Constant Domains Research Articles

TRBC1-CAR T cell therapy in peripheral T cell lymphoma: a phase 1/2 trial.

Relapsed/refractory peripheral T cell lymphomas (PTCLs) are aggressive tumors with a poor prognosis. Unlike B cell lymphomas, treatment of PTCL has not benefited from advances in immunotherapy. This is largely due to a lack of suitable target antigens that discriminate malignant from normal T cells, thus avoiding severe immunosuppression consequent to depletion of the entire T cell compartment. We recently described a targeting strategy based on the mutually exclusive expression of T cell antigen receptor beta-chain constant domain (TRBC) 1 and 2. Selective targeting of the T cell antigen receptor beta-chain expressed by the (clonal) malignancy spares normal T cells expressing the other chain. The LibraT1 study is an ongoing, multicenter, international, single-arm phase 1/2 study of TRBC1-directed autologous chimeric antigen receptor (CAR) T cells (AUTO4) in relapsed/refractory TRBC1-positive PTCL. Primary objectives were assessment of safety and tolerability of AUTO4 infusion. Key secondary endpoints included efficacy, CAR T cell expansion and persistence. Here we describe the findings from dose escalation in LibraT1 in the first ten patients, in a non-prespecified interim analysis. AUTO4 resulted in low frequency of severe immunotoxicity, with one of ten patients developing grade 3 cytokine release syndrome. Complete metabolic response was observed in four of ten evaluable patients, with remissions being durable beyond 1 year in two patients. While an absence of circulating CAR T cells was observed, CAR T cells were readily detected in lymph node biopsy samples from sites of original disease suggesting homing to tumor sites. These results support the continuing exploration of TRBC1 targeting in PTCL. ClinicalTrials.gov registration: NCT03590574 .

Development of imaged capillary isoelectric focusing as a platform mispairing byproduct testing method for asymmetric WuXiBody-based bispecific antibodies

This study explores the application of imaged capillary isoelectric focusing (icIEF) to distinguish and quantify mispairing byproducts in asymmetric WuXiBody-based bispecific antibodies (AsWXbsAbs). Bispecific antibody (BsAb), developed using Knobs-into-Holes (KiH) technology, often result in byproducts such as knob-knob (KK) and hole-hole (HH) homodimers, which share similar sizes with the target BsAb, complicating their separation by traditional methods like size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Our approach leverages the unique pI differences introduced by substituting the CH1/CL domain with the T cell receptor (TCR) constant domain in AsWXbsAbs. This modification enables icIEF to effectively differentiate between the KK and HH homodimers and the target BsAb. Through the construction and expression of heavy and light chain variants, we validated that the experimental pI values aligned with theoretical predictions, confirming icIEF’s capability in distinguishing these entities. Enrichment of in-process K-related and H-related species was achieved, allowing for high-purity samples necessary for icIEF method development. The method was qualified and showed good specificity and linearity, with a quantitation limit of 4 % for K-related species (R2 = 0.9919) and 1 % for H-related species (R2 = 0.9805). This method was used effectively as an in-process test and release assay, proving its simple and quick utility in multiple AsWXbsAbs projects.

Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format.

Rabbit monoclonal antibodies are attractive reagents for research, and have also found use in diagnostic and therapeutic applications. This is owed to their high affinity and specificity, along with their ability to recognize epitopes conserved between mouse and human antigens. Phage display is a powerful method for the de novo generation, affinity maturation, and humanization of rabbit monoclonal antibodies from naive, immune, and synthetic antibody repertoires. Using phagemid family pComb3, a preferred phage display format is chimeric rabbit/human Fab, which consists of rabbit variable domains (VH, Vκ, and Vλ) fused to human constant domains. The human constant domains, CH1 of IgG1 and CL (Cκ or Cλ), not only provide established purification and detection handles but also facilitate higher expression in Escherichia coli compared to the corresponding rabbit constant domains. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of chimeric rabbit/human Fab libraries with randomly combined rabbit variable domains of high sequence diversity, starting from the preparation of total RNA from rabbit spleen and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent chimeric rabbit/human Fab clones. As such, it can be used, for instance, for the generation of either specialized immune or large naive rabbit antibody libraries.

Generation of Antibody Libraries for Phage Display: Human Fab Format.

Phage display is a powerful method for the de novo generation and affinity maturation of human monoclonal antibodies from naive, immune, and synthetic antibody repertoires. The pComb3 phagemid family of phage display vectors facilitates the selection of human monoclonal antibody libraries in the monovalent Fab format, which consists of human variable domains VH and VL (Vκ or Vλ), fused to the human constant domains CH1 of IgG1 and CL (Cκ or Cλ), respectively. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of human Fab libraries with randomly combined human variable domains (VH, Vκ, and Vλ) of high sequence diversity, starting from the preparation of mononuclear cells from blood and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent human Fab clones. As such, it can be used, for instance, for the generation of a large naive human Fab library from healthy individuals or for the generation of a specialized immune human Fab library from individuals with an endogenous antibody response of interest.

Development, physico-chemical characterization, and in vivo stability of a novel aglycosylated monoclonal antibody targeting FAM19A5

AbstractIntroducing aglycosylation into therapeutic monoclonal antibodies (mAbs) can prevent side effects associated with fragment crystallizable (Fc)-mediated effector functions. This modification induces structural changes in the heavy chain constant domain 2-constant domain 3 within Fc regions, which decreases antibody stability at acidic pH and high temperature. In this study, NS101, a novel aglycosylated mAb targeting family with sequence similarity 19, A5 (FAM19A5) for neurological diseases was evaluated with respect to its developability and in vivo stability as therapeutics. When recombinant CHO cells producing NS101 were cultivated using a fed-batch mode in a 500 L bioreactor, cell growth and mAb production profiles were consistent across three consecutive runs. NS101, thus produced, features an additional intra-disulfide bond in the heavy chain complementarity-determining region 3, contributing strong and sophisticated binding to the cryptic epitope. The melting temperature (Tm) of NS101 was lower than that of commercial glycosylated therapeutic mAbs, but NS101 showed better stability at 4 °C for 36 months. The binding affinity of NS101 to FAM19A5 and neonatal Fc receptor were comparable to those of glycosylated NS101. In addition, in three human cohort groups receiving 6, 12, and 24 mg/kg of NS101, the mean half-life was 22 days, and NS101 exhibited in vivo stability, considering that the half-lives of commercialized therapeutic mAbs and endogenous IgGs are 2–4 weeks and 21 days, respectively. Taken together, the results obtained here demonstrate that NS101, a novel aglycosylated mAb, has potential as a therapeutic agent for neurological diseases.

Conformational Differences in the Light Chain Constant Domain of Immunoglobulin G and Free Light Chain May Influence Proteolysis in AL Amyloidosis

Immunoglobulin light chain amyloidosis (AL) is a life-threatening disease caused by the deposition of light chain (LC) and its fragments containing variable (VL) and portions of constant (CL) domains. AL patients feature either monoclonal free LCs (FLCs) circulating as covalent and noncovalent homodimers, or monoclonal immunoglobulin (Ig) wherein the LC and heavy chain (HC) form disulfide-linked heterodimers, or both. The role of full-length Ig in AL amyloidosis is unclear as prior studies focused on FLC or VL domain. We used a mammalian cell-based expression system to generate four AL patient-derived full-length IgGs, two non-AL IgG controls, and six corresponding FLC proteins derived from an IGLV6-57 germline precursor. Comparison of proteins’ secondary structure, thermal stability, proteolytic susceptibility, and disulfide link reduction suggested the importance of local vs. global conformational stability. Analysis of IgGs vs. corresponding FLCs using hydrogen–deuterium exchange mass spectrometry revealed major differences in the local conformation/dynamics of the CL domain. In all IgGs vs. FLCs, segments containing β-strand and α-helix βAC-αACBC were more dynamic/exposed while segment βDC-βEC was less dynamic/exposed. Notably, these segments overlapped proteolysis-prone regions whose in vivo cleavage generates LC fragments found in AL deposits. Altogether, the results suggest that preferential cleavage in segments βAC-αACBC of FLC or βDC-βEC of LC in IgG helps generate amyloid protein precursors. We propose that protecting these segments using small-molecule stabilizers, which bind to the interfacial cavities CL-CL in FLC and/or CL-CH1 in IgG, is a potential therapeutic strategy to complement current approaches targeting VL-VL or VL-CL stabilization of LC dimer.

Functional comparison of Fc-engineering strategies to improve anti-HIV-1 antibody effector functions

Substantial reduction of the intact proviral reservoir is essential towards HIV-1 cure. In vivo administration of broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) trimer can decrease the viral reservoir, through Fc-mediated killing of infected cells. In this study, we compared three commonly used antibody engineering strategies to enhance Fc-mediated effector functions: (i) glyco-engineering, (ii) protein engineering, and (iii) subclass/hinge modifications in a panel of anti-HIV-1 antibodies. We found that antibody-dependent cellular phagocytosis (ADCP) was improved by elongating the hinge domain and switching to an IgG3 constant domain. In addition, potent NK cell activation and ADCC activity was observed for afucosylated antibodies and antibodies bearing the GASDALIE mutations. The combination of these engineering strategies further increased NK cell activation and induced antibody dependent cytotoxicity (ADCC) of infected cells at low antibody concentrations. The bNAb N6 was most effective at killing HIV-1 infected cells, likely due to its high affinity and optimal angle of approach. Overall, the findings of this study are applicable to other antibody formats, and can aid the development of effective immunotherapies and antibody-based treatments for HIV-1 cure strategies.

A genetically engineered mouse/human chimeric antibody targeting CD99 enhances antibody-dependent cellular phagocytosis against human mantle cell lymphoma Z138 cells

Background: Mantle cell lymphoma (MCL) is an aggressive form of B-cell nonHodgkin lymphoma. The elimination of MCL cells via phagocytosis is essential for cancer eradication. Therefore, discovering novel targeted antibodies that can induce phagocytosis is needed. We have demonstrated that our in-houseproduced mouse anti-CD99 mAb clone MT99/3 could induce potent anticancer activities against MCL cell lines in both in vitro and in vivo mouse xenograft models. Nevertheless, for use in humans, the mouse mAb needs to be transformed into a mouse/human chimeric mAb that contains a human Fc region to activate human immune effector functions, especially macrophage-mediated phagocytosis. Antibody-dependent cellular phagocytosis (ADCP) mediated by mouse/human chimeric mAb MT99/3 against MCL has not been previously reported. Objective: This study aimed to genetically engineer a mouse/human chimeric antibody against human CD99 derived from mouse mAb MT99/3 and to evaluate its effect in mediating the ADCP mechanism for eradicating MCL cells in vitro using monocyte-derived macrophages. Materials and methods: The expression plasmid to produce chimeric anti-CD99 antibody, ChAbMT99/3, was constructed by fusing the variable domains of mouse mAb MT99/3 with the constant domains of human IgG1 and the constant domains of kappa light chain. ChAbMT99/3 was expressed in the stable human expression system based on HEK293T cells. ChAbMT99/3 was purified from the culture supernatant of ChAbMT99/3-expressing HEK293T cells using Protein G chromatography. The purity and structure of ChAbMT99/3 were verified by SDS-PAGE and western blotting. The binding specificity and activity were determined by staining with cells expressing recombinant and native human CD99. The anticancer activity of ChAbMT99/3 in mediating the ADCP mechanism against MCL cell line Z138 using human monocytederived macrophages was evaluated. Results: We successfully constructed the plasmid to produce ChAbMT99/3. Human HEK293T cells stably expressing ChAbMT99/3 were established. The ChAbMT99/3- expressing HEK293T cells could secrete ChAbMT99/3 into the culture supernatant. The high purity and complete IgG structure of ChAbMT99/3 were obtained from the purification process. Crucially, this chimeric antibody retained its binding reactivity to recombinant and native human CD99. In addition, the produced ChAbMT99/3, upon binding to MCL cells, significantly enhanced ADCP against MCL cell line Z138 in a dose-dependent manner. Conclusion: The production of a mouse/human chimeric antibody against human CD99 derived from mouse mAb MT99/3 was successful. The engineered antibody could mediate ADCP activity against MCL cells. The produced ChAbMT99/3 might be a promising therapeutic candidate for MCL treatment.

Nanobody-based heavy chain antibodies and chimeric antibodies.

Nanobodies are the products of an intriguing invention in the evolution of immunoglobulins. This invention can be traced back approximately 45 million years to the common ancestor of extant dromedaries, camels, llamas, and alpacas. Next to conventional heterotetrameric H2L2 antibodies, these camelids produce homodimeric nanobody-based heavy chain antibodies, composed of shortened heavy chains that a lack the CH1 domain. Nanobodies against human target antigens are derived from immunized animals and/or synthetic nanobody libraries. As a robust, highly soluble, single immunoglobulin domain, a nanobody can easily be fused to another protein, for example to another nanobody and/or the hinge and constant domains of other immunoglobulins. Nanobody-derived heavy chain antibodies hold promise as a new form of immunotherapeutics.

Characterization of IgD and IgT with their expressional analysis following subtype II megalocytivirus vaccination and infection in rock bream (Oplegnathus fasciatus)

In this study, heavy chain genes of IgD and IgT were sequenced and characterized their gene expression in rock bream (Oplegnathus fasciatus). Rock bream (RB)-IgD cDNA is 3319 bp in length and encodes a leader region, variable domains, a μ1 domain, and seven constant domains (CH1–CH7). A membrane-bound (mIgT) and secretory form (sIgT) of RB-IgT cDNAs are 1902 bp and 1689 bp in length, respectively, and encode a leader region, variable domains, four constant domains (CH1–CH4) and C-terminus. Their predicted 3D-structure and phylogenetic relation were similar to those of other teleost. In healthy fish, RB-IgD and mIgT gene expressions were higher in major lymphoid organs and blood, while RB-sIgT gene was more highly expressed in midgut. IgT expressing cells were detected in melano-macrophage centers (MMC) of head kidney in immunohistochemistry analysis. Under immune stimulation in vitro, RB-IgD and IgT gene expressions were upregulated in head kidney and spleen cells by bovine serum albumin or a rock bream iridovirus (RBIV) vaccine. In vivo, their expressions were significantly upregulated in head kidney, blood, and gill upon vaccination. Especially, RB-mIgT gene expression in head kidney and blood was upregulated at day 3 after vaccination while upregulated at earlier time point of day 1 by challenge with RBIV. This may suggest that memory cells might be produced during the primary response by vaccination and rapidly proliferated by secondary immune response by viral infection. RB-sIgT gene expression was highly upregulated in peripheral blood in vaccinated fish after viral infection, indicating that IgT plays an important role in systemic immune response as well as mucosal immune system. Our findings provide information on the role of RB-IgT in adaptive immunity during vaccination and viral infection in the vaccinated fish.

Overcompensation of transient and permanent death rate increases in age-structured models with cannibalistic interactions

There has been renewed interest in understanding the mathematical structure of ecological population models that lead to overcompensation, the process by which a population recovers to a higher level after suffering a permanent increase in predation or harvesting. Here, we apply a recently formulated kinetic population theory to formally construct an age-structured single-species population model that includes a cannibalistic interaction in which older individuals prey on younger ones. Depending on the age-dependent structure of this interaction, our model can exhibit transient or steady-state overcompensation of an increased death rate as well as oscillations of the total population, both phenomena that have been observed in ecological systems. Analytic and numerical analysis of our model reveals sufficient conditions for overcompensation and oscillations. We also show how our structured population partial integrodifferential equation (PIDE) model can be reduced to coupled ODE models representing piecewise constant parameter domains, providing additional mathematical insight into the emergence of overcompensation.

The constant domain of CRTAM is essential for high-affinity interaction with Nectin-like 2

CRTAM (Class-I MHC restricted T cell-associated molecule) is a member of the Nectin-like family, composed of two extracellular domains, one constant domain (IgC) and another variable domain (IgV), expressed in activated CD8 T cells, epithelial cells, natural killer (NK) cells, and in a subpopulation of CD4 T cells. CRTAM recognizes the ligand Nectin-like 2 (Necl2) through the IgV domain. However, the role of the IgC domain during this ligand recognition has yet to be understood. In this study, we show the purification of soluble-folded Ig domains of CRTAM, and we demonstrate that the IgC domain forms a homodimer in solution via hydrophobic interactions. By surface plasmon resonance (SPR) analysis, we also demonstrate that CRTAM binds to Necl2 with an affinity of 2.16 nM. In conclusion, CRTAM's IgC is essential for a high-affinity interaction with Necl-2.

The reggeon model with the pomeron and odderon: renormalization group approach

The Regge–Gribov model of the pomeron and odderon in nontrivial transverse space is studied by the renormalization group technique. The single-loop approximation is adopted. Five real fixed points are found, and the high-energy behavior of the propagators is correspondingly calculated. As without the odderon, the asymptotic is modulated by logarithms of energy in certain rational powers. Movement of coupling constants away from the fixed points is investigated both analytically (close to the fixed points) and numerically (far away). In the former case, attraction occurs only in restricted domains of initial coupling constants. More generally, in one third of the cases the coupling constants instead grow large, indicating the breakdown of the single-loop approximation.

Helical superstructures between amyloid and collagen in cardiac fibrils from a patient with AL amyloidosis

Systemic light chain (LC) amyloidosis (AL) is a disease where organs are damaged by an overload of a misfolded patient-specific antibody-derived LC, secreted by an abnormal B cell clone. The high LC concentration in the blood leads to amyloid deposition at organ sites. Indeed, cryogenic electron microscopy (cryo-EM) has revealed unique amyloid folds for heart-derived fibrils taken from different patients. Here, we present the cryo-EM structure of heart-derived AL amyloid (AL59) from another patient with severe cardiac involvement. The double-layered structure displays a u-shaped core that is closed by a β-arc lid and extended by a straight tail. Noteworthy, the fibril harbours an extended constant domain fragment, thus ruling out the variable domain as sole amyloid building block. Surprisingly, the fibrils were abundantly concatenated with a proteinaceous polymer, here identified as collagen VI (COLVI) by immuno-electron microscopy (IEM) and mass-spectrometry. Cryogenic electron tomography (cryo-ET) showed how COLVI wraps around the amyloid forming a helical superstructure, likely stabilizing and protecting the fibrils from clearance. Thus, here we report structural evidence of interactions between amyloid and collagen, potentially signifying a distinct pathophysiological mechanism of amyloid deposits.

Diagnostics of power cable insulation by using the methods of dielectric spectroscopy: overview of physical basis and features of practical application

The article provides an overview of the physical foundations and features of the application of diagnostic methods for the insulation of power cables using dielectric spectroscopy methods in the frequency and time domains. The relationship between the depolarization currents and the frequency spectrum of the complex dielectric constant is shown. An analysis of the factors determining the frequency dependence of the tangent angle of dielectric losses of paper and polyethylene insulation of power cables is given. The main types of reaction of dielectric materials with different levels of development of water channels at the level and frequency of the applied voltage are listed. According to the results of the analysis, it is shown that for high-voltage measurements, one of the factors that complicates the diagnosis of the insulation of power cables by analyzing the frequency dependences of the capacitance and the tangent of the dielectric loss angle is the distortion of the sinusoidal current through the dielectric due to the nonlinear dependence of the complex dielectric constant on the level of the applied voltage. A comparative analysis of dielectric spectroscopy methods in the frequency domain, involving high-voltage and low-voltage measurements of the complex dielectric constant, was carried out. The correlation between the tangent of the dielectric loss angle of paper insulation and its mechanical characteristics is shown

Maximal L p -regularity for evolution equations governed by non-autonomous forms in weighted spaces

We consider the maximal regularity for non-autonomous Cauchy problem u ′ ( t ) + A ( t ) u ( t ) = f ( t ) , t -a.e. , u ( 0 ) = u 0 . Each operator A ( t ) arises from a time depending on a sesquilinear form a ( t ) on a Hilbert space H with a constant domain V . We show maximal L p − regularity result in temporally weighted L p -spaces for 2 < p < ∞ and other regularity properties for the solution of the previous problem under sufficient regularity assumption on the forms and the initial value u 0 , which is significantly weaker than those from previous contributions. Our results are motivated by boundary value problems.

On a sharper bound on the stability of non-autonomous Schrödinger equations and applications to quantum control

We study the stability of the Schrödinger equation generated by time-dependent Hamiltonians with constant form domain. That is, we bound the difference between solutions of the Schrödinger equation by the difference of their Hamiltonians. The stability theorem obtained in this article provides a sharper bound than those previously obtained in the literature. This makes it a potentially useful tool for time-dependent problems in Quantum Physics, in particular for Quantum Control. We apply this result to prove two theorems about global approximate controllability of infinite-dimensional quantum systems. These results improve and generalise existing results on infinite-dimensional quantum control.

Perturbations of non-autonomous second-order abstract Cauchy problems

In this paper we present time-dependent perturbations of second-order non-autonomous abstract Cauchy problems associated to a family of operators with constant domain. We make use of the equivalence to a first-order non-autonomous abstract Cauchy problem in a product space, which we elaborate in full detail. As an application we provide a perturbed non-autonomous wave equation.

Development of an enabling platform biotechnology for the production of proteins.

Protein-based drugs are a mainstay of modern medicine. In contrast to antibodies, most of these need highly individualized production processes which often limits their development. Here, we develop an immunoglobulin domain tag (i-Tag), which can be fused to any protein of interest. This tag is made of a linear arrangement of antibody light chain constant domains. It enhances expression as well as secretion of the fusion partner and allows for simple purification of several structurally and functionally distinct fusion proteins. Furthermore, it improves the biophysical characteristics of most fusion proteins tested, is inert, and does not compromise the fusion partners' functionality. Taken together, the i-Tag should facilitate the development of biopharmaceuticals and diagnostic proteins otherwise lacking a common structural element.

Systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus)

Systemic amyloid light-chain (AL) amyloidosis is an infrequent disease in which amyloid fibrils derived from the immunoglobulin light chain are deposited in systemic organs, resulting in functional impairment. This disease has been notably uncommon in animals, and nonhuman primates have not been reported to develop it. In this study, we identified the systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus) and analyzed its pathogenesis. Amyloid deposits were found severely in the submucosa of the large intestine, lung, mandibular lymph nodes, and mediastinal lymph nodes, with milder lesions in the liver and kidney. Mass spectrometry-based proteomic analysis revealed an abundant constant domain of the immunoglobulin kappa chain in the amyloid deposits. Immunohistochemistry further confirmed that the amyloid deposits were positive for immunoglobulin kappa chains. In this animal, AL amyloidosis resulted in severe involvement of the gastrointestinal submucosa and lymph nodes, which is consistent with the characteristics of AL amyloidosis in humans, suggesting that AL amyloid may have a similar deposition mechanism across species. This report enhances the pathological understanding of systemic AL amyloidosis in animals by providing a detailed characterization of this disease based on proteomic analysis.