Next Generation Of Therapeutic Antibodies Research Articles

Next-Generation Therapeutic Antibodies for Cancer Treatment: Advancements, Applications, and Challenges.

The field of cancer treatment has evolved significantly over the last decade with the emergence of next-generation therapeutic antibodies. Conventional treatments like chemotherapy pose significant challenges, including adverse side effects. Monoclonal antibodies have paved the way for more targeted and effective interventions. The evolution from chimeric to humanized and fully human antibodies has led to a reduction in immunogenicity and enhanced tolerance in vivo. The advent of next-generation antibodies, including bispecific antibodies, nanobodies, antibody-drug conjugates, glyco-engineered antibodies, and antibody fragments, represents a leap forward in cancer therapy. These innovations offer increased potency, adaptability, and reduced drug resistance. Challenges such as target validation, immunogenicity, and high production costs exist. However, technological advancements in antibody engineering techniques provide optimism for addressing these issues. The future promises a paradigm shift, where ongoing research will propel these powerful antibodies to the forefront, revolutionizing the fight against cancer and creating new preventive and curative treatments. This review provides an overview of three next-generation antibody-based molecules, namely bispecific antibodies, antibody-drug conjugates, and nanobodies that have shown promising results in cancer treatment. It discusses the evolution of antibodies from conventional forms to next-generation molecules, along with their applications in cancer treatment, production methods, and associated challenges. The review aims to offer researchers insights into the evolving landscape of next-generation antibody-based cancer therapeutics and their potential to revolutionize treatment strategies.

Molecular basis for antibody recognition of multiple drug–peptide/MHC complexes

The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.

Abstract 2925: In vitro characterization of next generation therapeutic antibodies

Abstract Therapeutic antibodies are an established drug class. Checkpoint inhibitors, such as anti-PD-1/PD-L1 and anti-CTLA-4 antibodies for cancer indications, and anti-TNF-α monoclonal antibody against autoimmune disorders represent some of most successful therapeutics in the past decade. Over 100 therapeutic targets suitable for antibody therapy have been identified for various disease indications, with several currently in late-stage clinical trials. In addition, there has been an explosion in the various types of therapeutic antibody modalities due to the increasing number of antibody engineering platforms. The new emerging bi-specific T-cell engaging antibodies (BiTEs) along with antibody-drug conjugates (ADCs) have demonstrated superior therapeutic qualities over the traditional monoclonal antibodies. Here, we describe a pipeline to extensively characterize the various antibody therapeutics in vitro utilizing multiple innovative technologies. Assays to determine target expression and binding potency provide a thorough understanding of the target engagement. Furthermore, we present data from an innovative technology platform, the z-Movi, which measures cell avidity to predict invitro effector functions of T-cell engaging therapies, enabling more robust read-outs and could drive the lead selection of said therapies. Several mechanisms of action of different antibodies were investigated and relevant case studies describing the innate immune effector functions and the signaling pathway modulation are presented. Using a unique label-free approach, the xCELLigence, we report on the kinetics of target cell killing mediated by lymphocytes upon recognition of antigen-antibody complexes. Finally, safety assessment studies focusing on on-target/off-tumor effects of antibodies on primary cells were conducted. The cytokine profiling and cell viability assays to assess potential risk due to exaggerated levels of cytokine, reiterate the specificity and safety of the next generation of antibody therapeutics and support Investigational New Drug (IND) applications. In conclusion, we provide a comprehensive report on the in vitro characterization of the advanced antibody therapeutics, showcasing a pipeline for development and functional characterization of various antibody modalities, including bispecific T-cell engagers. This workflow can be used to progress a wide range of engineered antibodies from pre-clinical to the clinical phase of the drug discovery process. NJ and GM contributed equally to the work. Citation Format: Benita Quist, Hamed Janbazacyabar, David Cobeta Lopez, Namrata Jayanth, Gemma Moiset. In vitro characterization of next generation therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2925.

Production of afucosylated antibodies in CHO cells by coexpression of an anti-FUT8 intrabody.

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.

Engineered mRNA and the Rise of Next-Generation Antibodies.

Monoclonal antibodies are widely used as therapeutic agents in medicine. However, clinical-grade proteins require sophisticated technologies and are extremely expensive to produce, resulting in long lead times and high costs. The use of gene transfer methods for in vivo secretion of therapeutic antibodies could circumvent problems related to large-scale production and purification and offer additional benefits by achieving sustained concentrations of therapeutic antibodies, which is particularly relevant to short-lived antibody fragments and next-generation, Fc-free, multispecific antibodies. In recent years, the use of engineered mRNA-based gene delivery has significantly increased in different therapeutic areas because of the advantages it possesses over traditional gene delivery platforms. The application of synthetic mRNA will allow for the avoidance of manufacturing problems associated with recombinant proteins and could be instrumental in consolidating regulatory approvals for next-generation therapeutic antibodies.

A general Fc engineering platform for the next generation of antibody therapeutics.

Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform.Methods: By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8+ T cell response model in FcγR/CD40-humanized mice.Results: Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the in vitro and in vivo models.Conclusions: This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics.

An IgA mimicry of IgG that binds Polymeric Immunoglobulin Receptor for mucosa transcytosis.

Most pathogens establish infection through mucosa, where secretory immunoglobulin A (sIgA) plays an ‘immune exclusion’ role in humoral defense. Extravasation of intravenously (i.v.) administrated therapeutic immunoglobulin G (IgG) mainly relies on convection and/or neonatal Fc receptor-mediated transcytosis from circulation into interstitial space. Active transport of interstitial IgG further across epithelium into mucosa, like sIgA, is a much desired feature for the next generation of therapeutic antibodies, especially for anti-infection purposes. For the first time, we report the engineering of an IgA mimicry of IgG, with its Fc portion in fusion with the 18-aa tail piece (tp) of sIgA and the J chain, possessing sIgA’s full binding activity towards polymeric immunoglobulin receptor that mediates mucosa transcytosis. In a diphtheria toxin receptor (DTR) knockin mouse model, i.v. injected anti-diphtheria toxin (DT) IgG(tp)J protected DTR+ cells from deletion upon DT injection. The compact design of IgG(tp)J opens new revenues for more effective therapeutic IgG mimicking some of the important biological functions of IgA.

Development of Antibody-Drug Conjugates Using DDS and Molecular Imaging.

Antibody-drug conjugate (ADC), as a next generation of antibody therapeutics, is a combination of an antibody and a drug connected via a specialized linker. ADC has four action steps: systemic circulation, the enhanced permeability and retention (EPR) effect, penetration within the tumor tissue, and action on cells, such as through drug delivery system (DDS) drugs. An antibody with a size of about 10 nm has the same capacity for passive targeting as some DDS carriers, depending on the EPR effect. In addition, some antibodies are capable of active targeting. A linker is stable in the bloodstream but should release drugs efficiently in the tumor cells or their microenvironment. Thus, the linker technology is actually a typical controlled release technology in DDS. Here, we focused on molecular imaging. Fluorescent and positron emission tomography (PET) imaging is useful for the visualization and evaluation of antibody delivery in terms of passive and active targeting in the systemic circulation and in tumors. To evaluate the controlled release of the ADC in the targeted area, a mass spectrometry imaging (MSI) with a mass microscope, to visualize the drug released from ADC, was used. As a result, we succeeded in confirming the significant anti-tumor activity of anti-fibrin, or anti-tissue factor-ADC, in preclinical settings by using DDS and molecular imaging.

Next Generation Antibody Therapeutics Using Bispecific Antibody Technology.

Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

Therapeutic Antibodies by Phage Display.

Antibody phage display is a major technological platform for the generation of fully human antibodies for therapeutic purposes. The in vitro binder selection by phage display allows researchers to have more extensive control over binding parameters and facilitates the isolation of clinical candidate antibodies with desired binding and/or functional profiles. Since the invention of antibody phage display in late 1980s, significant technological advancements in the design, construction, and selection of the antibody libraries have been made, and several fully human antibodies generated by phage display are currently approved or in various clinical development stages. In this review, the background and details of antibody phage display technology, and representative antibody libraries with natural or synthetic sequence diversity and different construction strategies are described. The generation, optimization, functional and biophysical properties, and preclinical and clinical developments of some of the phage display-derived therapeutic antibodies approved for use in patients or in late-stage clinical trials are also discussed. With evolving novel disease targets and therapeutic strategies, antibody phage display is expected to continue to play a central role in the development of the next generation of therapeutic antibodies.

Cetuximab Resistance in Squamous Carcinomas of the Upper Aerodigestive Tract Is Driven by Receptor Tyrosine Kinase Plasticity: Potential for mAb Mixtures.

Squamous cell carcinomas (SCC) arising in upper parts of the aerodigestive tract are among the leading causes of death worldwide. EGFR has been found to play an essential role in driving the malignancy of SCC of the upper aerodigestive tract (SCCUAT), but, despite this, clinical results using a range of different EGFR-targeted agents have been disappointing. Cetuximab is currently the only EGFR-targeted agent approved by the FDA for treatment of SCCUAT. However, intrinsic and acquired cetuximab resistance is a major problem for effective therapy. Thus, a better understanding of the mechanisms responsible for cetuximab resistance is valuable for development of the next generation of antibody therapeutics. In order to better understand the underlying mechanisms of cetuximab resistance in SCCUAT, we established from cetuximab-sensitive models cell lines with acquired resistance to cetuximab by continuous selective pressure in vitro and in vivo Our results show that resistant clones maintain partial dependency on EGFR and that receptor tyrosine kinase plasticity mediated by HER3 and IGF1R plays an essential role. A multitarget mAb mixture against EGFR, HER3, and IGF1R was able to overcome cetuximab resistance in vitro To our surprise, these findings could be extended to include SCCUAT cell lines with intrinsic resistance to cetuximab, suggesting that the triad consisting of EGFR, HER3, and IGF1R plays a key role in SCCUAT. Our results thus provide a rationale for simultaneous targeting of EGFR, HER3, and IGF1R in SCCUAT. Mol Cancer Ther; 15(7); 1614-26. ©2016 AACR.

Structural analysis of Fc/FcγR complexes: a blueprint for antibody design.

The number of studies and the quality of the structural data of Fcγ receptors (FcγRs) has rapidly increased in the last few years. Upon critical examination of the literature, we have extracted general conclusions that could explain differences in affinity and selectivity of FcγRs for immunoglobulin G (IgG) based on structural considerations. FcγRs employ a little conserved asymmetric surface of domain D2 composed of two distinct subsites to recognize the well-conserved lower hinge region of IgG1-Fc. The extent of the contact interface with the antibody in subsite 1 of the receptor (but not in subsite 2), the geometrical complementarity between antibody and receptor, and the number of polar interactions contribute decisively toward strengthening the binding affinity of the antibody for the receptor. In addition, the uncertain role of the N-linked glycan of IgG for the binding and effector responses elicited by FcγRs is discussed. The available data suggest that not only the non-covalent interactions between IgG and FcγRs but also their dynamic features are essential for the immune response elicited through these receptors. We believe that the integration of structural, thermodynamic, and kinetic data will be critical for the design and validation of the next generation of therapeutic antibodies with enhanced effector capabilities.

Abstract 3585: HER3 and IGF1R are major mediators of both acquired and intrinsic cetuximab resistance in head and neck squamous cell carcinomas

Abstract Head and neck squamous cell carcinoma (HNSCC) is among the leading causes of death worldwide. HNSCC originates from the squamous epithelium of the upper aerodigestive tract, including the lip, oral cavity, pharynx, larynx and paranasal sinuses. The epidermal growth factor receptor (EGFR) has been found to play an essential role in driving the malignancy of HNSCC. The monoclonal antibody targeting EGFR, cetuximab, has been approved for clinical use in HNSCC patients, in combination with either radiation or chemotherapy. However, intrinsic and acquired cetuximab resistance is a major problem for effective therapy. Thus, a better understanding of the mechanisms responsible for cetuximab resistance is valuable for development of the next generation of antibody therapeutics. In order to investigate the molecular mechanisms behind cetuximab resistance in HNSCC, we screened a panel of cell lines for sensitivity to cetuximab. As expected, the cell lines exhibited a varied response to cetuximab. The most sensitive cell line, HN5, was used to establish a number of cetuximab-resistant clones. These resistant clones had lower EGFR levels but remained partially dependent on EGFR, as demonstrated by EGFR gene knockdown and treatment with a mixture of two anti-EGFR antibodies. HER3 and IGF1R are known to be major drivers of resistance to EGFR targeting therapies, and therefore we investigated the role of the two receptors in resistant clones with a mixture of antibodies against EGFR, HER3 and IGF1R. The antibody mixture, which simultaneously blocked EGFR, HER3 and IGF1R signaling, resulted in near total growth inhibition of cetuximab resistant clones. These results were extended to HNSCC cell lines with intrinsic resistance to cetuximab and also in these cell lines the three target mAb mixture led to effective growth inhibition. In conclusion, our results demonstrate that HNSCC cell lines have a heterogeneous response to cetuximab and that HER3 and IGF1R effectively compensate for EGFR inhibition in both the acquired and intrinsic cetuximab resistant setting. Our results thus provide a rationale for simultaneous targeting of EGFR, HER3, and IGF1R in HNSCC. Citation Format: Ida Kjaer, Trine Lindsted, Camilla Fröhlich, Ivan D. Horak, Michael Kragh, Jesper V. Olsen, Mikkel W. Pedersen. HER3 and IGF1R are major mediators of both acquired and intrinsic cetuximab resistance in head and neck squamous cell carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3585. doi:10.1158/1538-7445.AM2015-3585

Functionalization of Bispecific Therapeutic Antibodies Based on Protein Engineering

Although antibodies have been used as molecularly targeted agents for difficult-to-treat diseases such as cancers, the high production costs associated with mammalian expression systems continue to be a drawback. In addition, the clinical efficacy of conventional IgG antibodies is limited. Several types of recombinant antibody (e.g. fused with anticancer drugs, multivalent, or multispecific) have been designed in efforts to develop next-generation antibodies with higher functionality. We used protein engineering to construct several anticancer recombinant antibodies by developing bispecific antibodies that induced specific antitumor effects against cancer cells through the recruitment of lymphocytes. We found that a humanized small bispecific antibody (Ex3) that targets epidermal growth factor receptor on tumor cells and CD3 on T lymphocytes had marked anticancer activity. Furthermore, the function of Ex3 was enhanced by fusion with the human Fc region, domain rearrangement, multimerization, and affinity maturation; a combination of these modifications showed at least additive cytotoxic effects. Interestingly, merely rearranging the domain order of Ex3 induced substantial cytotoxic enhancements, even though the structural format remained the same. Here, we describe our efforts to develop highly functional bispecific antibodies as next-generation therapeutic antibodies using protein engineering.

Asymmetrical Fc Engineering Greatly Enhances Antibody-dependent Cellular Cytotoxicity (ADCC) Effector Function and Stability of the Modified Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

Antibody–Drug Conjugate Drug Development: Where are The Challenges?

BioanalysisVol. 5, No. 12 EditorialAntibody–drug conjugate drug development: where are the challenges?Laura Wright, Sharon Baughman & Ashleigh WakeLaura Wright* Author for correspondenceIntertek Pharmaceutical Services, 3985 Sorrento Valley Blvd Suite C, San Diego, CA 92121, USA. Search for more papers by this authorEmail the corresponding author at laura.wright@intertek.com, Sharon BaughmanProPharma Services, LLC, 1050 Driftwood Lane, Ventura, CA 93001, USASearch for more papers by this author & Ashleigh WakeIntertek Pharmaceutical Services, Hexagon Tower, PO Box 42, Crumpsall Vale, BlackleyManchester, M9 8ZS, UKSearch for more papers by this authorPublished Online:24 Jun 2013https://doi.org/10.4155/bio.13.104AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: antibody–drug conjugatebiopharmaceuticalsdrug-to-antibody ratioPKReferences1 Shapiro M, Chen X. Regulatory considerations when developing assays for the characterization and quality control of antibody–drug conjugates. American Laboratory, 27 August (2012).Google Scholar2 US FDA. ICH Guideline Q11 on Development and Manufacture of Drug Substances (Chemical Entities and Biotechnological/Biological Entities). US FDA, Washington, DC, USA (2012).Google Scholar3 European Medicines Agency. Guideline on Development, Production, Characterization and Specifications For Monoclonal Antibodies And Related Products, EMEA/CHMP/BWP/157653/2007. European Medicines Agency, London, UK (2008).Google Scholar4 Wakankar A, Chen Y, Gokarn Y, Jacobson FS. Analytical methods for physiochemical characterization of antibody drug conjugates. mAbs3(2),161–172 (2011).Crossref, Medline, Google Scholar5 Beck A, Wurch T, Bailly C, Corvaia N. Strategies and challenges for the next generation of therapeutic antibodies. Nat. Rev. Immunol.10(5),345–352 (2010).Crossref, Medline, CAS, Google Scholar6 Sanderson RJ, Hering MA, James SF et al.In vivo drug–linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin. Cancer Res.11,843 (2005).Medline, CAS, Google Scholar7 Xiao Y, Halford A, Hayes R. Meeting challenges for analysis of antibody–drug conjugates. BioPharm Int.25(10),60–63 (2012).CAS, Google Scholar8 Kaur S, Xu K, Saad O, Dere R, Carrasco-Triguero M. Bioanalytical assay strategies for the development of antibody–drug conjugate biotherapuetics. Bioanalysis5(2),201–206 (2013).Link, CAS, Google Scholar101 Wyatt Technology. Technical note: antibody drug conjugate analysis (ADC). www.wyatt.com/files/literature/Antibody_Drug_Conjugate_(ADC)_Analysis.pdfGoogle ScholarFiguresReferencesRelatedDetails Vol. 5, No. 12 Follow us on social media for the latest updates Metrics Downloaded 641 times History Published online 24 June 2013 Published in print June 2013 Information© Future Science LtdKeywordsantibody–drug conjugatebiopharmaceuticalsdrug-to-antibody ratioPKFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

SAT0129 Incidence and clinical outcome of anti-drug antibodies in infliximab-treated patients

BackgroundInfliximab (chimeric monoclonal antibody against TNF-α) reduces the severity of symptoms and induces remission of active disease. However, some patients may experience adverse drug reactions (ADR) or loss of efficacy...

Abstract 5647: Receptor tyrosine kinase plasticity as a mechanism of acquired resistance to cetuximab in vitro: potential for co-targeting with antibody mixtures.

Abstract Cetuximab is an EGFR-blocking antibody that belongs to the first generation of monoclonal antibodies approved for treatment of head and neck squamous cell carcinoma and metastatic colorectal cancer. Although anti-EGFR therapy is clinically successful, development of cetuximab resistance is an increasing problem in the clinic. Thus, a better understanding of the mechanisms responsible for development of cetuximab resistance is valuable for development of the next generation of antibody therapeutics. Here we report the establishment of a number of cetuximab-resistant clones derived from the cetuximab-sensitive head and neck squamous carcinoma cell line HN5. Initial characterization of the cetuximab-resistant cells shows that the total level of EGFR is lower compared to the parental cells, but that the cetuximab binding is unaltered. However, cetuximab resistant clones remain dependent on EGFR for growth and proliferation, as a mixture of antibodies targeting non-overlapping epitopes on EGFR is able to partially overcome the cetuximab-induced resistance. Inititial results indicate that the antibody mixture overcomes cetuximab resistances by inducing efficient EGFR internalization followed by lysosomal degradation of the receptor. Removal of EGFR from the cell surface blocks interactions of EGFR with other receptor tyrosine kinases (RTKs) and intracellular proteins as compensatory response to cetuximab inhibition. The observed level of inhibition of the resistant clones by the anti-EGFR antibody mixture did not, however, induce as efficient growth inhibition as in the parental cells, indicating additional resistance mechanisms. Indeed we were able to show that adding antibodies to other RTKs to the anti-EGFR antibody mixture restored sensitivity of the resistant cells to a level similar to parental cells. In conclusion, our results demonstrate that cetuximab resistant clones remain partially dependent on EGFR for growth and proliferation. Additional RTKs are activated as a compensatory response to the EGFR inhibition, demonstrating the plasticity of the RTK family. Our results provide a rationale for targeting multiple RTKs to overcome acquired resistance to currently approved mAbs or to delay the emerge of resistance if used upfront. An update will be presented. Citation Format: Ida Kjaer, Michael Kragh, Ivan D. Horak, Mikkel Wandahl Pedersen. Receptor tyrosine kinase plasticity as a mechanism of acquired resistance to cetuximab in vitro: potential for co-targeting with antibody mixtures. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5647. doi:10.1158/1538-7445.AM2013-5647

Generation and characterization of monospecific and bispecific hexavalent trimerbodies

Here, we describe a new class of multivalent and multispecific antibody-based reagents for therapy. The molecules, termed “trimerbodies,” use a modified version of the N-terminal trimerization region of human collagen XVIII noncollagenous 1 domain flanked by two flexible linkers as trimerizing scaffold. By fusing single-chain variable fragments (scFv) with the same or different specificity to both N- and C-terminus of the trimerizing scaffold domain, we produced monospecific or bispecific hexavalent molecules that were efficiently secreted as soluble proteins by transfected mammalian cells. A bispecific anti-laminin x anti-CD3 N-/C-trimerbody was found to be trimeric in solution, very efficient at recognizing purified plastic-immobilized laminin and CD3 expressed at the surface of T cells, and remarkably stable in human serum. The bispecificity was further demonstrated in T cell activation studies. In the presence of laminin-rich substrate, the bispecific anti-laminin x anti-CD3 N-/C-trimerbody stimulated a high percentage of human T cells to express surface activation markers. These results suggest that the trimerbody platform offers promising opportunities for the development of the next-generation therapeutic antibodies, i.e., multivalent and bispecific molecules with a format optimized for the desired pharmacokinetics and adapted to the pathological context.

Abstract 4562: Superior targeting of the human epidermal growth factor receptor 2 (HER-2) with recombinant monoclonal antibody mixtures

Abstract Human epidermal growth factor receptor 2 (HER2) is involved in development and maintenance of malignant phenotypes of several human cancers and therefore represent an attractive therapeutic target. Trastuzumab is currently the only anti-HER2 antibody approved for treatment of human cancers and although succesful more efficacious treatments are warranted. Mixtures of recombinant monoclonal antibodies are promising candidates as the next generation of antibody therapeutics due to their multipotent activities. The aim of the present study was to identify mixtures of anti-HER2 antibodies with superior activity to existing monoclonal antibodies. Anti-HER2 antibodies were raised in mice by immunizations with HER-2 in different antigen presenting formats. A large antibody repertoire consisting of approximately 150 unique anti-HER-2 antibodies was cloned from the mice using the mSymplex™ technology. Based on a thorough sequence and binding analysis, 40 antibodies were selected for functional evaluation. The 40 antibodies were tested as individual antibodies and in mixtures of two and three for the ability to inhibit the growth of four human cancer cell lines using a standard viability assay. In total, more than 1300 mixtures were evaluated for ability to inhibit growth of the four cell lines. The 20 mixtures with the highest levels of growth inhibition were further characterized with regard to potency (IC50) and ability to engage in ADCC and CDC. The results demonstrated that HER2 mixtures were superior to trastuzumab, pertuzumab and the mixture of the two at inhibiting the growth of seven of the 10 cell lines investigated. Interestingly, the mixtures also inhibited the growth of the trastuzumab resistant breast cancer cell line HCC202, reflecting the differentiated mechanisms of anti-HER2 antibody mixtures. In vivo, anti-HER2 mixtures had superior activity compared to the monoclonal anti-HER2 antibody trastuzumab in two models of human gastric cancer. Based on these results, a candidate mixture was selected and referred to as Sym005. In conclusion, these data demonstrate that the novel antibody mixture Sym005 has a unique set of HER2 inhibitory mechanisms, which translates into superior anti-cancer activity in HER2-positive tumor xenografts. Furthermore, the results provide a clear rationale for evaluation of Sym005 in clinical trials on patients with HER2 positive 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 4562. doi:10.1158/1538-7445.AM2011-4562