Therapeutic Use Of Monoclonal Antibodies Research Articles

B-165 The EXENT System reliably distinguishes daratumumab from endogenous monoclonal immunoglobulins in multiple myeloma patients

Abstract Background The use of therapeutic monoclonal antibodies (t-mAbs) in the treatment of multiple myeloma (MM) has created challenges in the clinical laboratory. Co-migration of t-mAbs (predominantly IgGK) and the endogenous M-protein during serum protein electrophoresis can result in inability to distinguish between a patient’s M protein and the t-mAb. This can hinder accurate assessment of therapeutic responses or may lead to extensive workup with immunofixation electrophoresis and gel-shift assays for accurate confirmation of a complete response, or to exclude the emergence of a new clone. There is a need for an analytically specific routine method to identify t-mAb interference. MALDI-TOF mass spectrometry is an especially promising tool, as due to its ability to identify M proteins’ molecular mass, it has the potential to recognize any t-mAbs with known amino acid composition. We have used the EXENT® Immunoglobulin Isotypes (GAM) Assay for the EXENT Analyzer (The Binding Site, part of Thermo Fisher Scientific) to assess the reproducibility of daratumumab (anti-CD38, IgGK) detection in an analytical study, and assessed the EXENT System’s ability to detect daratumumab in a cohort of MM patients. Methods The expected mass to charge ratio (m/z) of the +2-charge state of daratumumab’s kappa light chain was calculated based on published information. Daratumumab was spiked into normal human serum at 1.0 g/L, and this material was tested in 5 replicates a day over 5 days. In addition, 109 samples from 34 MM patients receiving daratumumab, carfilzomib, lenalidomide and dexamethasone (Dara-KRd) treatment were also tested. Samples were obtained after 14 or 28 days following daratumumab administration in various treatment cycles, ranging from cycle 4 to end-of-treatment. Results The expected m/z for the +2-charge state of daratumumab’s kappa light chain was calculated as 11,691.01 to 11,693.1. In the reproducibility experiment, the median m/z was 11691.3, within the expected range. The difference between the lowest and highest m/z was 1.3 across the 25 replicates. Daratumumab was detected in 103 out of 109 samples (97.3%): 51/57 (89.5%) in IgGK samples, and in all IgGL (23) and IgA (29) samples. In two IgGK patients, where daratumumab was not detected in 2 out of 4 samples and in 1 out of 3 samples, respectively, the patients’ own M proteins were in the proximity (24.8 and 27.1 m/z) of daratumumab’s m/z. In the other three cases where daratumumab was not modeled by the algorithm, the daratumumab peak overlapped with the endogenous M-protein's adduct peak (estimated m/z 11,688) in one sample; it was masked by the shoulder of a large (8.42 g/L) IgGK M-protein in another sample; and no obvious reason was identified in the third sample. There was no difference in the detection rate between 14 days and 28 days post administration. Conclusions The EXENT Immunoglobulin Isotypes (GAM) Assay has successfully identified the presence of daratumumab in approximately 90% of IgGK patient samples in addition to all other (non-IgGK) MM samples. These results demonstrate the potential of the EXENT Immunoglobulin Isotypes (GAM) Assay for recognizing known t-mAb interference.

A nanowell platform to identify, sort and expand high antibody-producing cells

Increased use of therapeutic monoclonal antibodies and the relatively high manufacturing costs fuel the need for more efficient production methods. Here we introduce a novel, fast, robust, and safe isolation platform for screening and isolating antibody-producing cell lines using a nanowell chip and an innovative single-cell isolation method. An anti-Her2 antibody producing CHO cell pool was used as a model. The platform; (1) Assures the single-cell origin of the production clone, (2) Detects the antibody production of individual cells and (3) Isolates and expands the individual cells based on their antibody production. Using the nanowell platform we demonstrated an 1.8–4.5 increase in anti-Her2 production by CHO cells that were screened and isolated with the nanowell platform compared to CHO cells that were not screened. This increase was also shown in Fed-Batch cultures where selected high production clones showed titers of 19–100 mg/L on harvest day, while the low producer cells did not show any detectable anti-Her2 IgG production. The screening of thousands of single cells is performed under sterile conditions and the individual cells were cultured in buffers and reagents without animal components. The time required from seeding a single cell and measuring the antibody production to fully expanded clones with increased Her-2 production was 4–6 weeks.

Biodistribution of Drug/ADA Complexes: The Impact of Immune Complex Formation on Antibody Distribution

The clinical use of therapeutic monoclonal antibodies (mAbs) for the treatment of cancer, inflammation, and other indications has been successfully established. A critical aspect of drug-antibody pharmacokinetics is immunogenicity, which triggers an immune response via an anti-drug antibody (ADA) and forms drug/ADA immune complexes (ICs). As a consequence, there may be a reduced efficacy upon neutralization by ADA or an accelerated drug clearance. It is therefore important to understand immunogenicity in biological therapies. A drug-like immunoglobulin G (IgG) was radiolabeled with tritium, and ICs were formed using polyclonal ADA, directed against the complementary-determining region of the drug-IgG, to investigate in vivo biodistribution in rodents. It was demonstrated that 65% of the radioactive IC dose was excreted within the first 24 h, compared with only 6% in the control group who received non-complexed 3H-drug. Autoradiographic imaging at the early time point indicated a deposition of immune complexes in the liver, lung, and spleen indicated by an increased radioactivity signal. A biodistribution study confirmed the results and revealed further insights regarding excretion and plasma profiles. It is assumed that the immune complexes are readily taken up by the reticuloendothelial system. The ICs are degraded proteolytically, and the released radioactively labeled amino acids are redistributed throughout the body. These are mainly renally excreted as indicated by urine measurements or incorporated into protein synthesis. These biodistribution studies using tritium-labeled immune complexes described in this article underline the importance of understanding the immunogenicity induced by therapeutic proteins and the resulting influence on biological behavior.Graphical Created with BioRender.com

High throughput purification of monoclonal recombinant antibodies using a Protein-A coated membrane plate system

The therapeutic use of monoclonal antibodies (mAbs) ranges from cancer treatment to immune-mediated conditions, covering infectious and cardiovascular disorders, among others. The development of improved methods for therapeutic antibody discovery has accelerated the identification of numerous mAbs: a discovery campaign can be deeply mined, resulting in hundreds, even thousands, of potential antibody leads for a given target of interest. High throughput mAb expression and purification methods are required for the rapid validation of those leads. In this work, we describe the implementation of a Protein-A coated membrane plate system, the Purexa™ AHT membrane plate, for robust preparative purification of hundreds of recombinant mAbs, without the need for automation. The high efficiency (>80%) recovery generated sufficient mAb for downstream screening analyses such as ELISA and surface plasmon resonance (SPR). This new system allows the functional validation of hundreds of lead antibodies from discovery campaigns in a timely manner regardless of operational size.

Analysis of Adherence to anti-PCSK9 Antibody Therapy among Patients from Italy.

Hypercholesterolemia is one of the main risk factors associated with atherosclerotic cardiovascular disease and coronary heart disease. Statins are the standard cholesterollowering treatment; however, they have shown, in clinical practice, a reduced adherence to therapy (<50%) and a modest achievement of the expected outcomes for treatment. This condition prompt scientific research to develop drugs with different mechanisms of action. In this regard, excellent results have been achieved with therapeutic use of monoclonal antibodies against PCSK9, enzyme involved in recycling of Low density lipoprotein receptors (LDLR) on the hepatocytes surface. Indeed, the reduction in receptor density caused by PCSK9 is associated with increased serum LDL levels. After the data extraction of all Local Health Authority (ASL) of Foggia patients (302) who received, in 2021, at least one administration of Alirocumab or Evolocumab, the therapeutic adherence was calculated, for each individual patient, by indirect method (calculation of the Medication Possession Ratio - MPR). According to scientific literature, patients were classified into: adherents (MPR>80%), average adherents (MPR between 40% and 80%) and non-adherents (MPR<40%). Patients were then stratified by gender and age groups (0-18, 19-49, 50-64, >65). The results show that, for both drugs (Alirocumab and Evolocumab), women are more adherent than men and the group of young adults (19-49 years old) is the one with the lowest adherence to therapy, 69% for Alirocumab and 56% for Evolocumab. According to Italian Drug Agency (AIFA), poor therapeutic adherence is the main cause of ineffectiveness of drug therapies, and it is associated with increased hospitalizations, morbidity and mortality. Data obtained from this study allow to detect the categories of patients who need specific programs about the correct use of drugs, in order to increase therapeutic adherence and facilitate the achievement of the expected outcomes for treatment.

Activity of Sotrovimab against BQ.1.1 and XBB.1 Omicron sublineages in a hamster model

The successive emergence of SARS-CoV-2 Omicron variants has completely changed the modalities of use of therapeutic monoclonal antibodies. Recent in vitro studies indicated that only Sotrovimab has maintained partial activity against BQ.1.1 and XBB.1. In the present study, we used the hamster model to determine whether Sotrovimab retains antiviral activity against these Omicron variants in vivo. Our results show that at exposures consistent with those observed in humans, Sotrovimab remains active against BQ.1.1 and XBB.1, although for BQ.1.1 the efficacy is lower than that observed against the first globally dominant Omicron sublineages BA.1 and BA.2.

Integrating a new knowledge organisation system for monoclonal antibodies for therapeutic use authorised in Europe into HeTOP terminology-ontology server

Monoclonal antibodies (MAs) are increasingly used in the therapeutic arsenal. Clinical Data Warehouses (CDWs) offer unprecedented opportunities for research on real-word data. The objective of this work is to develop a knowledge organization system on MAs for therapeutic use (MATUs) applicable in Europe to query CDWs from a multi-terminology server (HeTOP). After expert consensus, three main health thesauri were selected: the MeSH thesaurus, the National Cancer Institute thesaurus (NCIt) and the SNOMED CT. These thesauri contain 1,723 MAs concepts, but only 99 (5.7 %) are identified as MATUs.The knowledge organisation system proposed in this article is a six-level hierarchical system according to their main therapeutic target. It includes 193 different concepts organised in a cross lingual terminology server, which will allow the inclusion of semantic extensions. Ninety nine (51.3 %) MATUs concepts and 94 (48.7 %) hierarchical concepts composed the knowledge organisation system. Two separates groups (an expert group and a validation group) carried out the selection, creation and validation processes.Queries identify, for unstructured data, 83 out of 99 (83.8 %) MATUs corresponding to 45,262 patients, 347,035 hospital stays and 427,544 health documents, and for structured data, 61 out of 99 (61.6 %) MATUs corresponding to 9,218 patients, 59,643 hospital stays and 104,737 hospital prescriptions. The volume of data in the CDW demonstrated the potential for using these data in clinical research, although not all MATUs are present in the CDW (16 missing for unstructured data and 38 for structured data).The knowledge organisation system proposed here improves the understanding of MATUs, the quality of queries and helps clinical researchers retrieve relevant medical information. The use of this model in CDW allows for the rapid identification of a large number of patients and health documents, either directly by a MATU of interest (e.g. Rituximab) but also by searching for parent concepts (e.g. Anti-CD20 Monoclonal Antibody).

Antigenic Characterization of Human Monoclonal Antibodies for Therapeutic Use against H7N9 Avian Influenza Virus.

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14, and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications. IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

Logic-gated antibody pairs that selectively act on cells co-expressing two antigens

The use of therapeutic monoclonal antibodies is constrained because single antigen targets often do not provide sufficient selectivity to distinguish diseased from healthy tissues. We present HexElect®, an approach to enhance the functional selectivity of therapeutic antibodies by making their activity dependent on clustering after binding to two different antigens expressed on the same target cell. lmmunoglobulin G (lgG)-mediated clustering of membrane receptors naturally occurs on cell surfaces to trigger complement- or cell-mediated effector functions or to initiate intracellular signaling. We engineer the Fc domains of two different lgG antibodies to suppress their individual homo-oligomerization while promoting their pairwise hetero-oligomerization after binding co-expressed antigens. We show that recruitment of complement component C1q to these hetero-oligomers leads to clustering-dependent activation of effector functions such as complement mediated killing of target cells or activation of cell surface receptors. HexElect allows selective antibody activity on target cells expressing unique, potentially unexplored combinations of surface antigens.

Multi-Terminology Thesaurus for Monoclonal Antibodies for Therapeutic Use in Europe: Facilitating Queries within Clinical Data Warehouses

Multi-Terminology Thesaurus for Monoclonal Antibodies for Therapeutic Use in Europe: Facilitating Queries within Clinical Data Warehouses

Glycan Profile Analysis of Engineered Trastuzumab with Rationally Added Glycosylation Sequons Presents Significantly Increased Glycan Complexity.

Protein aggregation constitutes a recurring complication in the manufacture and clinical use of therapeutic monoclonal antibodies (mAb) and mAb derivatives. Antibody aggregates can reduce production yield, cause immunogenic reactions, decrease the shelf-life of the pharmaceutical product and impair the capacity of the antibody monomer to bind to its cognate antigen. A common strategy to tackle protein aggregation involves the identification of surface-exposed aggregation-prone regions (APR) for replacement through protein engineering. It was shown that the insertion of N-glycosylation sequons on amino acids proximal to an aggregation-prone region can increase the physical stability of the protein by shielding the APR, thus preventing self-association of antibody monomers. We recently implemented this approach in the Fab region of full-size adalimumab and demonstrated that the thermodynamic stability of the Fab domain increases upon N-glycosite addition. Previous experimental data reported for this technique have lacked appropriate confirmation of glycan occupancy and structural characterization of the ensuing glycan profile. Herein, we mutated previously identified candidate positions on the Fab domain of Trastuzumab and employed tandem mass spectrometry to confirm attachment and obtain a detailed N-glycosylation profile of the mutants. The Trastuzumab glycomutants displayed a glycan profile with significantly higher structural heterogeneity compared to the HEK Trastuzumab antibody, which contains a single N-glycosylation site per heavy chain located in the CH2 domain of the Fc region. These findings suggest that Fab N-glycosites have higher accessibility to enzymes responsible for glycan maturation. Further, we have studied effects on additional glycosylation on protein stability via accelerated studies by following protein folding and aggregation propensities and observed that additional glycosylation indeed enhances physical stability and prevent protein aggregation. Our findings shed light into mAb glycobiology and potential implications in the application of this technique for the development of “biobetter” antibodies.

In vitro evaluation of potential interference of lokivetmab with protein electrophoresis and immunofixation.

In humans, misdiagnoses of monoclonal gammopathy after use of therapeutic monoclonal antibodies has been documented. This triggers concerns for similar misdiagnoses in animals treated with monoclonal antibodies. The aim of this study was to evaluate if lokivetmab interferes with serum protein electrophoresis and immunofixation electrophoresis in dogs. Residual sera from 25 client-owned, healthy blood donor dogs from 2 veterinary hospitals in Germany were used. The residual sera were analysed with serum protein electrophoresis and immunofixation electrophoresis before and after being spiked with lokivetmab at a concentration of 10 µg/ml (corresponding to the mean peak serum concentration after a subcutaneous injection of 2 mg/kg lokivetmab). No monoclonal gammopathy was observed on serum protein electrophoresis and all proteins had a normal distribution pattern without any pathologic bands on immunofixation electrophoresis. The absolute γ-globulin values of spiked samples, however, were significantly higher than in the native sera although they remained within the reference interval. No other globulin fractions were significantly different. This study suggests that lokivetmab at a dose of 2 mg/kg is not detected as a monoclonal peak on serum protein electrophoresis or immunofixation electrophoresis, and thus is unlikely to lead to a misdiagnosis of other diseases that are characterised by monoclonal gammopathies.

Abstract 543: Monoclonal antibodies against thyimidine kinase 1 for the immunotargeting of lung and breast cancer cells, a preclinical evaluation

Abstract This study explores the potential therapeutic use of monoclonal antibodies against the tumor proliferation biomarker TK1 for the immunotargeting of lung and breast cancer cells. Recent clinical trials have led to the approval of monoclonal antibodies for the treatment of several solid tumors including lung and breast tumors. However, a common limitation that antibody-based therapies face is that a significant portion of the current tumor targets are expressed on normal tissues, thus creating off-target tumor effects. TK1 is a tumor proliferation biomarker that is up-regulated in malignant tissues. Recently, we have reported the expression of TK1 on the cell membrane of several cancer cell lines, mononuclear cells (MNC) from patients with leukemia and cells from breast and colon tumors. No membrane expression of TK1 was found on normal cells. To further explore the potential of TK1 as an immunotherapeutic target, we evaluated the capacity of a novel panel of anti TK1 antibodies to target TK1 on the cell membrane of lung and breast cancer cells and to elicit an antibody-dependent cell-mediated-cytotoxicity (ADCC) response in vitro. Antibodies previously developed in our lab were validated to specifically target six different regions of the TK1 molecule. Four different antibodies targeting three different regions in the TK1 molecule were selected for ADCC experiments. Nuclear restricted GFP versions of the NCI-H460 and MDA-MB-231 cell lines were utilized in conjunction with the real-time cell imaging system ImageXpress® Pico to measure the ADCC response. Mono nuclear cells (MNCs) from healthy donors were co-cultured with target cells and 10ug/ml of each anti-TK1 antibody were added to the media. An antibody with the same isotype than the TK1 antibodies, but that doesn't bind to any human surface antigen was utilized as a control. All ADCC responses elicited by the anti-TK1 antibodies were observed between 24-72hrs. The highest ADCC responses were elicited by antibodies 4G10 and 1B12 in both breast and lung cancer cell lines while a minimal response was observed with antibodies 7D1 and 3B2E11. A 50-60% increment in the ADCC response against NCI-H460 cells was observed with antibody 4G10 in comparison with the isotype control (p= 0.001). A 40% increase in the ADCC response against MDA-MB-231 cells was observed using antibody 1B12 (p = 0.003) compared to the its isotype control. This preliminary data suggests that anti TK1 monoclonal antibodies can be used for the immunotargeting of tumor cells expressing membrane associated TK1. The further exploration of TK1 as a tumor target could lead to the development of new TK1-based immunotherapies. Citation Format: Edwin J. Velazquez, Jordan D. Cress, Kathryn R. Smith, Taylor D. Brindley, Gajendra Shrestha, Richard A. Robison, Kim L. O'Neill. Monoclonal antibodies against thyimidine kinase 1 for the immunotargeting of lung and breast cancer cells, a preclinical evaluation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 543.

Efficacy, tolerability, and safety of erenumab for the preventive treatment of persistent post-traumatic headache attributed to mild traumatic brain injury: an open-label study

BackgroundCalcitonin gene-related peptide (CGRP) has recently been implicated in the pathogenesis of post-traumatic headache (PTH), which raises the prospect for therapeutic use of monoclonal antibodies targeting CGRP or its receptor. Therefore, we decided to assess the efficacy, tolerability, and safety of erenumab for prevention of persistent PTH attributed to mild traumatic brain injury.MethodsA single-center, non-randomized, single-arm, open-label study of erenumab for adults aged 18–65 years with persistent PTH. Patients were assigned to receive 140-mg erenumab monthly by two subcutaneous 1-mL injections, given every 4 weeks for 12 weeks. The primary outcome measure was the mean change in number of monthly headache days of moderate to severe intensity from baseline (4-week pretreatment period) to week 9 through 12. Tolerability and safety endpoints were adverse events (i.e. number and type).ResultsEighty-nine of 100 patients completed the open-label trial. At baseline, the mean monthly number of headache days of moderate to severe intensity was 15.7. By week 9 through 12, the number was reduced by 2.8 days. The most common adverse events were constipation (n = 30) and injection-site reactions (n = 15). Of 100 patients who received at least one dose of erenumab, two patients discontinued the treatment regimen due to adverse events.ConclusionsAmong patients with persistent PTH, erenumab resulted in a lower frequency of moderate to severe headache days in this 12-week open-label trial. In addition, erenumab was well-tolerated as discontinuations due to adverse events were low. Placebo-controlled randomized clinical trials are needed to adequately evaluate the efficacy and safety of erenumab in patients with persistent PTH.Trial registrationClinicalTrials.Gov, NCT03974360. Registered on April 17, 2019 - Retrospectively registered

Future role of proprotein convertase subtilisin/kexin type 9 inhibitors in preventive cardiology.

The use of therapeutic monoclonal antibodies to target proprotein convertase subtilisin/kexin type 9 (PCSK9) represents a novel approach to the management of hypercholesteremia and prevention of atherosclerotic cardiovascular disease. We review the most recent literature relevant to PCSK9 inhibition with emphasis on how recent results and ongoing trials have and will continue to shape the use of this new therapeutic class in preventive cardiology. PCSK9 inhibitors reduce plasma lipoprotein(a) concentrations but a mechanistic understanding remains elusive. Evaluation of evolocumab for use in patients without prior myocardial infarction or stroke is underway (NCT03872401). Concerns regarding the cost-effectiveness of PCSK9 inhibitors have continued to thwart access to these drugs, though innovative models of care delivery and price reductions have improved this situation. Inclisiran, a small interfering ribonucleic acid (siRNA), reduces translation of PCSK9 and demonstrates more durable reductions in low-density lipoprotein-cholesterol (LDL-C). It is currently evaluated in the context of a phase III cardiovascular outcome trial in patients with established vascular disease (NCT03705234). The current scope of PCSK9 inhibitor therapy in preventive cardiology is limited to patients with familial hypercholesterolemia and/or established atherosclerotic cardiovascular disease. Future cardiovascular outcome trial results with PCSK9 blocking antibodies in primary prevention and with siRNA to PCSK9 in secondary prevention, improved understanding of the drivers of lipoprotein(a) reduction with PCSK9 inhibition, and cost-effectiveness will determine the future role of this therapeutic class.

Abstract SY42-02: Inactivating STK11/LKB1 genomic alterations are a major driver of primary resistance to PD-1 axis blockade in non-squamous non-small cell lung cancer

Despite improvements in overall survival (OS) and clinical responses of unprecedented duration with the use of therapeutic monoclonal antibodies that target PD-1 or PD-L1, the majority of patients with non-small cell lung cancer (NSCLC) fail to respond to PD-1/PD-L1 axis inhibitors. The genomic determinants of PD-1/PD-L1 inhibitor efficacy in NSCLC are incompletely understood and likely key to the development of novel personalized immunotherapy approaches. Through integrative analysis of multiple independent datasets of patients with KRAS-mutant lung adenocarcinoma (LUAC) - the most prevalent oncogenic driver event in LUAC- we identified three major disease subsets defined on the basis of co-occurring genomic events in STK11/LKB1 (the KL subgroup), TP53 (KP) and CDKN2A/B, the latter coupled with low expression of the TTF1 transcription factor (KC) (Skoulidis F et al., Cancer Discovery, 2015). STK11/LKB1 is somatically mutated in ~17% of LUAC and encodes a serine/threonine kinase with an established role in the regulation of cellular metabolism, growth and polarity. Gene set enrichment analysis of RNASeq data from the TCGA LUAC cohort indicated striking de-enrichment of multiple gene expression signatures pertaining to anti-tumor immunity in the KL subgroup, raising the possibility that inactivation of STK11/LKB1 may promote establishment of a non-T-cell inflamed immune microenvironment in lung adenocarcinoma. Indeed, STK11/LKB1-mutant LUAC exhibited significantly lower density of infiltrating CD3+ (P=0.0019) and CD8+ (P=0.0072) T-lymphocytes by automated quantitative immunohistochemistry (IHC) in a tumor microarray encompassing early stage, surgically resected, chemotherapy-naive tumors with available whole exome sequencing (PROSPECT cohort). In an independent, unbiased analysis of a large dataset of 924 LUAC with comprehensive NGS-based genomic profiling and available tumor cell PD-L1 expression (Foundation Medicine dataset) seeking to identify candidate genomic drivers of immune evasion and immunotherapy resistance, STK11/LKB1 genomic alterations emerged as the only significantly enriched gene in PD-L1Negative;TMBIntermediate/High compared to PD-L1High Positive;TMBIntermediate/High tumors (adjusted P 0). Therefore, quantitative IHC for STK11/LKB1 can capture STK11/LKB1-deficient tumors with intact STK11/LKB1 genomic locus and may enhance the predictive utility of STK11/LKB1 genomic alterations. Importantly, the impact of STK11/LKB1 mutations on clinical outcomes with PD-1/PD-L1 inhibitors extended to PD-L1 positive tumors. Among 66 PD-L1-positive (≥1%) non-squamous NSCLC treated with PD-1/PD-L1 inhibitors, STK11/LKB1-mutated tumors exhibited significantly lower objective response rates to PD-1/PD-L1 blockade compared with NSCLC with intact STK11/LKB1 (ORR 0% vs. 34.5%, P = 0.026) and dramatically shorter PFS (HR 4.76; 95% CI, 2.0-11.1, P = 0.00012, log-rank test) and OS (HR 14.3; 95% CI, 3.4-50.0, P Citation Format: Ferdinandos Skoulidis. Inactivating STK11/LKB1 genomic alterations are a major driver of primary resistance to PD-1 axis blockade in non-squamous non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY42-02.

A multiplex liquid chromatography tandem mass spectrometry method for the quantification of seven therapeutic monoclonal antibodies: Application for adalimumab therapeutic drug monitoring in patients with Crohn's disease

The use of therapeutic monoclonal antibodies (mAbs) is steadily increasing. Previous studies have reported the clinical interest of mAb therapeutic-drug monitoring (TDM), including that of adalimumab, for patients with Crohn's disease (CD). Proof of concept mAb-quantification studies by liquid chromatography mass spectrometry (LC-MS/MS) have been published, but a specific and reliable routine-suited multiplex quantification method is still needed to facilitate mAb TDM.We describe an electrospray ionization LC-MS/MS method for the simultaneous quantification of seven mAbs (adalimumab, cetuximab, infliximab, rituximab, secukinumab, tocilizumab, and trastuzumab) in human plasma. Sample preparation was performed using protein-G purification and trypsin digestion to obtain proteotypic peptides. We retrospectively measured the adalimumab concentration in 65 plasma samples from 56 CD patients and determined the adalimumab therapeutic cut-off concentration associated with biological remission.Calibration curves were linear from 1 to 100 μg mL−1, except for rituximab (5–100 μg mL−1). This method was reproducible, repeatable, and accurate (coefficient of variation and bias < 20%), with no cross contamination. Adalimumab concentrations were significantly higher (p = 0.0198) for patients with biological remission (median: 11.3 μg mL−1 [4.6; 18.3]) than that for patients without a biological response (9.5 μg mL−1 [3.94;17.0]). An adalimumab cut-off concentration of 8.0 μg mL−1 correctly discriminated patients with or without biological remission (sensitivity: 74.1%, specificity: 57.9%).This validated LC-MS/MS routine-suited method is the first allowing simultaneous quantification of up to seven mAbs acting against different pharmacological targets. It opens the field of TDM to numerous mAbs.

Carcinoid Heart Disease: Review of Current Knowledge.

Carcinoid heart disease is the collective term for all cardiac manifestations in patients who have carcinoid syndrome. Carcinoid heart disease has a multifactorial pathophysiology, and the right side of the heart is usually involved. Symptoms and signs vary depending upon the affected cardiac components; most typical is right-sided heart failure secondary to diseased tricuspid and pulmonary valves. Despite no single ideal diagnostic test, strong suspicion, coupled with serologic and imaging results, usually enables diagnosis. Advances in imaging, such as speckle-tracking echocardiography and cardiac magnetic resonance, have improved the diagnostic yield. Treatment is challenging, warrants a multidisciplinary approach, and can be medical or surgical depending on the cardiac manifestations. Investigators are exploring the therapeutic use of monoclonal antibodies and new somatostatin analogues. In this review, we cover current knowledge about the pathophysiology, diagnosis, and treatment of carcinoid heart disease.

Half-Chain Cetuximab Nanoconjugates Allow Multitarget Therapy of Triple Negative Breast Cancer

The use of therapeutic monoclonal antibodies (mAbs) has revolutionized cancer treatment. The conjugation of mAbs to nanoparticles has been broadly exploited to improve the targeting efficiency of drug nanocarriers taking advantage of high binding efficacy and target selectivity of antibodies for specific cell receptors. However, the therapeutic implications of nanoconjugation have been poorly considered. In this study, half-chain fragments of the anti-EGFR mAb cetuximab were conjugated to colloidal nanoparticles originating stable nanoconjugates that were investigated as surrogates of therapeutic mAbs in triple negative breast cancer (TNBC). Three TNBC cell lines were selected according to EGFR expression, which regulates activation of MAPK/ERK and PI3K/Akt pathways, and to distinctive molecular profiling including KRAS, PTEN, and BRCA1 mutations normally associated with diverse sensitivity to treatment with cetuximab. The molecular mechanisms of action of nanoconjugated half-chain mAb, including cell targeting, interference with downstream signaling pathways, proliferation, cell cycle, and apoptosis, along with triggering of ADCC response, were investigated in detail in sensitive and resistant TNBC cells. We found that half-chain mAb nanoconjugation was able to enhance the therapeutic efficacy and improve the target selectivity against sensitive, but unexpectedly also resistant, TNBC cells. Viability assays and signaling transduction modulation suggested a role of BRCA1 mutation in TNBC resistance to cetuximab alone, whereas its effect could be circumvented using half-chain cetuximab nanoconjugates, suggesting that nanoconjugation not only improved the antibody activity but also exerted different mechanisms of action. Our results provide robust evidence of the potential of half-chain antibody nanoconjugates in the treatment of TNBC, which could offer a new paradigm for therapeutic antibody administration, potentially allowing improved curative efficiency and reduced minimal effective dosages in both sensitive and resistant tumors.

Current Mass Spectrometric Tools for the Bioanalyses of Therapeutic Monoclonal Antibodies and Antibody-Drug Conjugates.

The increase in the use of therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) has made the detailed bioanalysis of these drugs essential not only for planning optimal therapeutic programs for clinical practice, but also for evaluating the biological equivalencies in the development of other biosimilars. The ligand binding assays that are widely in use now are being replaced rapidly by the highly accurate, sensitive, and selective analytical method using a mass spectrometer. This review will discuss the progress in and challenges observed during the development of a mass spectrometry-based bioanalytical method for therapeutic mAbs and ADCs.