With more than 20 molecules in clinical use, monoclonal antibodies have finally come of age as therapeutics, generating a market value of $11 billion in 2004, expected to reach $26 billion by 2010. While delivering interesting results in the treatment of several major diseases including autoimmune, cardiovascular and infectious diseases, cancer and inflammation, clinical trials and research are generating a wealth of useful information, for instance about associations of clinical responses with Fc receptor polymorphisms and the infiltration and recruitment of effector cells into targeted tissues. Some functional limitations of therapeutic antibodies have come to light such as inadequate pharmacokinetics and tissue accessibility as well as impaired interactions with the immune system, and these deficiencies point to areas where additional research is needed. This review aims at giving an overview of the current state of the art and describes the most promising avenues that are being followed to create the next generation of antibody-based therapeutic agents.

This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009

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Therapeutic antibodies: successes, limitations and hopes for the future.

Formulation and delivery issues for monoclonal antibody therapeutics

In the year 2003 there was a 17% increase in the number of publications citing work performed using optical biosensor technology compared with the previous year. We collated the 962 total papers for 2003, identified the geographical regions where the work was performed, highlighted the instrument types on which it was carried out, and segregated the papers by biological system. In this overview, we spotlight 13 papers that should be on everyone's 'must read' list for 2003 and provide examples of how to identify and interpret high-quality biosensor data. Although we still find that the literature is replete with poorly performed experiments, over-interpreted results and a general lack of understanding of data analysis, we are optimistic that these shortcomings will be addressed as biosensor technology continues to mature.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jmr.753

Survey of the year 2003 commercial optical biosensor literature

Heterogeneity of Monoclonal Antibodies

Summary
A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of β1,2-xylosylation and core α1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous β1,2-xylosyltransferase (XylT) and α1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core α1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core α1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and α1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.

Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure.

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor

The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.

Production of human monoclonal antibody in eggs of chimeric chickens


 Mutations in the KRAS gene are among the most common driver mutations in cancer. Targeting KRAS G12C mutations has shown some promise in clinical studies, though acquired resistance mutations pose a challenge for small molecule-based monotherapies. Vaccines targeting KRAS mutations remain highly desirable for durable tumor control and clinical benefit to patients with solid tumors. While cytotoxic CD8 T cells are critical to tumor control and clearance, CD4 T cell activation is a key component of a durable anti-tumor response. Cancer neoepitopes with homology to infectious diseases antigens (mimotopes) were associated with long-term clinical benefit following CTLA-4 blockade. In this study, we screened healthy donor PBMC samples via ex vivo interferon-gamma (IFNγ) ELISpot and identified KRAS G12C-specific T cell responses associated with cross-reactive TCRs recognizing a bacterial lipoprotein peptide sequence with homology to KRAS G12C. Healthy donors with T cell responses to both KRAS G12C and mimotope peptides shared common HLA-DR alleles, and KRAS G12C-specific T cell responses were CD4 dependent via depletion assays. KRAS G12C-reactive CD4 healthy donor T cells were polyfunctional as assessed by intracellular cytokine staining, and killing of class II target cells presenting G12C peptides assessed by Incucyte assay was CD4-dependent. We further identified vaccine-induced KRAS G12C specific CD4 T cell responses in a patient with KRAS G12C-positive non-small cell lung cancer (NSCLC) receiving a shared neoantigen-targeting cancer vaccine consisting of Chimpanzee adenovirus prime and self-amplifying mRNA (samRNA) boost vaccinations in combination with nivolumab 480 mg IV (NCT03953235). This patient had previously progressed on prior checkpoint inhibitor therapy and showed signs of clinical benefit with molecular response (e.g., ctDNA reduction) and tumor shrinkage following vaccination. Patient PBMCs were analyzed by ex vivo IFNγ ELISpot, and G12C-specific T cell responses were detectable at baseline and increased following vaccination. T cell receptor (TCR) and transcriptome single cell sequencing analyses showed a large clonal population of CD4 effector memory cells in the post-vaccination samples. Functional analyses of patient-derived TCR clonotypes from post-vaccination samples via recombinant TCR screening assays identified two functional clonotypes recognizing KRAS G12C epitopes in the context of HLA-DR on target cells. Studies to further elucidate KRAS G12C vaccine-induced TCR functionality in primary cells and transgenic mouse models are ongoing. This study provides important insight into both naturally occurring bacterial mimotope-based and vaccine-induced T cell responses against KRAS G12C neoepitopes that informs future therapeutic approaches targeting KRAS G12C tumors.
 Citation Format: Christine D. Palmer, Meghan G. Hart, Sonia Kounlavouth, Harshni Venkatraman, Lauren D. Kraemer, Martina Marrali, Calixto Dominguez, Fatema Z. Chowdhury, Jason R. Jaroslavsky, Lindsey Arcebuche, Lorenzo Hernandez, Bukola Adeoye, Severino Cuison, Amy R. Rappaport, Greg Boucher, Monica Lane, Melissa Rotunno, Kenneth Avocetien, Leiliane Sousa, Chris Puccia, Molly Likes, Rahul Vegesna, Rita Zhou, Alexis Mantilla, Matthew J. Davis, Ankur Dhanik, Melissa Johnson, Andrew R. Ferguson, Karin Jooss. HLA-DR-restricted CD4 T cell responses to KRAS G12C in healthy donors linked to bacterial mimotope: lessons for KRAS neoantigen vaccines in cancer patients [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 2267.

Abstract 2267: HLA-DR-restricted CD4 T cell responses to KRAS G12C in healthy donors linked to bacterial mimotope: lessons for KRAS neoantigen vaccines in cancer patients

Severino Cuison

Papers

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor

Production of human monoclonal antibody in eggs of chimeric chickens

Abstract 2267: HLA-DR-restricted CD4 T cell responses to KRAS G12C in healthy donors linked to bacterial mimotope: lessons for KRAS neoantigen vaccines in cancer patients