This study also provides data concerning the validation of the hybridisation dot blot assay and the total DNA Threshold(trade mark)assay.

Human Respiratory Syncytial Virus (hRSV) is a major pathogen responsible for bronchiolitis and severe pulmonary disease. BBG2Na is a promising subunit vaccine candidate against this pathogen [1]. It is a well defined recombinant protein [2], composed of G2Na(130–230), the central domain of RSV G glycoprotein [3] and BB, an albumin binding domain of streptococcal protein G [4], The G2Na part contains 4 Cys and several protective epitopes [5], while the BB part has shown carrier properties [4]. Phase I and II clinical trials were very promising [6], and phase III is on going. BBG2Na (pi = 9.3) was recently shown to be strongly adsorbed by A1PO4 (pZ0 = 4.0) and surprisingly by Al(OH)3 (pZ0 = 11.1) [7]. These unexpected properties could be due to the presence of 2 independent structural domains with opposite charges (BB, pI = 5.5 and G2Na, pi = 10.0) that is not reflected by the pi of the whole protein. G2Na(130–230) was generated by CNBr cleavage of BBG2Na and purified. The Cys pairings were established after enzymatic digestion and LC-MS. On the other hand, all 3 possible isomers of the 4 Cys containing peptide G4a(172–187) were obtained using various oxidation conditions [3]. G4a(Cys173-Cys186/Cys176-Cys183), which corresponded to the native isomer, was investigated by 2D NMR in solution and a three-dimensional structure was established by molecular modeling.

Chemokines are small, secreted peptides that control the migration of leukocytes along a chemical gradient of ligand, especially during immune and inflammatory reactions. They are divided into two major subfamilies, CC and CXC, based on the position of their NH2-terminal cysteine residues, and bind to G protein coupled receptors, whose two major sub families are designated CCR and CXCR. More than 50 human chemokines and 18 chemokine receptors have been discovered so far. The chemokine receptor CXCR4 and its ligand Stromal cell-derived factor-1 (SDF-1) play a central role in various physiological and pathological processes, including cancer. CXCR4 is over-expressed in a large number of tumors: colon, breast, prostate, lung, ovary, pancreas. CXCR4/SDF-1 axis is directly implicated in migration, invasion leading to metastases, cell proliferation and angiogenesis. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. A novel humanized monoclonal antibody hz515H7 was raised against the human CXCR4. It displayed efficacious antagonist properties for all major pathways associated with SDF-1-induced CXCR4 signaling in vitro. Its antitumor activity was investigated in vivo using several human tumor models. Materials and Methods: CHOK1 and NIH3T3 cells were stably transfected with human CXCR4. [125I]SDF1 and [35S]GTP S binding assays were performed on cell membranes containing CXCR4, using SPA-WGA beads. Calcium mobilization was monitored using Fluo-4NW dye. BRET assays were developed upon genetic fusion of CXCR4 to Rluc and β-arrestin-2 to YFP and transient co-expression in HEK293 cells. Xenograft model: 10.106 Ramos cells (B-cell lymphoma) were implanted s.c. into the right flank region of each SCID mouse and allowed to grow to the designated size before administration of antibodies. The mice were followed twice a week for the observation of xenograft growth. Tumor volume was calculated using the formula: π/6 × length × width × height. Results: Hz515H7 Mab was found to strongly inhibit SDF-1 binding, Gα protein activation and -arrestin-2 recruitment. It also inhibited calcium release, and constrained by itself CXCR4 homodimers conformation. Hz515H7 reduced SDF-1-induced cell migration in vitro. Moreover we also demonstrated that hz515H7 Mab was able to significantly inhibit growth of xenograft tumors in mice. Altogether, the data demonstrate that Mab hz515H7 behaves as a potent and efficacious antagonist of all major CXCR4-controlled signaling pathways and suggest that targeting CXCR4 is a promising way for the treatment of tumors. 285-286 C57 Targeting stromal platelet-derived growth factor receptorα (PDGFRα) inhibits lung cancer growth independent of tumor cell PDGFRα expression. Colleen Burns1, David Gerber2, Puja Gupta2, Michael T. Dellinger2, Jason E. Toombs2, Michael Peyton2, Inga Dunignan1, Jennifer Malaby1, Timothy Bailey1, Rolf A. Brekken2, Nick Loizos1. 1ImClone Systems, New York, NY; 2University of Texas Southwestern Medical Center, Dallas, TX. Platelet-derived growth factor receptor alpha (PDGFRα) is a type III receptor tyrosine kinase that is normally expressed on cells of mesenchymal origin (e.g. fibroblasts and smooth muscle cells) as well as on a variety of tumor types. In lung cancer, PDGFRα is expressed frequently by tumor associated stromal cells and, in a subset of tumors, by cancer cells themselves. The anti-human PDGFRα mAb, IMC-3G3, blocks the binding of PDGF-AA, -BB & - CC to PDGFRα, and was previously shown to inhibit tumor growth of glioblastoma, prostate, and sarcoma xenografts in mice. In our analysis, IMC-3G3 administered to mice 2× per week at 40 mg/kg significantly inhibited the growth of human H1703 non-small cell lung cancer (NSCLC) tumors with a T/C value of 35%. The PDGFRα-positive H1703 lung cancer line contains both PDGFRα and PDGF-CC gene amplifications and shows a co-dependency on this axis for proliferation. Co-dependence of this kind is a rare event in human lung cancer cell lines, suggesting that only a limited number of lung tumor patients might benefit from PDGFRα inhibition. However, this experimental system does not account for the potential therapeutic effects of stromal PDGFRα inhibition. In tumor stroma, the PDGF-PDGFRα axis functions in fibroblast activation, modulation of tumor interstitial pressure, and production and secretion of vascular endothelial growth factor (VEGF). To target stromal PDGFRα, an anti-mouse PDGFRα mAb (1E10) was generated and shown to inhibit PDGF-AA from binding to murine PDGFRα with an IC50 of 8.51 × 10−9M. MAb 1E10 inhibited PDGF-AA-induced phosphorylation of mouse PDGFRα but not human PDGFRα in cell-based assays; thus demonstrating the species specificity of 1E10. Tumor xenograft studies were performed using the PDGFRα-negative NSCLC cell lines Calu-6, H1993, and A549. MAb 1E10 treatment attenuated the tumor growth of A549 and Calu-6 xenografts in nude mice with T/C values of 67% and 51%, respectfully. In Calu-6 xenografts, the combination of 1E10 plus an anti-VEGF antibody (S12) significantly inhibited tumor growth compared to control. Microvessel density was significantly decreased by both 1E10 and S12 given alone or in combination. MAb 1E10 enhanced the anti-tumor activity of cisplatin-gemcitabine chemotherapy in A549 xenografts. However, MAb 1E10 monotherapy or when combined with cisplatin-gemcitabline chemotherapy showed no anti-tumor effect on H1993 xenograft growth. PDGF-AA and VEGF-A levels were determined in cell lines resistant and sensitive to 1E10 in vivo treatment. Elevated cancer cell expression of PDGF-AA and low expression of VEGF were associated with response to stromal PDGFRα targeting. Specifically, sensitive cell lines had relatively low VEGF-A expression (VEGF-A/PDGF-AA ratio 1.96 in Calu-6 cells and 10.87 in A549 cells), while the resistant cell line H1993 had elevated VEGF-A (VEGF-A/PDGF-AA ratio 65.86) when grown in cell culture. This difference may suggest that the 1E10-sensitive tumors have a relatively greater dependence on PDGF-AA-induced production of stromal VEGF. Therefore, inhibition of stromal PDGFRα represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression. 286 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C56.

Chemokines are small, secreted peptides that control the migration of leukocytes along a chemical gradient of ligand, especially during immune and inflammatory reactions. They are divided into two major subfamilies, CC and CXC, based on the position of their NH2-terminal cysteine residues, and bind to G protein coupled receptors, whose two major sub families are designated CCR and CXCR. More than 50 human chemokines and 18 chemokine receptors have been discovered so far. Chemokine receptor CXCR4/[Stromal cell-derived factor (SDF)-1] axis plays a central role in various physiological and pathological processes, including cancer. CXCR4 is over-expressed in a large number of tumors: colon, breast, prostate, lung, ovary, pancreas. CXCR4/SDF-1 axis is directly implicated in migration, invasion leading to metastases, cell proliferation and angiogenesis. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. A novel monoclonal antibody (Mab 515H7) was raised against the human CXCR4. It displayed efficacious antagonist properties for all major pathways associated with SDF-1-induced CXCR4 signaling in vitro. It was able to efficiently bind CXCR4 on both transfected cells and human tumor cell lines. It was found to strongly inhibit SDF-1 binding, Gα protein activation and beta-arrestin2 recruitment. It also blocked second messenger release (calcium, cAMP) and constrained by itself the formation of CXCR4 homodimers. It also inhibits SDF-1-induced cell migration in vitro. 515H7 Mab antitumor activities were also investigated in vivo using several human tumor models. We demonstrated that 515H7 Mab was able to significantly inhibit growth of xenograft tumors in mice. In addition to its effect on tumor growth, 515H7 Mab was also able to significantly improve mice survival in the lethal U937 model. The herewith data demonstrate that Mab 515H7 behaves as a potent and efficacious antagonist of all major CXCR4-controlled signaling pathways and suggest that targeting CXCR4 is a promising way for the treatment of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4571. doi:10.1158/1538-7445.AM2011-4571

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 described h7C10 (also named MK-0646), a humanized anti-IGF-1R Mab, that exhibits a potent anti-tumor activity both in vitro and in vivo. It blocks IGF-1 and IGF-2 induced signaling and blocks in vivo tumor growth of IGF-IR expressing tumors. In this report, we examined the in vitro effects of this selective IGF-1R Mab in combination with a panel of chemotherapeutic agents. The effect of drugs and h7C10 on the growth of human breast cancer cell line MCF-7 was determined by 3H-thymidine incorporation in DNA. The combined effect of drugs and h7C10 on MCF-7 cell line was evaluated by the combination index (CI) analysis method described by Chou and Talalay. We showed that the combination of h7C10 with a panel of drugs namely, paclitaxel, docetaxel, doxorubicin and gemcitabine induced synergistic inhibitory effects on MCF-7 breast cancer cell proliferation in vitro. These findings demonstrated that h7C10 combined with established cancer therapeutics is highly effective against cancer cells that expressed IGF-1R and support clinical evaluation of h7C10 in combination with these drugs in the treatment of human cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B131.