Comparing the pharmacokinetics, safety, and immunogenicity of HLX02 to US- and EU-approved trastuzumab in healthy Chinese male subjects: A Phase I, randomized, double-blind, parallel-group study.

BACKGROUND: HLX02, the first China-manufactured trastuzumab biosimilar, is approved in Europe (EU) and China. This study evaluated bioequivalence between HLX02 and US-approved trastuzumab (US-trastuzumab). METHOD: In this double-blind, parallel-group, Phase I study, healthy Chinese men were randomized (1:1:1) to receive a single 6 mg/kg dose of HLX02, reference US-trastuzumab, or reference EU-approved trastuzumab (EU-trastuzumab). Equivalence in PK profiles was demonstrated if the 90% confidence intervals (CIs) for the geometric mean ratio (GMR) for the difference between the least square means of the area under the curve (AUC) from time 0 to infinity (AUC∞) were 0.8-1.25. RESULTS: Pharmacokinetic profiles of the three trastuzumab products were similar in 111 Chinese men. Equivalence was confirmed between HLX02 and US-trastuzumab (GMR for AUC∞ 1.009, 90% CI 0.950-1.072); HLX02 and EU-trastuzumab (GMR for AUC∞ 1.068, 90% CI 1.005-1.135); and EU- and US-trastuzumab (GMR for AUC∞ 0.945, 90% CI 0.889-1004). Exploratory analysis of all other PK parameters also demonstrated equivalence between any two of the three trastuzumab products. HLX02 had similar safety and immunogenicity profiles to US- and EU-trastuzumab. CONCLUSION: HLX02 is bioequivalent to US-trastuzumab and EU-trastuzumab, with similar safety and immunogenicity profiles. US- and EU-trastuzumab were also bioequivalent to each other.

Preparation of an antigen-responsive fluorogenic immunosensor by tyrosine chemical modification of the antibody complementarity determining region.

Full-length pharmaceutical antibodies, trastuzumab and rituximab, were chemically modified into Quenchbody, a fluorescent immunosensor, using a two-step reaction: (1) selective tyrosine residue modification of antibody complementarity determining regions (CDRs), and (2) introduction of fluorescent dye molecules by Cu-free click reaction. Without the need for genetic manipulation and time-consuming examination of protein expression conditions, the antibody-dye combination with good antigen response efficiency was examined in a simple two-hour operation.

A randomized, double-blind, single-dose study (LAVENDER) to assess the safety, tolerability, pharmacokinetics, and immunogenicity of a combined infusion of ABP 980 and pertuzumab in healthy subjects.

PURPOSE: ABP 980 (KANJINTI™) is a biosimilar to reference product HERCEPTIN® (trastuzumab RP). The goal of this study was to characterize the safety, tolerability, and immunogenicity of ABP 980 plus pertuzumab (PERJETA®) when co-administered in a single infusion bag in healthy subjects. METHODS: This randomized, double-blind, single-dose, 2-arm, parallel-group study (LAVENDER Study) evaluated an intravenous (IV) infusion of ABP 980 (6 mg/kg) plus pertuzumab (420 mg) combined in a single infusion bag relative to an IV infusion of trastuzumab RP (6 mg/kg) plus pertuzumab (420 mg) combined in a single infusion bag given over 60 min. The subjects were followed for 92 days post dosing. RESULTS: A total of 42 subjects were enrolled in the study and treated with investigational product. Due to an operational issue during dosing, the first 6 subjects enrolled in the study were replaced. A total of 36 randomized subjects, n = 18 for ABP 980 plus pertuzumab and n = 18 for trastuzumab RP plus pertuzumab, were treated. Resulting serum concentrations of ABP 980 and trastuzumab RP were similar. There were no serious adverse events, no deaths, and no cardiac disorders during the study. No subject developed anti-drug antibodies throughout the study. CONCLUSIONS: This study demonstrated the safety and tolerability of ABP 980 and pertuzumab admixture in a single infusion bag. The safety profiles and pharmacokinetic parameters of ABP 980 and pertuzumab were consistent with what is known for trastuzumab RP and pertuzumab. CLINICAL TRIAL LISTING: EudraCT 2018-002903-33.

TROIKA-1: A double-blind, randomized, parallel group, study aimed to demonstrate the equivalent pharmacokinetic profile of HD201, a potential biosimilar candidate to trastuzumab, versus EU-Herceptin® and US-Herceptin® in healthy male subjects.

Prestige Biopharma Ltd (Singapore) has developed HD201, a proposed biosimilar to reference product trastuzumab. As a part of the stepwise approach to ensure comparability between the biosimilar candidate and the reference medicinal product, a phase I study in healthy subjects was conducted to demonstrate the pharmacokinetic (PK) equivalence (NCT03776240). The primary objective of the study was to demonstrate (PK) equivalence of HD201, EU-Herceptin® , and US-Herceptin® given at 6 mg/kg as a 90-min i.v. infusion to healthy male subjects. A pairwise comparisons based on the primary endpoint AUC0-inf and secondary PK endpoints, AUC0-last and Cmax were undertaken. PK equivalence was to be concluded if the 90% confidence interval (CI) for the ratio of geometric means for each criterion were within the equivalence margin of 80% to 125%. Secondary objectives included assessment of other PK parameters, safety, tolerability, and immunogenicity in the three arms. A total of 105 healthy male subjects (35/treatment) were randomized in this study. The 90% CI for the ratios of AUC0-inf , Cmax and AUC0-last , were within 80%-125% for the comparisons of HD201 to EU-Herceptin® or US-Herceptin® and EU-Herceptin® to US-Herceptin® . The frequency of subjects with TEAEs of special interest was slightly lower in the HD201 group (20.0%) compared to the other treatment groups (EU-Herceptin® : 34.3%; US-Herceptin® : 31.4%). Only 1 subject (EU-Herceptin® group) developed anti-drug antibodies prior to dosing. Overall, HD201 demonstrates PK similarity to both EU-Herceptin® and US-Herceptin® . The three study drugs also demonstrated similar safety profiles.

Optimization of therapeutic antibodies by predicting antigen specificity from antibody sequence via deep learning.

The optimization of therapeutic antibodies is time-intensive and resource-demanding, largely because of the low-throughput screening of full-length antibodies (approximately 1 × 103 variants) expressed in mammalian cells, which typically results in few optimized leads. Here we show that optimized antibody variants can be identified by predicting antigen specificity via deep learning from a massively diverse space of antibody sequences. To produce data for training deep neural networks, we deep-sequenced libraries of the therapeutic antibody trastuzumab (about 1 × 104 variants), expressed in a mammalian cell line through site-directed mutagenesis via CRISPR-Cas9-mediated homology-directed repair, and screened the libraries for specificity to human epidermal growth factor receptor 2 (HER2). We then used the trained neural networks to screen a computational library of approximately 1 × 108 trastuzumab variants and predict the HER2-specific subset (approximately 1 × 106 variants), which can then be filtered for viscosity, clearance, solubility and immunogenicity to generate thousands of highly optimized lead candidates. Recombinant expression and experimental testing of 30 randomly selected variants from the unfiltered library showed that all 30 retained specificity for HER2. Deep learning may facilitate antibody engineering and optimization.

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding.

Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

Trastuzumab immunogenicity development in patients' sera and in laboratory animals.

BACKGROUND: Immunogenicity is a major challenge in drug development and patient care. Clinicians and regulators are familiar with immunogenicity concerns of monoclonal antibody (mAb) therapeutics, growth factors and enzyme replacements. Although most small therapeutic molecules are unlikely to trigger undesirable immunogenic responses against themselves upon their administration, the biological therapeutic agents are likely to induce such kind of immunogenicity. This imparts a problem that has to be considered upon judging their risk-benefit ratio. In this article, we tested the immunogenicity developed in patients' sera due to the use of trastuzumab and that developed in laboratory animals injected with this recombinant humanized IgG1 monoclonal antibody. METHODS: We studied trastuzumab immunogenicity by: I in vitro detection of anti-trastuzumab antibody (Ab) levels in patient's serum samples withdrawn at different points during trastuzumab treatment course; I.1 using an Affinity Capture Elution (ACE) assay, the assay is both sensitive and highly tolerant to free drug; I.2 using MTT cytotoxicity method against MCF-7 cell line as confirmatory method used in sample showed high level of anti-trastuzumab Ab and to determine neutralizing activity of the anti-trastuzumab Ab. II in vivo immunogenicity testing of trastuzumab in lab animals. RESULTS: In vitro analysis of patients' sera for antibodies developed against trastuzumab revealed that this monoclonal antibody has low immunogenicity since most samples showed low levels of anti-trastuzumab antibodies that decreased progressively along the treatment course. Only 1% of samples showed high levels of anti-trastuzumab antibodies which might affect treatment course. In vivo immunogenicity testing in mice showed also low immunogenicity of trastuzumab that could support the in vitro clinical assessment applied in our study. CONCLUSIONS: The study gives an evidence for the low trastuzumab immunogenicity when assessed in Egyptian patients under treatment with this biological therapeutic agent. This supports its prescription and continuous use across the approved indications as biological therapeutic agent.

An Efficient Conjugation Approach for Coupling Drugs to Native Antibodies via the PtII Linker Lx for Improved Manufacturability of Antibody-Drug Conjugates.

The PtII linker [ethylenediamineplatinum(II)]2+ , coined Lx, has emerged as a novel non-conventional approach to antibody-drug conjugates (ADCs) and has shown its potential in preclinical in vitro and in vivo benchmark studies. A crucial improvement of the Lx conjugation reaction from initially <15 % to ca. 75-90 % conjugation efficiency is described, resulting from a systematic screening of all relevant reaction parameters. NaI, a strikingly simple inorganic salt additive, greatly improves the conjugation efficiency as well as the conjugation selectivity simply by exchanging the leaving chloride ligand on Cl-Lx-drug complexes (which are direct precursors for Lx-ADCs) for iodide, thus generating I-Lx-drug complexes as more reactive species. Using this iodide effect, we developed a general and highly practical conjugation procedure that is scalable: our lead Lx-ADC was produced on a 5 g scale with an outstanding conjugation efficiency of 89 %.

Cytokine-induced killer cells-assisted tumor-targeting delivery of Her-2 monoclonal antibody-conjugated gold nanostars with NIR photosensitizer for enhanced therapy of cancer.

Maximizing the accumulation of anticancer medicine in the tumor is the priority to achieve minimal invasive cancer therapy, which raises high demands on tumor-targeting ability of drug delivery systems. Herein, we adopted an emerging "cell-drug" strategy via the nanoplatform construction to achieve high aggregation and intratumoral distribution. We fabricated gold nanostars (GNSs) with HER-2 monoclonal antibody (trastuzumab) and near-infrared region (NIR) photosensitizer indocyanine green (ICG) to obtain GNS@ICG-Ab, which combined the photothermal therapy with photodynamic therapy (PTT/PDT) that rely on enhanced photothermal conversion efficiency of GNS and 1O2 generator ICG under the exposure of a NIR laser. Tumor-tropism CIK cells loaded with GNS@ICG-Ab were able to migrate into tumors and make a difference in efficient accumulation and uniform distribution of the GNS@ICG-Ab-CIK nanoplatform inside tumors based on fluorescence, photoacoustic (PA), and computed tomography (CT) imaging observations. Encouraged by the improvements in tumor targeting and retention presented by real-time imaging, we employed the novel nanoplatform to synergistically inhibit the progression of tumors in SK-BR-3 tumor-bearing mice via PTT/PDT and immunotherapy-implemented by CIK cells for activating the immune response, and with the specific linkage between trastuzumab and SK-BR-3 tumor cells, our platform could exert a precise strike of PDT/PTT. Taken together, the integrating tri-modal imaging with tri-modal therapy endows CIK-GNS@ICG-Ab with promising potential in cancer theranostics and lays a solid foundation for the development of immune cell application in nanomedicine delivery.

Antibody Clicking as a Strategy to Modify Antibody Functionalities on the Surface of Targeted Cells.

We established a methodology for initiating cross-linking of antibodies selectively on the cell surface through intermolecular copper-free click reactions facilitated by increased effective concentrations of antibodies binding to target antigens. Upon cross-linking of tetrazine- and bicyclononyne-modified trastuzumab on the surface of HER2-overexpressing cells, increased antibody uptake and activation of intracellular signaling were observed. Our findings demonstrate that the cross-linking reaction can significantly alter the biophysical properties of proteins, activating their unique functionalities on targeted cells to realize an increased cargo delivery and synthetic manipulation of cellular signaling.

Generation and testing of engineered multimeric Fabs of trastuzumab.

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells. Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody. Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates.

Rapid desensitization to antineoplastic drugs in an outpatient immunoallergology clinic: Outcomes and risk factors.

BACKGROUND: Hypersensitivity reactions to antineoplastic agents may lead to discontinuation of first-line treatments. Rapid drug desensitization (RDD) allows for a safe reintroduction in patients who are allergic to them. OBJECTIVE: To evaluate the safety and efficacy of the Brigham and Women's Hospital's 12-step RDD in a Portuguese patient population with cancer and to identify markers associated with breakthrough reactions (BTRs) to platins. METHODS: We conducted a retrospective review of desensitizations undertaken at the Immunoallergology Day-Care Unit of the Santa Maria Hospital in Lisbon, Portugal, from July 2008 to July 2019. Adult patients with cancer and with immediate hypersensitivity reactions were included. Skin testing was performed to platins, trastuzumab, and cetuximab. The 12-step protocol was used for most patients, and a shorter protocol was used in 9 patients who were taxane-reactive to resume regular infusions. RESULTS: A total of 1471 RDDs were performed in 272 patients to 136 platins, 124 taxanes, 13 monoclonal antibodies, and 10 other drugs. Skin test results were positive in 127 of patients who were platin-reactive (95.3%) and negative in patients who were cetuximab- and trastuzumab-reactive. There were 141 BTRs during RDD (9.6% of infusions), 79.4% induced by platins with the majority having mild reactions (68.8%). There were 8 patients who were paclitaxel-reactive, and who completed a shorter protocol and resumed regular infusions successfully. Multiple platin infusions (cutoff: ≥10) and total immunoglobulin E greater than or equal to 100 U/mL were identified as independent risk factors for BTRs in patients who were platin-reactive. CONCLUSION: This large single-center study confirmed the safety and efficacy of the 12-step RDD protocol in a diverse cancer population, providing evidence of its universal applications. Total immunoglobulin E is a potentially useful biomarker to identify high-risk patients who are platin-reactive.

Hyphenation of strong cation exchange chromatography to native mass spectrometry for high throughput online characterization of charge heterogeneity of therapeutic monoclonal antibodies.

Characterization of charge heterogeneity in monoclonal antibodies (mAbs) is needed during developability assessment and downstream development of drug candidates. Charge heterogeneity can come from post-translational modifications like deamidation, isomerization, and sialylation. Elucidation of charge variants with mass spectrometry (MS) has historically been challenging. Due to the nonvolatility and high ionic strength of conventional buffer systems, labor-intensive offline fractionation followed by MS analysis is routinely used. Here, we describe an alternative strategy that directly couples strong cation exchange (SCX) chromatography to high-resolution Orbitrap MS for online native MS analysis (SCX-MS). A combined pH and salt gradient was used for universal separation of mAbs from a wide range of pI values (6.38 ~ 9.2), including infliximab (Remicade®, chimeric IgG1/kappa), NISTmab (humanized IgG1/kappa) and trastuzumab (Herceptin®, humanized IgG1/kappa), without tailoring of chromatographic profiles. Liquid chromatography and MS parameters were optimized to achieve high-quality spectra and enhanced detection of low abundant species under high flow rate conditions. Genedata Expressionist, a vendor agnostic software, was used for data processing. This integrated strategy allows unbiased characterization of numerous charge variant species and low molecular weight fragments (<0.05%) without post-column flow splitting. The application was further expanded with middle-up approaches for subdomain analysis, which demonstrated the versatility of the strategy for analysis of various construct types. With our analysis of mAbs during developability assessment and forced degradation studies, which aimed at assessing potential critical quality attributes in antibody drug molecules, we provide, for the first time, direct visualization of molecular alterations of mAbs at intact level. Furthermore, strong correlation was observed between this novel MS approach and analysis by capillary isoelectric focusing.

Trastuzumab derived HER2-specific CARs for the treatment of trastuzumab-resistant breast cancer: CAR T cells penetrate and eradicate tumors that are not accessible to antibodies.

HER2-targeted monoclonal antibodies improve the outcome for advanced breast cancer patients; however, resistance to therapy is still frequent. Epitope masking and steric hindrance to antibody binding through matrix components are thought to be the major mechanism. We asked whether tumors resistant to trastuzumab can still be eliminated by CAR T cells redirected by the same antibody domain. While saturating doses of trastuzumab in the presence of CD16.176V.NK-92 effector cells and trastuzumab derived CAR T cells equally well recognized and killed HER2-positive tumor cells in a monolayer, only CAR T cells penetrated into the core region of tumor spheroids and exhibited cytotoxic activity in vitro, whereas antibodies failed. In NSG mice treatment with trastuzumab and CD16.176V.NK-92 cells only transiently retarded tumor growth but did not induce regression of clinically trastuzumab-resistant breast cancer xenografts. In contrast, one dose of HER2-specific CAR T cells eradicated established tumors resulting in long-term survival. Data indicate that CAR T cells can successfully combat antibody resistant tumors by targeting the same epitope suggesting that CAR T cells can penetrate the tumor matrix which is a barrier for antibodies.

A Small Number of HER2 Redirected CAR T Cells Significantly Improves Immune Response of Adoptively Transferred Mouse Lymphocytes against Human Breast Cancer Xenografts.

HER2 positive JIMT-1 breast tumors are resistant to trastuzumab treatment in vitro and develop resistance to trastuzumab in vivo in SCID mice. We explored whether these resistant tumors could still be eliminated by T cells redirected by a second-generation chimeric antigen receptor (CAR) containing a CD28 costimulatory domain and targeting HER2 with a trastuzumab-derived scFv. In vitro, T cells engineered with this HER2 specific CAR recognized HER2 positive target cells as judged by cytokine production and cytolytic activity. In vivo, the administration of trastuzumab twice weekly had no effect on the growth of JIMT-1 xenografts in SCID mice. At the same time, a single dose of 2.5 million T cells from congenic mice exhibited a moderate xenoimmune response and even stable disease in some cases. In contrast, when the same dose contained 7% (175,000) CAR T cells, complete remission was achieved in 57 days. Even a reduced dose of 250,000 T cells, including only 17,500 CAR T cells, yielded complete remission, although it needed nearly twice the time. We conclude that even a small number of CAR T lymphocytes can evoke a robust anti-tumor response against an antibody resistant xenograft by focusing the activity of xenogenic T cells. This observation may have significance for optimizing the dose of CAR T cells in the therapy of solid tumors.

A Novel Anti-HER2 Bispecific Antibody With Potent Tumor Inhibitory Effects In Vitro and In Vivo.

Overexpression of HER2 has been reported in many types of cancer, making it a perfect candidate for targeted immunotherapy. The combination of two FDA approved monoclonal antibodies (mAbs), trastuzumab and pertuzumab, has more robust anti-tumor activity in patients with HER2-overexpressing breast cancer. We recently produced a new humanized anti-HER2 mAb, hersintuzumab, which recognizes a different epitope than trastuzumab and pertuzumab on HER2. This mAb, in combination with trastuzumab, exhibits more potent anti-tumor activity than each parental mAb alone. Here we have developed a novel bispecific anti-HER2 antibody (BsAb) designated as trasintuzumab, composed of trastuzumab and hersintuzumab, using dual variable domain immunoglobulin (DVD-Ig) technology. Both variable domains of trasintuzumab are fully functional and have similar affinities to the parental mAbs and are also able to bind to natural HER2 on the surface of several HER2-expressing cell lines. Trasintuzumab was found to inhibit the growth of different types of tumor cell lines through suppression of the AKT and ERK signaling pathways as efficiently as the combination of the parental mAbs. It also induced tumor regression as potently as the combination of the two mAbs in nude mice bearing ovarian and gastric cancer xenografts. Our data suggest that trasintuzumab may be a promising BsAb therapeutic candidate for the treatment of HER2-overexpressing cancers.

Application of proteometric approach for identification of functional mutant sites to improve the binding affinity of anticancer biologic trastuzumab with its antigen human epidermal growth factor receptor 2.

The aim of the present study was to develop a linear regression model aiding to a quick scan of the most important sites for mutation of an anticancer biologic trastuzumab. The important sites identified on trastuzumab can be used to carry out site-directed mutagenesis to improve the binding affinity of the drug towards its antigen, human epidermal growth factor receptor 2 (HER2). This will lead to low dosage requirement of the drug for treating cancer patients, which in turn help to cut the cost and combat development of resistance. A quantitative structure-activity relationship (QSAR) model was built by multiple linear regressions using genetic algorithm-based feature selection (GA-MLR) method using 48 dependent variables (dissociation constant Kd ) and 226 independent variables (theoretical descriptors generated using a proteometrics approach). The final QSAR model selected in the study was more on the basis of ability to predict accurately independent test data and generalization ability of the model rather than mere statistical significance of the model. With combined analysis of descriptors presented in final QSAR model and most frequent descriptors pooled from all solution models, it was demonstrated that the modeling procedure was able to bring on the factors important for antigen-antibody interactions with an example of HER2-trastuzumab interaction reported in previous experimental studies. This paper will allow the prediction of the most preferable site to mutate for improving the binding affinity of trastuzumab with HER2 and also will be helpful in selecting most preferable amino acids to substitute in the selected site for mutations. This is the novel report on proteometrics approach with autocorrelation formalism for antibody engineering, which can be extended to other antibody-antigen pairs.

Responsive Antibody Conjugates Enable Quantitative Determination of Intracellular Bond Degradation Rate.

Degradable crosslinkers that respond to intracellular biological stimuli are a critical component of many drug delivery systems. With numerous stimuli-responsive drug delivery systems in development, it is important to quantitatively study their intracellular processing. Herein we report a framework for quantifying the rate of intracellular bond degradation in the endocytic pathway. Toward this end, we devised and synthesized a reduction-sensitive FRET-based crosslinker that can be readily conjugated to a variety of targeting ligands. This crosslinker was conjugated to trastuzumab, a humanized monoclonal antibody against the HER2 receptor. We developed a model based on mass-action kinetics to describe the intracellular processing of this conjugate. The kinetic model was developed in conjunction with live-cell experiments to extract the rate constant for intracellular disulfide bond degradation. This framework may be applied to other endocytosis pathways, bond types, and cell types to quantify this fundamental degradation rate parameter.

Efficient Screening and Design of Variable Domain of Heavy Chain Antibody Ligands Through High Throughput Sequencing for Affinity Chromatography to Purify Fab Fragments.

To design an affinity ligand for purification of antigen-binding fragment (Fab) antibody, variable domain of heavy chain antibody (VHH) phage libraries were constructed from Fab-immunized Alpaca and subjected to biopanning against Fabs. To find the specific binders, we directly applied high-throughput sequencing (HTS) analysis of the VHH sequences in the panned phages on next-generation sequencer. The efficiently enriched sequences were aligned for construction of the phylogenetic tree to be categorized into five groups. VHHs from three major groups were first selected to analyze their properties as an affinity ligand. However, those VHHs were not suitable as an affinity ligand because of lack of resistance against alkaline pH and/or difficulty in acidic elution from the affinity column. So, we further searched the candidates from minor group sequences. Among five, one VHH showed the binding ability but with low affinity against Fabs. Therefore, we improved its affinity-by-affinity maturation through error-prone PCR library techniques. The final designed VHH showed highly alkaline pH resistance and easy acidic elution together with high affinity to Fabs. These results indicate that HTS techniques combined with biopanning and followed by error-prone PCR library techniques is powerful in designing specific binders with desired properties.

Site-Specific Conjugation of Thiol-Reactive Cytotoxic Agents to Nonnative Cysteines of Engineered Monoclonal Antibodies.

Antibody-drug conjugates (ADCs) have been proven to be a successful therapeutic concept, allowing targeted delivery of highly potent active pharmaceutical ingredients (HPAPIs) selectively to tumor tissue. So far, HPAPIs have been mainly attached to the antibody via a chemical reaction of the payload with lysine or cysteine side chains of the antibody backbone. However, these conventional conjugation technologies result in formation of rather heterogeneous products with undesired properties. To overcome the limitations of heterogeneous ADC mixtures, several site-specific conjugation technologies have been developed over the last years. Originally pioneered by scientist from Genentech with their work on THIOMABs, several engineered cysteine mAb ADCs (ECM-ADCs) are now investigated in clinical trials. Here, we describe in detail how to engineer additional cysteines into antibodies and efficiently use them as highly site-specific conjugation sites for HPAPIs.