Framework Regions Research Articles

Molecular Insights of Nickel Binding to Therapeutic Antibodies as a Possible New Antibody Superantigen

The binding of nickel by immune proteins can manifest as Type IV contact dermatitis (Ni-specific T cells mediated) and less frequently as Type I hypersensitivity with both mechanisms remaining unknown to date. Since there are reports of patients co-manifesting the two hypersensitivities, a common mechanism may underlie both the TCR and IgE nickel binding. Focusing on Trastuzumab and Pertuzumab IgE variants as serendipitous investigation models, we found Ni-NTA interactions independent of Her2 binding to be due to glutamine stretches. These stretches are both Ni-inducible and in fixed pockets at the antibody complementarity-determining regions (CDRs) and framework regions (FWRs) of both the antibody heavy and light chains with influence from the heavy chain constant region. Comparisons with TCRs structures revealed similar interactions, demonstrating the possible underlying mechanism in selecting for Ni-binding IgEs and TCRs respectively. With the elucidation of the interaction, future therapeutic antibodies could also be sagaciously engineered to utilize such nickel binding for biotechnological purposes.

The Prognostic Significance of the Complete IgH Rearrangement Pattern Using the BIOMED-2 Protocol in Diffuse Large B-Cell Lymphoma

▪Introduction: In several B-cell malignancies, such as chronic lymphocytic leukemia and mantle cell lymphoma, the somatic hypermutation (SHM) status of immunoglobulin heavy chain (IgH) variable region (VH) significantly correlates with clinical outcome. Diffuse large B-cell lymphoma (DLBCL) usually contains a number of VH SHMs, with considerably variable frequency. However, there have been few reports about the association between VHSHM frequency and clinical outcome in DLBCL.The BIOMED-2 multiplex PCR protocol is one of the standardized methods for detecting IgH gene rearrangements, and has been used to prove clonality in B-cell lymphomas. VH SHMs in the germinal center prevent proper annealing of the applied PCR primers, rendering clonal VH rearrangements undetectable by this protocol. Thus, the complete IgH rearrangement pattern produced by the BIOMED-2 protocol is expected to reflect the frequency of VH SHMs. In this study, we investigated the prognostic impact of complete IgH rearrangement pattern on patients with DLBCL, and analyzed the correlation between complete IgH rearrangement pattern and VH SHM frequency by Sanger sequencing.Materials and Methods: We retrospectively identified 386 patients diagnosed with de novo DLBCL that received anthracycline-based immunochemotherapy at Kobe City Medical Center General Hospital from January 2005 to June 2015. We excluded 83 patients because fresh samples were not available for PCR analysis, and a total of 303 patients were enrolled. Based on the BIOMED-2 protocol, complete IgH rearrangement was detected by three PCR reactions using three VH forward primer sets: framework region (FR) 1, FR2, and FR3. The primary endpoint was 5-year overall survival (5-yr OS). The secondary endpoint was 5-year progression-free survival (5-yr PFS). OS and PFS were estimated using the log-rank test. Multivariate analysis using the Cox proportional hazard ratio was performed for 5-yr OS. For VH sequencing analysis, the cryopreserved PCR products of 42 available samples were sequenced directly using both forward and reverse primers, and for each sequenced sample, the percentage of VH identity to the closest germline gene was calculated, using the immunoglobulin gene database (IMGT/V-QUEST).Results: Of the 303 patients enrolled (median age: 69 years old), 183 patients (60.3%) had advanced stage disease and 148 (48.8%) had a high-intermediate or high International Prognostic Index (IPI) score. The median follow-up was 60.6 months (2.0-133.8 months). Complete IgH rearrangement was detected in 176 (58.1%), 172 (56.8%), and 113 (37.2%) of patients using the FR1, FR2, and FR3 primer sets, respectively. In total, 80 patients (26.4%) were positive for all three PCR reactions, 82 (27.1%) for two, 57 (18.8%) for one, and 84 (27.7%) had completely negative results. We compared the 5-yr OS and PFS among these subgroups (Figure 1), and observed that the probability of survival decreased with accumulation of positive PCR results. As shown in Figure 2, both the 5-yr OS and PFS were significantly worse for patients with all three reactions positive for complete IgH rearrangement (OS: 54.2% vs 73.2% p=0.002; PFS: 34.4% vs. 59.3%, p<0.001). In multivariate analysis adjusted by IPI score, complete IgH rearrangement detected by all three PCR reactions was significantly correlated with poor prognosis (hazard ratio: 2.13; 95% CI: 1.38-3.26; p<0.001).This classification also allowed subgroups with unfavorable prognosis to be identified, even among patients with a high-intermediate or high IPI score (5-yr OS: 28.0% vs. 62.9%, p<0.001). Moreover, VH sequencing analysis of 42 available samples confirmed that patients with complete IgH rearrangement in all three PCR reactions had significantly lower VH gene SHMs than other groups (median value of VH identity; 91.9% vs 84.0%, p<0.001).Conclusion: Complete IgH rearrangement detected by all three primer sets in the BIOMED-2 PCR protocol was associated with poor prognosis in patients with DLBCL. Combined with the additional sequencing analysis, this suggests that the lower levels of VH SHMs may be associated with unfavorable prognosis in DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An antibody engineering platform using amino acid networks: A case study in development of antiviral therapeutics

We present here a case study of an antibody-engineering platform that selects, modifies, and assembles antibody parts to construct novel antibodies. A salient feature of this platform includes the role of amino acid networks in optimizing framework regions (FRs) and complementarity determining regions (CDRs) to engineer new antibodies with desired structure-function relationships. The details of this approach are described in the context of its utility in engineering ZAb_FLEP, a potent anti-Zika virus antibody. ZAb_FLEP comprises of distinct parts, including heavy chain and light chain FRs and CDRs, with engineered features such as loop lengths and optimal epitope-paratope contacts. We demonstrate, with different test antibodies derived from different FR-CDR combinations, that despite these test antibodies sharing high overall sequence similarity, they yield diverse functional readouts. Furthermore, we show that strategies relying on one dimensional sequence similarity-based analyses of antibodies miss important structural nuances of the FR-CDR relationship, which is effectively addressed by the amino acid networks approach of this platform.

The EPO Guidelines They Are a-Changin’

The EPO Guidelines They Are a-Changin’

Characterization of DNA G-Quadruplex Structures in Human Immunoglobulin Heavy Variable (IGHV) Genes.

Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region.

Development of an Anti‐LMP2 Disulfide‐stabilized Recombinant Immunotoxin Based on Pseudomonas Exotoxin A

Recombinant immunotoxins (RITs) are fusion proteins consisting of a toxin attached to an antibody fragment. Unlike traditional chemotherapy, RITs used in cancer therapy can be specifically targeted to tumor cells while sparing healthy cells. For this project, we constructed and purified an anti-LMP2 disulfide-stabilized RIT to target cells infected by Epstein-Barr virus (EBV). EBV is a common herpesvirus that infects over 90% of the world's population. It is the causative agent of mononucleosis, known as “mono,” but is also linked to the development of cancers such as Burkitt's lymphoma and nasopharyngeal cancer. We hope to develop a RIT that can eliminate EBV-induced cancers. EBV-infected cells express a protein called LMP2 that is unique to EBV, and RITs targeted to LMP2 should exhibit minimal off-target effects. Previous work has developed an antibody against LMP2. We have taken the variable fragment sequence from the antibody and engineered a disulfide bond into the framework region in order to stabilize it. The Fv was then attached recombinantly to the toxin Pseudomonas exotoxin A (PE), which has been engineered in several different forms for use in RITs. We plan to test a PE24 and a PE38 version of the toxin, which differ by their size and sequence composition. We have purified both versions of the RIT and are evaluating their cytotoxicities against cells that express LMP2.

Selected Articles from This Issue

Chimeric antigen receptor (CAR) T cell therap. has shown great efficacy against CD19 positive hematological cancers. However, cytokine release syndrome (CRS) in patients treated with CD19 CAR-T cell therapy remains a significant toxicity to address. Dwivedi and colleagues developed a novel anti-CD19 CAR T cell containing a humanized framework region of scFv from a murine FMC63 monoclonal antibody. The humanized CAR T cell released significantly less cytokines (IFN-γ, TNF-α), reduced IL-6 induction from monocytes, and produced a more even CD4+ and CD8+ T cell distribution. Furthermore, CAR T cells eradicated NALM6 tumor burden in vivo. Taken together, these modified features might be beneficial for reducing toxicity of CAR T cells.Mutations occurring in the ligand binding domain of the androgen receptor (AR) lead to therapy resistance. In particular, the AR F877L mutation leads to an antagonist-to-agonist switch in response to second-generation inhibitors such as enzalutamide and apalutamide. Branch and colleagues describe JNJ-63576253, a next-generation AR pathway inhibitor of wild-type, AR F877L, and other clinically detected ligand binding domain mutants. JNJ-63576253 inhibited downstream target gene expression of AR and impeded tumor growth in an enzalutamide-resistant F887L xenograft model. Their results support the ongoing Phase 2 clinical development of JNJ-63576253 (NCT02987829).IL-2 and IL-15 are both able to stimulate the proliferation of T cells, promote the synthesis of immunoglobulin, and preserve the survival of NK cells. However, IL-2 also maintains peripheral regulatory T cells (Tregs), while IL-15 instead stimulates the survival of CD8+ memory T cells. In this manuscript, Corbellari and colleagues describe two IL-15-diabody fusions with the F8 antibody (F8-F8-IL-15 and F8-F8-SD-IL-15). These antibody-interleukin-15 fusion proteins showed favorable distribution profiles and displayed potent anti-cancer activity in a mouse model of lung metastasis. These studies reinforce the many clinical studies investigating the use of targeted IL-15 therapies.In two related manuscripts Greenman, Pizam, and colleagues performed an extensive study of the functional significance of the biophysical properties of chimeric antigen receptor engineered T cells (CAR T cells). They generated an experimental system in which affinity, avidity and antigen density are controllable within a single experimental model of a specific HLA-peptide complex targeted with TCR-like antibodies of single defined specificity. In the second manuscript they built an evolving phenotypic model of CAR T-cell regulation, which suggested that receptor down-modulation is a key determinant of CAR T-cell function. These results have a potential to predict and therefore advance the rational design of CAR T-cell therapy.

The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants

In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting “hot regions,” i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two “designed” site-directed mutagenesis (A and B) targeted the N-terminal 1–10 positions of the VH framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (Ka, 3.6 × 108 M−1). Mutagenesis A substituted the amino acids at the position 1–3, 5–7, 9 and 10 with a limited set of substitutions to generate only 1,536 different members, while mutagenesis B inserted 1–6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17–31-fold increased affinity with > 109 M−1Ka values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB1-3#130 (with a single residue insertion) showed the greatest Ka value, 1.1 × 1010 M−1.

Rational Engraftment of Quaternary-Interactive Acidic Loops for Anti-HIV-1 Antibody Improvement.

Broadly neutralizing antibodies (bNAbs) are the focus of increasing interest for human immunodeficiency virus type 1 (HIV-1) prevention and treatment. Although several bNAbs are already under clinical evaluation, the development of antibodies with even greater potency and breadth remains a priority. Recently, we reported a novel strategy for improving bNAbs against the CD4-binding site (CD4bs) of gp120 by engraftment of the elongated framework region 3 (FR3) from VRC03, which confers the ability to establish quaternary interactions with a second gp120 protomer. Here, we applied this strategy to a new series of anti-CD4bs bNAbs (N49 lineage) that already possess high potency and breadth. The resultant chimeric antibodies bound the HIV-1 envelope (Env) trimer with a higher affinity than their parental forms. Likewise, their neutralizing capacity against a global panel of HIV-1 Envs was also increased. The introduction of additional modifications further enhanced the neutralization potency. We also tried engrafting the elongated CDR1 of the heavy chain from bNAb 1-18, another highly potent quaternary-binding antibody, onto several VRC01-class bNAbs, but none of them was improved. These findings point to the highly selective requirements for the establishment of quaternary contact with the HIV-1 Env trimer. The improved anti-CD4bs antibodies reported here may provide a helpful complement to current antibody-based protocols for the therapy and prevention of HIV-1 infection.IMPORTANCE Monoclonal antibodies represent one of the most important recent innovations in the fight against infectious diseases. Although potent antibodies can be cloned from infected individuals, various strategies can be employed to improve their activity or pharmacological features. Here, we improved a lineage of very potent antibodies that target the receptor-binding site of HIV-1 by engineering chimeric molecules containing a fragment from a different monoclonal antibody. These engineered antibodies are promising candidates for development of therapeutic or preventive approaches against HIV/AIDS.

Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo

The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin VH/VL regions of which one—employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66—showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (KD = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.

Occlusal marking film for accurate interference assessments of removable partial denture frameworks: A dental technique

A technique is described for detecting areas of interference for removable partial denture frameworks. An occlusal marking film is placed between the misfitted abutment teeth and framework region. Gentle pressure is applied to seat the framework, and the exact areas of interference are seen on removal. With this clean, rapid, cost-effective, and straightforward approach, areas of interference can be precisely adjusted for complete seating of the removable partial denture framework.

Robust Antitumor Activity and Low Cytokine Production by Novel Humanized Anti-CD19 CAR T Cells.

Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop life-threatening toxicities associated with cytokine release syndrome which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR T cells by humanizing the framework region of single-chain variable fragment (scFv) derived from a murine FMC63 mAb and combining it with CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain (h1CAR19-8BBζ). Docking studies followed by molecular dynamics simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared with murine scFv (mCAR19). Ex vivo studies with CAR T cells generated from healthy donors and patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable antitumor activity and proliferation. More importantly, h1CAR19-8BBζ T cells produced lower levels of cytokines (IFNγ, TNFα) upon antigen encounter and reduced the induction of IL6 cytokine from monocytes than mCAR19-8BBζ T cells. There was a comparable proliferation of h1CAR19-8BBζ T cells and mCAR19-8BBζ T cells upon repeated antigen encounter. Finally, h1CAR19-8BBζ T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.

Poorly Expressed Alleles of Several Human Immunoglobulin Heavy Chain Variable Genes are Common in the Human Population.

Extensive diversity has been identified in the human heavy chain immunoglobulin locus, including allelic variation, gene duplication, and insertion/deletion events. Several genes have been suggested to be deleted in many haplotypes. Such findings have commonly been based on inference of the germline repertoire from data sets covering antibody heavy chain encoding transcripts. The inference process operates under conditions that may limit identification of genes transcribed at low levels. The presence of rare transcripts that would indicate the existence of poorly expressed alleles in haplotypes that otherwise appear to have deleted these genes has been assessed in the present study. Alleles IGHV1-2*05, IGHV1-3*02, IGHV4-4*01, and IGHV7-4-1*01 were all identified as being expressed from multiple haplotypes, but only at low levels, haplotypes that by inference often appeared not to express these genes at all. These genes are thus not as commonly deleted as previously thought. An assessment of the 5’ untranslated region (up to and including the TATA-box), the signal peptide-encoding part of the gene, and the 3’-heptamer suggests that the alleles have no or minimal sequence difference in these regions in comparison to highly expressed alleles. This suggest that they may be able to participate in immunoglobulin gene rearrangement, transcription and translation. However, all four poorly expressed alleles harbor unusual sequence variants within their coding region that may compromise the functionality of the encoded products, thereby limiting their incorporation into the immunoglobulin repertoire. Transcripts based on IGHV7-4-1*01 that had undergone somatic hypermutation and class switch had mutated the codon that encoded the unusual residue in framework region 3 (cysteine 92; located far from the antigen binding site). This finding further supports the poor compatibility of this unusual residue in a fully functional protein product. Indications of a linkage disequilibrium were identified as IGHV1-2*05 and IGHV4-4*01 co-localized to the same haplotypes. Furthermore, transcripts of two of the poorly expressed alleles (IGHV1-3*02 and IGHV4-4*01) mostly do not encode in-frame, functional products, suggesting that these alleles might be essentially non-functional. It is proposed that the functionality status of immunoglobulin genes should also include assessment of their ability to encode functional protein products.

Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

Development of antibody-dependent cell cytotoxicity function in HIV-1 antibodies.

A prerequisite for the design of an HIV vaccine that elicits protective antibodies is understanding the developmental pathways that result in desirable antibody features. The development of antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) is particularly relevant because such antibodies have been associated with HIV protection in humans. We reconstructed the developmental pathways of six human HIV-specific ADCC antibodies using longitudinal antibody sequencing data. Most of the inferred naive antibodies did not mediate detectable ADCC. Gain of antigen binding and ADCC function typically required mutations in complementarity determining regions of one or both chains. Enhancement of ADCC potency often required additional mutations in framework regions. Antigen binding affinity and ADCC activity were correlated, but affinity alone was not sufficient to predict ADCC potency. Thus, elicitation of broadly active ADCC antibodies may require mutations that enable high-affinity antigen recognition along with mutations that optimize factors contributing to functional ADCC activity.

Hepcidin: Structure, Functions And Role In Hematological Disorders

Hepcidin is an essential iron regulatory protein encoded by human antimicrobial peptide gene. Structurally, hepcidin has three parts; the main structural framework, disulphide linkages and N-terminal region. Ferroportin, that allows the entry of iron into the blood, is degraded by hepcidin and inhibits this process. Transferrin receptor 1 and 2 play an important role along with hemochromatosis protein in sensing iron and increasing hepcidin expression. The role of hepcidin is instrumental in iron deficiency anemia. Iron-refractory iron deficiency anemia an autosomal recessive is associated with TMPRSS6 mutation that leads to increased concentration of serum hepcidin despite iron deficiency. In anemia of chronic disorder, hepcidin blocks the entry of iron from macrophages and duodenal enterocytes into blood, thus inhibiting the growth of microorganisms. In β-thalassemia major it has two effects; inhibitory and stimulatory. The inhibitory response is due to ineffective erythropoiesis and stimulatory response is due to multiple transfusions. In hereditary hemochromatosis type II hepcidin production is low due to gene mutation that leads to iron overload. This narrative review is compiled after relevant literature search from electronic databases. The key words used for searching relevant literature include iron, iron deficiency, iron deficiency anemia, iron metabolism, hepcidin, transferrin, causes of iron deficiency anemia and laboratory diagnosis of iron deficiency anemia. Reference hematology books were also consulted.

Modifications to the framework regions eliminate chimeric antigen receptor tonic signaling

Modifications to the framework regions eliminate chimeric antigen receptor tonic signaling

Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains

Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually lead to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody VL domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient VL domain with its nonamyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient VL domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the VL domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity.

Identification and characterization of carbonylation sites in trastuzumab biosimilars

Detection of metal catalyzed carbonylation in proteins is traditionally based on derivatization followed by detection and quantification via spectroscopy or immunodetection. However, these measure only cumulative carbonylation and do not identify the specific sites of modification within the protein. Recently, fluorescein thiosemicarbazide (FTC) based semi-microplate method was adapted for high throughput monitoring of carbonyl content during mAb process development, using size-exclusion chromatography followed by ultraviolet and fluorescence detection. Here, we have examined carbonylation in originators and 4 biosimilars of an IgG1 therapeutic monoclonal antibody, trastuzumab, a first line of therapy for HER2 positive breast cancer. The hyphenated RP-ESI-MS/MS detection was able to identify the location of each of the carbonylated amino acids for all products. The result is a comprehensive map of a total of 27 unique identified carbonylation sites of trastuzumab found across multiple batches of originator as well as marketed biosimilars. Our results demonstrate that although the different carbonylation sites are spread across different domains throughout the mAb sequence, the complementarity determining regions (CDRs) are free of carbonylation and all identified sites lie within the framework region of the variable domain. Moreover, the constant- heavy domain 3 (CH3) region seems to be particularly resistant to process induced carbonylation.

Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv.

e23sFv is a HER2-targeted single-chain variable fragment (scFV) that was characterized as the targeting portion of a HER2-targeted tumour proapoptotic molecule in our previous study. In vitro antibody affinity maturation is a method to enhance antibody affinity either by complementarity-determining region (CDR) mutagenesis or by framework region (FR) engraftment. In the present study, the affinity of e23sFv was enhanced using two strategies. In one approach, site-directed mutations were introduced into the FRs of e23sFv (designated EMEY), and in the other approach e23sFv FRs were substituted with FRs from the most homologous screened antibodies (designated EX1 and EX2). Notably, EX1 derived from the FR engraftment strategy demonstrated a 4-fold higher affinity for HER2 compared with e23sFv and was internalized into HER2-overexpressing cells; however, EMEY and EX2 exhibited reduced affinity for HER2 and decreased internalization potential compared with EX1. The 3D structure of EX1 and the HER2-EX1 complex was acquired using molecular homology modelling and docking and the HER2 epitopes of EX1 and the molecular interaction energy of the EX1-HER2 complex were predicted. In the present study, it was demonstrated that scFv affinity improvement based on sequence alignment was feasible and effective. Moreover, the FR grafting strategy was indicated to be more effective and simple compared with site-directed mutagenesis to improve e23sFv affinity. In conclusion, it was indicated that the affinity-improved candidate EX1 may present a great potential for the diagnosis and treatment of HER2-overexpressing tumours.